The present invention relates to the general fields of nucleic acid analysis in a biological sample, particularly the determination of genetic aberrations using a multiplex ligation-dependent method.

Nucleic acid analysis in a sample has many applications in both basic research and clinical settings. For example, nucleic acid analysis may be used to identify genetic aberrations in a patient blood sample. Genetic aberrations account for a large number of pathological conditions, including syndromic disorders (e.g., Down's syndrome) and diseases (e.g., breast cancer). Genetic aberrations may be, but is not limited to, single nucleotide polymorphisms (SNPs), gene copy number variants (CNVs), chromosomal rearrangements (e.g., insertions, deletions and duplications), gene mutations (e.g., single nucleotide changes, insertions, and deletions), nucleic acid modifications (e.g., methylation, acetylation and phosphorylations), gene over-expression (e.g., an oncogenes such as RAS), and gene under-expression (e.g., a tumor suppressor gene such as p53). In addition, nucleic acid analysis may be used to identify pathogens and transgenic organisms.

Many techniques have been developed for nucleic acid analysis. For one example, techniques such as oligonucleotide ligation assay (OLA) and ligation chain reaction (LCR) have been used to detect SNPs. See, e.g., Abravaya, et al., 1995, Detection of point mutations with a modified ligase chain reaction (Gap-LCR), Nucleic Acids Res. 23:675-82; Landegren et al., 1988, A ligase-mediated gene detection technique, Science. 241:1077-80; Schwartz et al., 2009, Identification of cystic fibrosis variants by polymerase chain reaction/oligonucleotide ligation assay, J Mol Diagn. 11:211-5. WO2010114599 A1. For another example, microarrays and high throughput DNA sequencing may be used to detect chromosome rearrangements and gene copy numbers. See, e.g., Agilent Human Genome CGH Microarray (Agilent Technologies, Inc., Santa Clara, CA), and the Illumina HiSeq DNA Sequencing Assays (Illumina, Inc., San Diego, CA). However, the nucleic acid analysis using these known techniques are either not easily multiplexed (e.g., the OLA and LCR methods), or time-consuming, expensive and/or inaccurate (e.g., microarrays and high throughput DNA sequencing).

Therefore, it is desirable to have a new technique that makes the nucleic acid analysis easily multiplexed and efficient. An object of the present invention is to provide methods and kits for multiplexed and efficient nucleic acid analysis in a sample. The methods and kits based on the present invention may be suitable for adaptation and incorporation into a compact device or instrument for use in a laboratory or a clinical setting, or in the field.

No reference cited in this background section is to be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the references cited herein do not constitute prior art under the applicable statutory provisions.

The present invention is defined by the appendent claims.

In one aspect, the present invention provides methods for multiplex nucleic acids analysis in a sample. In one embodiment, nucleic acids in a sample are assayed by adding a set of probes into the sample to form a mixture; denaturing nucleic acids in the mixture; hybridizing the set of probes to the complementary regions of target nucleic acids; performing a ligation reaction on the hybridized probes to form a third probe; amplifying the third probe with a set of primers to obtain an amplification product; and assaying the presence, absence or quantity of the target nucleic acids in the sample by determining the presence, absence or quantity of the third probe in the amplification product.

In each set of probes, there are at least a first probe having a first portion at least partially complementary to a first region of the target nucleic acid in the sample and a second portion forming a first primer binding site (herein also referred to as primer binding sequence); and a second probe having a first portion at least partially complementary to a second region of the target nucleic acid in the sample and a second portion forming a second primer binding site. The 5' end of the first probe is essentially adjacent to the 3' end of the second probe when both probes are hybridized to the target nucleic acid.

The set of primers used to amplify the ligation product comprises a first primer at least partially complementary to the first primer binding site; and a second primer at least partially complementary to the second primer binding site.

In some embodiments, the method is used to assay multiple target nucleic acids in a multiplexed manner. For example, the presence, absence or quantity of more than about 48, 96, 192, 384 or more target nucleic acids in the sample may be assayed in a multiplexed manner. The probes corresponding to all the target nucleic acids may be added to the sample and a single ligation reaction is performed to obtain ligation products for all the target nucleic acids.

The sample to be assayed may be a sample of a bodily fluid, a biopsy tissue, or a paraffin-embedded tissue from an animal. The bodily fluid may be blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, or genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, or combinations thereof from an animal, e.g., a human.

In some embodiments, nucleic acids are extracted from the sample before forming a mixture with the set of probes. The target nucleic acid may be DNA, RNA, or cDNA. The RNA may be reverse transcribed into cDNA before forming a mixture with the set of probes.

In some embodiments, at least one primer of the set of primers is labeled with a detectable moiety, e.g., an oligonucleotide tag or a fluorescent dye. The fluorescent dye may be FAM (5-or 6-carboxyfluorescein), VIC, NED, PET, Fluorescein, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, or Yakima Yellow. In other embodiments, at least one primer of the set of primers includes a stuffer sequence. The stuffer sequence in some primers may have about 10 to about 500 nucleotides. The stuffer sequence in other primers may be about 10 to about 60 nucleotides. In some preferred embodiments, no primer has more than about 125 nucleotides, preferably about 75 nucleotides. In still other embodiments, at least one primer of the set of primers includes an oligonucleotide comprising a sequence GTTTCTT or a functional equivalent variant of the oligonucleotide comprising a sequence GTTTCTT.

In further embodiments, at least one probe of the set of probes includes a stuffer sequence. In some instances, the stuffer sequence may have about 1 to about 200 nucleotides. In other instances, the stuffer sequence has about 1 to about 55 nucleotides. In some preferred embodiments, the third probe has no more than about 250 nucleotides. In other preferred embodiments, the third probe has no more than about 140 nucleotides. In still some preferred embodiments, no probe has more than about 125 nucleotides. In still some other preferred embodiments, no probe has more than about 70 nucleotides. In some further preferred embodiments, no probe has more than about 60 nucleotides.

In some embodiments, the multiplexity of nucleic acid analysis may result from the modifications that at least one primer of the set of primers is labeled with a detectable moiety and that at least one primer of the set of primers includes a stuffer sequence. In other embodiments, the multiplexity of nucleic acid analysis may result from the modifications that at least one primer of the set of primers is labeled with a detectable moiety and that at least one probe of the set of probes includes a stuffer sequence. In still other embodiments, the multiplexity of nucleic acid analysis may result from the modifications that at least one primer of the set of primers includes a stuffer sequence and that at least one probe of the set of probes includes a stuffer sequence. In some preferred embodiments, the multiplexity of nucleic acid analysis may result from the modifications that at least one primer of the set of primers is labeled with a detectable moiety, that at least one primer of the set of primers includes a stuffer sequence, and that at least one probe of the set of probes includes a stuffer sequence. In these embodiments, the determination of the presence, absence or quantity of the third probe in the amplification product may be carried out by measuring the presence, absence or quantity of the third probe in the amplification product on the basis of detectable moieties, fragment sizes, or both. The measurement may be carried out using capillary electrophoresis.

The method according to the present invention may be applied in many nucleic acid assays. For one example, single nucleotide polymorphisms in target nucleic acids may be detected using the method. For this purpose, an allele-specific probe corresponding to the single nucleotide polymorphism may be designed. In addition, a mismatch may be introduced at one or more positions, e.g., the second, third, or fourth nucleotide away from the polymorphic nucleotide in the allele-specific probe so that the specificity of the probe binding is enhanced.

For a second example, copy number variants in target nucleic acids may be detected using the method. In one instance, a deletion of one or more exons in the dystrophin gene may be determined using one or more probe pairs selected from SEQ ID NOs: 158-541.

For a third example, the method may be used to screen for an unknown point mutation, insertion, or deletion of nucleotides. In one instance, a point mutation in the dystrophin gene may be screened using one or more probe pairs selected from SEQ ID NOs: 158-541.

For a fourth example, the method may be used to measure the presence, absence or relative amount of messenger RNA, methylated DNA, a pathogen or a transgenic organism. A pathogen may be a virus or a bacterium. A transgenic organism may be a transgenic plant such as transgenic corn, transgenic rice, transgenic soybean and transgenic cotton, or a transgenic animal such as a transgenic cow, a transgenic pig, a transgenic sheep and a transgenic dog.

In some embodiments, the steps of denaturing, hybridization and ligation may be repeated about 1 to about 100 times. The denaturing step may be carried at about 90°C to about 99°C for about 5 seconds to about 30 minutes, and the hybridization and the ligation steps may be carried out simultaneously at about 4°C to about 70°C for about 1 minute to about 48 hours. In a preferred embodiment, the denaturing step is carried at about 95°C for about 30 seconds, and the hybridization and the ligation steps are carried out simultaneously at about 58°C for about 4 hours, and the steps of denaturing, hybridization and ligation are repeated 4 times.

In another aspect of the present invention, a method is provided for detecting small copy number changes. In one embodiment, for purpose of detecting small copy number changes of a target nucleic acid, two or more sets of probes are used to hybridize to two or more target sites in the same target nucleic acid, with each set of probes hybridizing to a different target site. The small copy number changes may be, for example, a quantitative variation of the target nucleic acid between two samples. In some instances, the quantitative variation of the target nucleic acid is about 0.1% to about 30%.

In some embodiments, for each set of probes hybridizing to each target site in the same target nucleic acid, one or more sets of reference probes are used to hybridize to one or more reference target sites, with each set of reference probes hybridizing to a different reference target site. For example, about 1 to about 100 sets of reference probes are used for an individual target site. In some instances, about 6 sets of reference probes are used for an individual target site.

In some embodiments, the same set of primers is used to amplify a group of ligation products (also referred to as the third probes). The ligation products may be formed from one or more sets of probes hybridizing to one or more target sites and from one or more sets of reference probes hybridizing to one or more reference target sites. For example, in one group of ligation products, there may be ligation products formed from about 1 to about 100 sets of probes hybridizing to gene target sites of interest and from about 1 to about 100 sets of reference probes hybridizing to reference target sites.

In other embodiments, about 50 to about 500 sets of probes hybridizing to about 50 to about 500 target sites on a target nuceic acid are used to detect small copy number variation of the target nucleic acid in a sample. In this case, multiple groups of ligation products formed from a plurality of probes for targets site and probes for reference sites may be obtained. For each group of ligation products, the same primer pair may be used to amply the ligation products in that group.

In some preferred embodiments, the target nucleic acids are from human chromosome 21, human chromosome 18, human chromosome 13, human chromosome region 22q11.2, or the pseudoautosomal regions of human chromosomes X or Y. As such, the method according to the present invention may be used to detect fetal aneuploidy for chromosomes 21, 18, 13, X, Y and 22q11.2 in a maternal blood or urine sample. In one instance, the method is used to detect fetal Down's syndrome in a maternal blood or urine sample using one or more probe pairs selected from SEQ ID NOs: 559-942.

In a further aspect of the present invention, a kit for assaying nucleic acids in a sample is provided. In one embodiment, the kit includes one or more sets of probes corresponding to a target nucleic acid; one or more sets of primers for amplifying the third probe; optionally reagents including a ligase, a buffer for a ligation reaction, a DNA polymerase, a buffer for polymerase chain traction or a combination thereof; and optionally a brochure containing instructions of using the kit.

In some embodiments, the set of probes includes a first probe having a first portion at least partially complementary to a first region of a target nucleic acid in the sample and a second portion forming a first primer binding site; and a second probe having a first portion at least partially complementary to a second region of the target nucleic acid in the sample and a second portion forming a second primer binding site. The 5' end of the first probe may be essentially adjacent to the 3' end of the second probe when both probes are hybridized to the target nucleic acid and the first and the second probes may be ligated to form a third probe. In some instances, at least one probe of the set of probes includes a stuffer sequence. The stuffer sequence may have about 1 to about 200 nucleotides.

In other embodiments, the set of primers includes a first primer at least partially complementary to the first primer binding site; and a second primer at least partially complementary to the second primer binding site. In some instances, at least one primer of the set of primers is labeled with a detectable moiety, e.g., an oligonucleotide tag or a fluorescent dye. The fluorescent dye may be FAM (5-or 6-carboxyfluorescein), VIC, NED, PET, Fluorescein, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, or Yakima Yellow. In still other embodiments, at least one primer of the set of primers includes a stuffer sequence. The stuffer sequence has about 10 to about 500 nucleotides.

In some embodiments, the kit is for detecting Duchenne muscular dystrophy and includes one or more sets of probes comprising probe pairs selected from SEQ ID NOs: 158-541. In other embodiments, the kit is for detecting fetal Down's syndrome in a maternal blood sample and includes one or more sets of probes comprising probe pairs selected from SEQ ID NOs: 559-942.

• Figure 1 is a schematic flowchart depicting the method of increasing the multiplexity by employing fluorescent dye labeled primers for amplifying ligation products.
• Figure 2 is a schematic flowchart depicting the method of increasing multiplexity by employing primers with stuffer sequences for amplifying ligation products.
• Figure 3 is a schematic flowchart depicting the method of increasing the fold of multiplexing by employing both fluorescent dye labeled primers and primers with stuffer sequences for amplifying ligation products.
• Figure 4 is a schematic flowchart depicting an exemplary analysis of 48 SNPs in a multiplex assay by employing both fluorescent dye labeled primers and primers with stuffer sequences for amplifying ligation products. X and Y refer to primer binding sites on the probes. LSHS refers to locus-specific hybridization sequences. Stuffer A refers to an allelic-specific stuffer sequence of 2 nucleotides in length. Stuffer L1 and stuffer L2 refer to stuffer sequences used for adjusting the size of the ligated probe. ASHS refers to allele-specific hybridization sequences. F1, F2, F3 and F4 refer to forward primers labeled with blue, green, yellow and red fluorescent dyes, respectively. R1 and R2 refer to reverse primers with different stuffer sequences. F1/R1(6) refers to 12 amplification products using the F1 and R1 primer pair based on 12 ligation products from 6 SNP loci with two SNP alleles per SNP locus. Similar interpretations stand for F2/R1(6), F3/R1(6), F4/R1(6), F1/R2(6), F2/R2(6), F3/R2(6), and F4/R2(6).
• Figure 5 is a schematic flowchart depicting an exemplary analysis of 96 CNVs in a multiplex assay by employing both fluorescent dye labeled primers and primers with stuffer sequences for amplifying ligation products. X and Y refer to primer binding sites on the probes. LSHS refers to locus-specific hybridization sequences. Stuffer L1 and stuffer L2 refer to stuffer sequences used for adjusting the size of the ligated probe. F1, F2, F3 and F4 refer to forward primers labeled with blue, green, yellow and red fluorescent dyes, respectively. R1 and R2 refer to reverse primers with different stuffer sequences. F1/R1(12) refers to 12 amplification products using the F1 and R1 primer pair based on 12 ligation products from 12 CNVs. Similar interpretations stand for F2/R1(12), F3/R1(12), F4/R1(12), F1/R2(12), F2/R2(12), F3/R2(12), and F4/R2(12).
• Figure 6 is a schematic flowchart depicting an exemplary mutations screening analysis of 96 fragments overlapping upon the target region in a multiplexed assay by employing both fluorescent dye labeled primers and primers with stuffer sequences for amplifying ligation products. The labels refer to the similar components as detailed in Figure 5.
• Figure 7 is a schematic flowchart depicting an exemplary gene expression analysis of 96 mRNAs in a multiplexed assay by employing both fluorescent dye labeled primers and primers with stuffer sequences for amplifying ligation products. The labels refer to the similar components as detailed in Figure 5.
• Figure 8 is a schematic flowchart depicting an exemplary analysis of 96 nucleic acid targets from pathogens or transgenic plants/animals in a multiplexed assay by employing both fluorescent dye labeled primers and primers with stuffer sequences for amplifying ligation products. The labels refer to the similar components as detailed in Figure 5.
• Figure 9 is a schematic flowchart depicting an exemplary analysis of 96 methylation target sites in a multiplexed assay by employing both fluorescent dye labeled primers and primers with stuffer sequences for amplifying ligation products. The labels refer to the similar components as detailed in Figure 5.
• Figures 10A, 10B, 10C, 10D, and 10E are electrophoresis chromatograms depicting the electrophoresis pattern of the amplification products in a multiplexed assay of 48 SNPs. Figure 10A is an electrophoresis chromatogram of all 96 amplification products. Figures 10B, 10C, 10D and 10E are electrophoresis chromatograms for 24 amplification products produced with primers labeled with blue, green, yellow and red fluorescent dyes, respectively.
• Figures 11A, 11B, 11C, 11D, 11E, 11F, 11G and 11H are electrophoresis chromatograms depicting the electrophoresis pattern of the amplification products in a multiplexed assay of 192 copy number variants. Figure 11A and 11B are electrophoresis chromatograms of all amplification products for the control sample panel A and panel B, respectively. Figure 11C and 11D are electrophoresis chromatograms of all amplification products for the patient sample panel A and panel B, respectively. Figures 11E and 11F are electrophoresis chromatograms of amplification products labeled with blue fluorescent dyes for the control and the patient samples, respectively. 11G and 11H are electrophoresis chromatograms for amplification products produced with primers labeled with green fluorescent dyes for the control and the patient samples, respectively. The arrows in Figures 11F and 11H refer to the missing peaks corresponding to gene target sites.
• Figure 12A shows a chart depicting the calculated copy numbers of all gene target sites in the dystrophin gene. The X axis refers to names of each gene target site. The Y axis refers to the calculated copy number for each gene target site. Figure 12B is a close-up view of a part of the chart in Figure 12A, showing the target sites E01A to E16B. Figure 12C shows the two chromatograms including the peak for DMD_E07B gene target site: one from a healthy control and the other from a male DMD patient. Figure 12D shows a chromatogram of DNA sequencing result of the region surrounding the DMD_07EB gene target site.
• Figures 13A, 13B, 13C, 13D, 13E, 13F, 113G, 13H, 13I, and 13J are electrophoresis chromatograms depicting the electrophoresis pattern of the amplification products for measuring human chromosome 21 copy number in a control DNA sample. Figure 13A and 13F are electrophoresis chromatograms of all 96 amplification products for the control sample panel A and panel B, respectively. Figures 13B, 13C, 13D, and 13E are electrophoresis chromatograms of the 24 amplification products from the control sample panel A separately labeled with blue, green, yellow and red fluorescent dyes, respectively. Figures 13G, 13H, 13I, and 13J are electrophoresis chromatograms of the 24 amplification products from the control sample panel B separately labeled with blue, green, yellow and red fluorescent dyes, respectively.
• Figure 14 shows a chart depicting the calculated human chromosome 21 copy number in the five DNA samples. The X axis refers to the five samples: M0, M2, M4, M8, and M16. The Y axis refers to the calculated copy numbers. For each DNA sample, the testing was repeated three times. The p values were derived from Student's t test with the average copy number for M0 DNA sample as the reference.
• Figure 15 shows a graph depicting the lineal correlation between the calculated copy number and the expected copy number for the five DNA samples.

In one aspect, the present invention provides methods for multiplex nucleic acid analysis. In one embodiment, the method for multiplex analysis of target nucleic acids in a sample includes the steps of preparing nucleic acids that are quantitatively and qualitatively correlated to a plurality of target nucleic acids in the sample and determining quantitatively and qualitatively the nucleic acids thus prepared.

As used herein, the phrase "multiplex" or grammatical equivalents refers to the quantitative and qualitative determination of more than one target nucleic acids of interest in a sample. In one embodiment "multiplex" refers to at least about 48 different target sequences. In another embodiment "multiplex" refers to at least about 96 different target sequences. In further embodiment "multiplex" refers to at least about 192 different target sequences. In still further embodiments, "multiplex" refers to at least about 384 different target sequences. In yet still further embodiment "multiplex" refers to at least about 500 to about 100,000 different target sequences.

As used herein, the sample in which target nucleic acids exist may be a sample from a subject, including, but not limited to, bodily fluids (e.g., blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid and combinations thereof); environmental samples (e.g., air, agricultural, water and soil samples); biological warfare agent samples; research samples; purified samples, such as purified genomic DNA, RNA, proteins, etc.; and raw samples (bacteria, virus, genomic DNA, etc.).

The term "subject" is intended to include all animals. In particular embodiments, the subject is a mammal, a human or nonhuman primate, a dog, a cat, a horse, a cow, other farm animals, or a rodent (e.g. mice, rats, guinea pig. etc.). A human subject may be a normal human being without observable abnormalities, e.g., a disease. A human subject may be a human being with observable abnormalities, e.g., a disease. The observable abnormalities may be observed by the human being himself, or by a medical professional. The term "subject", "patient", and "individual" are used interchangeably herein.

In one aspect, the present invention provides methods of preparing nucleic acids that are quantitatively and qualitatively correlated to a plurality of target nucleic acids in a sample. The correlation may be achieved through some mechanisms including, but not limited to, 1) specific hybridizations between complementary nucleic acids and 2) specific enzymatic recognition such as a ligation reaction to connect substantially adjacent nucleotides and a polymerase reaction to add specific nucleotide onto an existing polynucleotide based on a template sequence.

As used herein, "nucleic acid", "polynucleotide", and "oligonucleotide" are interchangeably used to indicate at least two nucleotides covalently linked together. Oligonucleotides may be generated by, e.g., chemical synthesis, restriction endonuclease digestion of plasmids or phage DNA, DNA replication, reverse transcription, or a combination thereof. One or more of the nucleotides can be modified e.g. by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag, or by using radioactive nucleotides.

A nucleic acid used in the present invention may contain phosphodiester bonds, although in some cases nucleic acid analogs are included that may have alternate backbones, including, e.g., phosphoramide, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. See, e.g., Pauwels et al., 1986, Chemica Scripta 26:141-9; U.S. Pat. No. 5,644,048; Briu et al., 1989, J. Am. Chem. Soc. 111:2321; and Carlsson et al., 1996, Nature 380:207. Other analog nucleic acids include those with positive backbones, non-ionic backbones, and non-ribose backbones. See, e.g., Denpcy et al., 1995, Proc. Natl. Acad. Sci. USA 92:6097; Jeffs et al., 1994, J. Biomolecular NMR 34:17; U.S. Pat. Nos. 5,386,023, 5,235,033 and 5,034,506. Modifications of the ribose-phosphate backbone may be done to facilitate the addition of labels, or to increase the stability and half-life of such molecules in physiological environments.

In some embodiments, peptide nucleic acids (PNA) may be used for nucleic acids in the present invention. The PNA backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages. First, the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (T_m) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4°C drop in T_m for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9°C. This allows for better detection of mismatches. Similarly, due to their non-ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration.

Nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. Nucleic acids may be DNA, genomic DNA, cDNA, RNA or a hybrid. Nucleic acids may contain any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine, hypoxanthine, isocytosine, isoguanine, etc. In one embodiment, nucleic acids utilize isocytosine and isoguanine in nucleic acids designed to be complementary to other probes, rather than target sequences, as this reduces non-specific hybridization, as is generally described in U.S. Patent No. 5,681,702. As used herein, the term "nucleoside" includes nucleotides as well as nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, "nucleoside" includes non-naturally occurring analog structures. Thus, for example, the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.

In some embodiments, the methods according to the present invention are directed to the multiplexed detection of target nucleic acids. The term "target nucleic acid" or grammatical equivalents herein refers to a specific nucleic acid sequence to be detected and/or quantified in the sample to be analyzed. The target nucleic acid may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, or RNA including mRNA and rRNA.

Complementary nucleic acids are capable of hybridizing to each other under normal hybridization conditions. The term "complementary" refers to sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In certain embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In other embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. As used herein, the pairing of complementary nucleic acids is referred to by the terms "hybridization" or "hybridizing".

Specific enzymatic recognition may be achieved through a ligation reaction to connect substantially adjacent nucleotides or a DNA or RNA polymerase reaction to add specific nucleotide onto an existing polynucleotide based on a template sequence.

Ligases are well known and may be used for specific enzymatic recognition. See, e.g., Lehman, 1974, Science, 186: 790-797; and Engler et al, DNA Ligases, pages 3-30 in Boyer, editor, The Enzymes, Vol. 15B (Academic Press, New York, 1982). Preferred ligases include T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq DNA ligase, Pfu ligase, and Tth ligase. Protocols for their use are well known. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition): Three-volume set, Cold Spring Harbor Laboratory Press; 4th edition (June 15, 2012) and the like. Generally, ligases require that a 5' phosphate group be present for ligation to the 3' hydroxyl of an adjacent strand.

In some embodiments, the preferred ligase is one which has the least mismatch ligation. The specificity of ligase can be increased by substituting the more specific NAD+-dependant ligases such as E. coli ligase and (thermostable) Taq ligase for the less specific T4 DNA ligase. The use of NAD analogues in the ligation reaction further increases specificity of the ligation reaction. See, e.g., U.S. Patent No. 5,508,179.

DNA or RNA polymerases can extend a nucleic acid sequence by adding nucleotides in the presence of a template. As is well known in the art, there are a wide variety of suitable polymerases. Suitable polymerases include, but are not limited to, DNA polymerases, including the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase, Phi29 DNA polymerase and various RNA polymerases such as from Thermus sp., Q beta replicase from bacteriophage, or SP6, T3, T4 and T7 RNA polymerases.

In some embodiments, preferred polymerases are those that are essentially devoid of a 5' to 3' exonuclease activity, so as to assure that the first probe will not be extended past the 5' end of the second probe. Exemplary enzymes lacking a 5' to 3' exonuclease activity include the Klenow fragment of the DNA Polymerase and the Stoffel fragment of DNA Taq Polymerase. (See e.g., Lawyer et al., 1989, J. Biol. Chem., 264:6427-6437; and Lawyer et al., 1993, PCR Meth. Appl., 2:275-287). Other mutant polymerases lacking 5' to 3' exonuclease activity have been generated for polymerases derived from T. vulgaris. See U.S. 6,632,645.

In other embodiments, preferred polymerases are those that lack a 3' to 5' exonuclease activity, which is commonly referred to as a proof-reading activity, and which removes bases which are mismatched at the 3' end of a primer-template duplex. Although the presence of 3' to 5' exonuclease activity provides increased fidelity in the strand synthesized, the 3' to 5' exonuclease activity found in thermostable DNA polymerases such as Tma (including mutant forms of Tma that lack 5' to 3' exonuclease activity) also degrades single-stranded DNA such as the primers used in the PCR, single-stranded templates and single-stranded PCR products. The integrity of the 3' end of an oligonucleotide primer used in a primer extension process is critical as it is from this terminus that extension of the nascent strand begins. Degradation of the 3' end leads to a shortened oligonucleotide which may result in a loss of specificity in the PCR reaction.

In still other embodiments, more preferred polymerases are thermostable polymerases. A thermostable enzyme is an enzyme that retains most of its activity after one hour at 40°C under optimal conditions. Examples of thermostable polymerases which lack both 5' to 3' exonuclease and 3' to 5' exonuclease include Stoffel fragment of Taq DNA polymerase. This polymerase lacks the 5' to 3' exonuclease activity due to genetic manipulation and no 3' to 5' activity is present as Taq polymerase is naturally lacking in 3' to 5' exonuclease activity.

The conditions for performing the addition of one or more nucleotides at the 3' end of a probe will depend on the particular enzyme used, and will generally follow the conditions recommended by the manufacturer of the enzymes used.

In some embodiments, the correlation may be achieved through a combination of different mechanisms, e.g., a combination of both specific hybridizations between complementary nucleic acids and a ligation reaction to connect substantially adjacent nucleotides; or a combination of specific hybridizations between complementary nucleic acids, a ligation reaction to connect substantially adjacent nucleotides, and a polymerase reaction to add specific nucleotide onto an existing polynucleotide based on a template sequence.

In some embodiments, the combination of both specific hybridizations between complementary nucleic acids and a ligation reaction to connect substantially adjacent nucleotides is applied to prepare nucleic acids that are quantitatively and qualitatively correlated with the target nucleic acids in a sample. As such, the method to prepare nucleic acids from a sample include the steps of 1) hybridizing probes to the target nucleic acids in the sample; and 2) ligating the hybridized probes to obtain a preparation of nucleic acids that are quantitatively and qualitatively correlated with the target nucleic acids in the sample.

In other embodiments, the combination of specific hybridizations between complementary nucleic acids, a ligation reaction to connect substantially adjacent nucleotides, and a polymerase reaction to add specific nucleotide onto an existing polynucleotide based on a template sequence is applied to prepare nucleic acids that are quantitatively and qualitatively correlated with the target nucleic acids in a sample. As such, the method to prepare nucleic acids from a sample include the steps of 1) hybridizing probes to the target nucleic acids in the sample; 2) extending one of the probes to close the gap between the probes so that the extended probes may be ligated to an adjacent probe; and 3) ligating the hybridized and extended probes to obtain a preparation of nucleic acids that are quantitatively and qualitatively correlated with the target nucleic acids in the sample.

In some embodiments, the target nucleic acid may be extracted from the sample before being hybridized to probes. Methods known for nucleic acid extraction in the art include the use of phenol/chloroform, the use of salting out procedure, the use of chaotropic salts and silica resins, the use of affinity resins, ion exchange chromatography and the use of magnetic beads. See, for example, U.S. Pat. Nos. 5,0574,26 and 4,923,978, EP Patents 0512767, WO 95/13368, WO 97/10331 and WO 96/18731. Conventional techniques of molecular biology, biochemistry, genetics, which are in the skill of the art, are explained fully in the literature. See, for instance, Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition): Three-volume set, Cold Spring Harbor Laboratory Press; 4th edition (June 15, 2012); Carson, Miller, and Witherow, Molecular Biology Techniques, Third Edition: A Classroom Laboratory Manual, Academic Press; 3 edition (November 21, 2011); Cheng and Zhang, Molecular Genetic Pathology, Humana Press; 1 edition (April 15, 2008).

In addition, when the target nucleic acids are preferred to be cut into a size that will facilitate handling and hybridization to the probes, particularly for genomic DNA, this may be accomplished by shearing the nucleic acid through mechanical forces (e.g. sonication) or by cleaving the nucleic acid using restriction endonucleases, or any other methods known in the art.

As used herein, "probe" refers to a known sequence of a nucleic acid that is capable of selectively binding to a target nucleic acid. More specifically, "probe" refers to an oligonucleotide designed to be sufficiently complementary to a sequence of one strand of a nucleic acid that is to be probed such that the probe and nucleic acid strand will hybridize under selected stringency conditions. Additionally, "probe" also refers to an end product which is derived by connecting one or more substantially adjacent oligonucleotides hybridized to substantially adjacent segments of a nucleic acid. For example, a "ligated probe" refers to the end product of a ligation reaction between a pair of probes.

As used herein, the term "substantially adjacent" is used in reference to nucleic acid molecules that are in close proximity to one another. The term also refers to a sufficient proximity between two nucleic acid molecules to allow the 5' end of one nucleic acid that is brought into juxtaposition with the 3' end of a second nucleic acid so that they may be ligated by a ligase enzyme. Nucleic acid segments are defined to be substantially adjacent when the 3' end of a first probe and the 5' end of a second probe, with the first probe hybridizing to one segment and the second probe to the other segment, are sufficiently near each other to allow connection of the ends of both probes to one another. Thus, two probes are substantially adjacent, when the ends thereof are sufficiently near each other to allow connection of the ends of both probes to one another.

As such, in some embodiments of the present invention, a set of probes including 2 or more probes are designed to hybridize to a target nucleic acid. The target nucleic acid may contain several target regions; for example, a first target region of the target nucleic acid may hybridize to a first probe or a portion of the first probe, a second target region of the target nucleic acid may hybridize a second probe or a portion of the second probe. In addition, the two target regions may be adjacent or separated. When the two regions are adjacent, e.g., a first probe hybridizing to a first target region and a second probe hybridizing to a second target region, the first and the second hybridized probes may be adjacent so that a ligation reaction may connect the two probes to form a third probe. The third probe may therefore be able to hybridize to both the first region and the second region of the target nucleic acid. When the two regions are separated by one or more nucleotides, the gap between the first and the second hybridized probes may be filled with the use of a polymerase and dNTPs to extend one of the probes so that the extended probe and the other hybridized probe may be substantially adjacent to form a third probe.

The terms "first" and "second" are not meant to confer an orientation of the sequences with respect to the 5'-3' orientation of the target sequence. For example, assuming a 5'-3' orientation of the complementary target nucleic acid, the first target region may be located either 5' to the second region, or 3' to the second region.

As such in one embodiment, the probes of the present invention are designed to be complementary to a target nucleic acid, such that the probes hybridize to the target nucleic acids. This complementarity need not be perfect; there may be any number of base pair mismatches that will interfere with hybridization between the target nucleic acid and the single stranded probe sequence. However, if the number of mismatches is so many so that no hybridization can occur under certain hybridization conditions, the sequence is not a complementary target sequence.

In addition, these probes may take on a variety of configurations and may have a variety of structural components. In some embodiments, a probe may be an allele specific probe or a locus specific probe. An allele specific probe includes an allele-specific hybridization sequence (ASHS) portion that hybridizes to a target nucleic acid and discriminates between alleles, or hybridizes to a target nucleic acid and is modified in an allele specific manner. A locus specific probe includes a locus-specific hybridization sequence (LSHS) portion that hybridizes to a target nucleic acid in a locus specific manner, but does not necessarily discriminate between alleles. A locus specific probe also may be modified, i.e. extended as described below, such that it includes information about a particular allele, but the locus specific primer itself does not discriminate between alleles. The length of the ASHS or LSHS may be designed to confer sufficient specificity for the probe to hybridize to the target nucleic acids. With the general guidance that the longer the ASHS or LSHS sequence, the more specific they binds to the target nucleic acids, a skilled person in the art has the knowledge to varying the length and decide the length of the ASHS or LSHS in order to achieve his/her testing goals as described in detail herein, e.g., in the Examples.

In other embodiments, a probe may include one or more segments in addition to ASHS or LAHS. In some instance, the additional segment of the probe may be a primer binding site, onto which a primer may bind in a polymerase chain reaction. In other instance, the additional segment of the probe may be a stuffer sequence, which may make the ligation product vary in length and thereby be distinguished on the basis of fragment sizes. The length of the primer binding site and the stuffer sequence may be designed by a person skilled in the art to suit his/her testing goals. For example, a primer binding site of the probes may be about 15-20 nucleotides in length, with 18 being especially preferred. For another example, the stuffer sequence of the probes may be about 2-100 nucleotides depending on the needs to distinguish different target nucleic acids. As such, the stuffer sequence is a nucleic acid that is generally not native to the target sequence, but is added or inserted in the probe sequence. Preferred stuffer sequences are those that are not found in a genome, e.g., a human genome, and they do not have undesirable structures, such as hairpin loops.

In some embodiments, double stranded target nucleic acids are denatured to render them single stranded so as to permit hybridization of the target nucleic acids and the probes. The denaturation may be achieved, among other suitable means, by treating the target nucleic acids with heat, alkali, or both heat and alkali. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition): Three-volume set, Cold Spring Harbor Laboratory Press; 4th edition (June 15, 2012). In some embodiments, the denaturing may be carried out at temperature about 90 °C to about 99 °C for about 5 seconds to 30 minutes. In a preferred embodiment, the denaturation may be carried out at about 98°C for 5 minutes.

The use of different stringency conditions such as variations in hybridization temperature and buffer composition may be used to determine the presence or absence of mismatches between a single stranded target nucleic acid and a probe. With regard to temperature, differences in the number of hydrogen bonds as a function of basepairing between perfect matches and mismatches can be exploited as a result of their different T_ms. Under a defined ionic strength, pH and nucleic acid concentration, the T_m is the temperature at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium. Accordingly, a hybridized nucleic acid comprising perfect complementarity will melt at a higher temperature than one comprising at least one mismatch, with all other parameters being equal. The other parameters include the length of the hybridized nucleic acid, the nature of the backbone (i.e. naturally occurring or nucleic acid analog), the assay solution composition, and the composition of the nucleic acid, e.g., the G-C content.

High stringency conditions are those that result in perfect matches remaining in hybridization complexes, while imperfect matches melt off. On the other hand, low stringency conditions are those that allow the formation of hybridization complexes with both perfect and imperfect matches. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.

A guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T_m) for the specific sequence at defined ionic strength and pH. Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3°C for short probes (e.g. 10 to 50 nucleotides) and at least about 6°C for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In another embodiment, less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art. See, e.g., Tijssen, supra.

Similarly, variations in buffer composition may be used to elucidate the presence or absence of a mismatch at the detection position. Suitable conditions include, but are not limited to, formamide concentration. Thus, for example, "low" or "permissive" stringency conditions include formamide concentrations of 0 to 10%, while "high" or "stringent" conditions utilize formamide concentrations of 40%. Low stringency conditions include NaCl concentrations of 1 M, and high stringency conditions include concentrations of 0.3 M. Furthermore, low stringency conditions include MgCl₂ concentrations of 10 mM, moderate stringency as 1-10 mM, and high stringency conditions include concentrations of 1 mM.

Ligase catalyzes the covalent bonding between two nucleotides adjacent to each other. The ligation reaction is facilitated by a complementary strand holding the two nucleotides comprising the 3' and 5' ends of two polynucleotides with no gaps between the two ends in close proximity. In addition, the ligation reaction requires that there is a phosphate group exposed on the 5' end or hydroxyl group exposed on the 3' end.

Ligation can be carried out using any enzyme capable of ligating nucleotides. In some embodiments, Taq DNA ligase is used to ligate the two adjacent oligonucleotide probes hybridized to the target nucleic acid. Taq DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini of two adjacent oligonucleotide probes which are hybridized to a complementary target DNA. The ligation will occur only if the oligonucleotides are perfectly paired to the complementary target DNA and have no gaps between them; therefore, a single-base substitution may be detected if no ligation product is generated.

The condition for the ligation reaction depends on what ligase is used. For a thermal stable ligase, the ligase retains activities at elevated temperatures. For example, Taq DNA ligase remains active at 45-65 °C. For a non-thermal stable ligase, the ligase is active at lower tempratures. For example, T4 DNA ligase is active at around 16°C. As such, in one embodiment, the temprature for ligation reaction may be from about 4 °C to about 70 °C depending on which ligase is used.

The amount of the ligase and the duration of the ligation reaction may depend on the amount of probes and target nucelic acids. A person skilled in the art may adjust the amount of the ligase added in a reaction and the duration of the reaction so that a desirable ligation product may be achieved. For example, in some embodiments of the present invention, 40 units of Taq DNA ligase is used for a 20µl ligation reaction. One unit is defined as the amount of enzyme required to give 50% ligation of the 12-base pair cohesive ends of 1 µg of BstEII-digested λ DNA in a total reaction volume of 50 µl in 15 minutes at 45°C.

In some embodiments, the duration of the ligation reaction is about 1 minute to 48 hours. In other embodiments, the duration of the ligation reaction is about 16 hours. In other embodiments, the steps of denaturation, hybridization, and ligation are repeated several time so that more probes may hybridize to the target nucleic acid or hybridize to the newly ligated probe. For example, the mixture of the probes and the target nucleic acids may be held at about 90 °C to about 99 °C for about 5 second to about 30 seconds for denaturation, and then at about 4°C to about 58 °C for about 1 miute to 48 hours for hybridization and ligation, and the cycles may be repeated to 100 times. In some preferred embodiments, for example, the mixture of the probes and the target nucleic acids may be held at about 95 °C for about 30 seconds for denaturation, and then at 58 °C for about 4 hours for hybridization and ligation, and the cycles may be repeated 4 times.

In other embodiments, there is a gap between the hybridized probes on the target nucleic acid. In some instances, the gap may be filled by another probe that is complementary to the gap in the target nucleic acid. This gap-filling probe is designed so that when it is hybridized to the nucleic acid, its two ends are substantially adjacent to the other two probes. In other instances, the gap may be filled by extending one of the probes hybridized to the target nucleic acid. This probe extension may be carried out using a DNA polymerase and dNTPs. A description of DNA polymerase is described supra. For both gap filling methods, a ligation reaction is performed to ligate the substantially adjacent gap-filling probe with the other two probes or ligate the substantially adjacent extended probe with the other probe to obtain ligation products. The ligation reaction may be carried out with suitable parameters including, but not limited to, the type of the ligase, the amount of the ligase, and the duration of the ligation reaction as describe above. Supra.

In some embodiments, the ligation products are then analyzed directly to determine the presence, absence, or quantity of target nucleic acids in the sample. In other embodiments, the ligation products are then amplified to obtain amplification products which are analyzed to determine the presence, absence, or quantity of target nucleic acids in the sample.

As used herein, "amplification" refers to the increase in the number of copies of a particular nucleic acid. Copies of a particular nucleic acid made in an amplification reaction are called "amplicons" or "amplification products".

Nucleic acid amplification methods include, without limitation, polymerase chain reaction (PCR) (U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, and 5,219,727) and its variants such as in situ polymerase chain reaction (U.S. Patent No. 5,538,871), quantitative polymerase chain reaction (U.S. Patent No. 5,219,727), and nested polymerase chain reaction (U.S. Patent No. 5,556,773).

In some embodiments, the amplification process is achieved through PCR. The PCR amplification process results in the exponential increase of discrete DNA fragments whose length is defined by the 5' ends of the oligonucleotide primers.

In some embodiments, universal primer binding sites are included in the probes and universal primers are used to amplify ligation products for all target nucleic acids. Alternatively, "sets" of universal primer binding sites are included in "sets" of corresponding probes, and "sets" of universal primers are used to amplify "sets" of the ligation products either simultaneously or sequentially to obtain "sets" of amplification products for further analysis.

Accordingly, in some embodiments of the present invention, "sets" of probes are provided for multiplex nucleic acid analysis with each set of probes including a first probe and a second probe. Multiplexity here refers to at least two target nucleic acids, with more than 10 being preferred, depending on the assay, sample and purpose of the test. In some embodiments the multiplexity refers to more than 48, 96, 192, or 384 target nucleic acids.

As used herein, the term "primer" refers to an oligonucleotide, sometimes produced synthetically, which is capable of acting as a point of initiation of nucleic acid sequence synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a template nucleic acid strand is induced, i.e. in the presence of different nucleotide triphosphates and a polymerase in an appropriate buffer and at a suitable temperature. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the template.

In some embodiments, an oligonucleotide GTTTCTT was included in the 5' portion of a primer sequence. The addition of the oligonucleotide GTTTCTT may help facilitate the non-templated addition of adenosine on the 3' end of PCR products when polymerases such as Taq polymerase are used in the PCR. See, e.g., Brownstein et al., Modulation of Non-Templated Nucleotide Addition by TaqDNA Polymerase: Primer Modifications that Facilitate Genotyping, BioTechniques 20:1004-1010 (June 1996). The consistent addition of adenosine may help to have most or all PCR products consistently have an adenosine end. As such, the genotyping result of these PCR products can be consistent. Without the consistent addition of adenosine, a PCR product may not vary in size by one base pair and the genotyping result of the PCR product may not be consistent. In some embodiments, the reverse primer used for amplifying the ligation products includes oligonucleotide GTTTCTT or its functional equivalents, e.g., GTTTCTTG.

In some embodiments, one or more of the nucleotides of the primer may be modified by adding a detectable moiety, e.g., a methyl group, a biotin or digoxigenin moiety, or a fluorescent tag. For some instances, the moiety is a fluorescent dye and the dye may be, but not limited to, FAM (5-or 6-carboxyfluorescein), VIC, NED, PET, Fluorescein, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, and Yakima Yellow. See, e.g., U.S. Patent Publication No. 20110151459 for fluorescent dyes that may be used to label primers in the present invention and the U.S. Patent Publication No. 20110151459.

In one embodiment, the multiplexity of nucleic acid analysis is increased by adding a detectable moiety to the primers used for amplifying ligation products. In one example, the moieties used for labeling the primers are FAM-blue, VIC-green, NED-yellow and PET-red (Life Technologies, Inc.). As shown in Figure 1, to analyze target nucleic acids T01, T02, T03, and T04 in a sample according to one embodiment of the present invention, two probes (here referred to as the right probe and the left probe as they appear in the figure) for each target site are designed so that each probe contains a locus specific hybridization sequence (LSHS) and a primer binding site. The right probe contains a primer binding site Y. The primer binding site Y may be shared by the right probes for all target nucleic acids. In contrast, the left probe for each target nucleic acid contains a unique primer binding sequence (X1, X2, X3 and X4). The primer binding sequences are incorporated in the ligation products. To amplify the ligation product, a pair of primers is designed for each ligation product corresponding to each target site. Because the left probes have four unique primer binding sites, four unique forward primers (F1, F2, F3 and F4) are designed corresponding to the four unique primer binding sites, respectively. In the example, the forward primer F1 is labeled with FAM-blue fluorescent dye, F2 with VIC-green, F3 with NED-yellow, and F4 with PET-red. The reverse primer R binds to the primer binding site Y. As such, the amplification product for target T01 is labeled with FAM-blue fluorescent dye. And the amplification products for targets T02, T03, and T04 are labeled with VIC-green, NED-yellow, and PET-red, respectively. The fluorescent labeled amplification products may then be analyzed by capillary electrophoresis on the basis of the different fluorescent dyes that each product is labeled with. The amplification products differentially labeled with fluorescent dyes may be distinguished even if the amplification products for different target nucleic acids are of the same length.

In another embodiment, the multiplexity of nucleic acid analysis is increased by varying the length of primers used for amplifying ligation products. To vary the length of primers, in one example, a stuffer sequence may be inserted into the primers. The stuffer sequence may be incorporated into the amplification product during the PCR reaction; i.e., the primer may be extended to form the amplification product to incorporate the stuffer sequence. As such, the addition of a stuffer sequence may help distinguish the amplification products on the basis of fragment sizes. In one embodiment as illustrated in Figure 2, a stuffer sequence is inserted in primer R2 so that the amplification product from target T02 has a bigger fragment size than the product from target T01. As shown in Figure 2, to detect target nucleic acids T01 and T02 in a sample according to one embodiment of the present invention, two probes (here referred to as the right probe and the left probe as they appear in the figure) are designed for each target nucleic acid so that each probe contain a locus specific hybridization sequence and a primer binding sequence. The left probe contains a primer binding sequence X, which is shared by left probes for both target sites. In contrast, the right probe contains a unique primer binding sequence (Y1 or Y2). The primer binding sequences may be incorporated into the ligation products. To amplify the ligation products, a pair of primers is designed for each ligation product corresponding to each target site. The forward primer binds to the primer binding site X shared by the two ligation products. The primers binding to the unique primer binding site Y1 and Y2 are designed to have a stuffer sequence inserted in the 5' portion. In one example, the reverse primer R2 contains a stuffer sequence so that R2 is longer than R1 and the amplification product from F/R2 is longer than from F/R1 if the ligation products for the two target sites are of the same length. In some embodiments, the stuffer sequence may have about 10 to about 500 nucleotides. In other embodiments, the stuffer sequence may have about 10 to about 60 nucleotides. In some preferred embodiments, the length of each and very primer is no more than 125 nucleotides. In other preferred embodiments, the length of each and every primer is no more than 75 nucleotides. The amplification products may then be analyzed by capillary electrophoresis on the basis of the different fragment size. The amplification products thus obtained may be distinguished even if the amplification products are labeled with the same fluorescent dye and the ligation products are of the same length for different target nucleic acids.

In still other embodiment, the multiplexity of nucleic acid analysis is increased by a combination of the use of primers labeled with fluorescent dyes and primers inserted with stuffer sequences. For example, as illustrated in Figure 3A, the forward primers are labeled with four fluorescent dyes: F1-FAM-blue, F2-VIC-green, F3, NED-yellow, and F4-PET-red. In addition, the reverse primer R2 contains a stuffer sequence but R1 does not contain a stuffer sequence. As such, the combination may give rise to an eight fold increase in terms of the multiplexity for determining the ligation products; eight ligation product of the same length may be distinguished by determining the corresponding amplification products on the basis of fragment sizes and fluorescent labels.

In still other embodiments, the multiplexity of the nucleic acid analysis according to the present invention may be further increased by inserting stuffer sequences in the probes to be hybridized to target nucleic acids and ligated to form ligation products. The insertion of stuffer sequences in probes may produce ligation products with unique sequence length corresponding to a particular target nucleic acid. For example, to distinguish 16 target nucleic acids T01-T16 in a sample according to one embodiment of the present invention, two locus-specific probes (here referred to as the right probe and the left probe as they appear in the figure) are designed for each target nucleic acid. The left probe contains a locus specific hybridization sequence and a universal primer binding sequence X. The right probe for each target nucleic acid contains locus specific hybridization sequence, a stuffer sequence and a universal primer binding sequence Y. The stuffer sequence for each target nucleic acid may vary in length, e.g., for T01, the right probe has no stuffer sequence; for T02, the right probe has a stuffer sequence of 2 nucleotides; for T03, 4 nucleotides; for T04, 6 nucleotides; for T05, 8 nucleotides; for T06, 10 nucleotides...and for T16, 30 nucleotides. Consequently, the ligation product for each target nucleic acid has a unique fragment size because the stuffer sequences in the probes are incorporated into the ligation product. The ligation products are optionally further amplified with a pair of universal primers that binds to the universal primer binding sites X and Y. As such, the fragment sizes of the amplification products match the fragment sizes of the ligation products and therefore may be determined on the basis of fragment sizes. As such, if without the stuffer sequences in the probes, the ligation products are of the same length, the addition of the stuffer sequences in the probes makes it possible to distinguish the ligation product and/or the amplification product for each target nucleic acid on the basis of fragment size. In this example, the multiplexity for target nucleic acid analysis increases 16 folds. Thus, as exemplified in this embodiment, the fold increase of multiplexity may depend on the number of stuffer sequences with different length that are inserted in the locus-specific probes. If 12 stuffer sequences of varying length are inserted in the locus-specific probes for each target nucleic acid, the multiplexity for target nucleic acid analysis may increase 12 fold.

Therefore, according to the present invention, the methods of increasing multiplexity by inserting stuffer sequences in probes may be combined with the method of increasing multiplexity by labeling the primers with detectable moieties and/or inserting stuffer sequences in primers. For example, by both labeling primers with fluorescent dyes and inserting stuffer sequences in probes, 64 amplification products corresponding to 64 target nucleic acids may be distinguished by capillary electrophoresis on the basis of fluorescent dyes and fragment size (see Figure 1, bottom chart). For another example, by inserting stuffer sequences in both primers and probes, 24 amplification products corresponding to 24 target nucleic acids may be distinguished by capillary electrophoresis on the basis of fragment size (see Figure 2, bottom chart). For still another example, by labeling primers with fluorescent dyes and inserting stuffer sequences in primers and probes, 96 amplification products corresponding to 96 target nucleic acids may be distinguished by capillary electrophoresis on the basis of fluorescent dyes and fragment size (see Figure 3, bottom chart).

The analysis of amplification products may be by any method that can separate DNA fragments on the basis of size, mass, fluorescent moieties, or other measurable properties. These methods include, but not limited to, agarose gel electrophoresis followed by capillary electrophoresis (CE), DNA sequencing, ethidium bromide or DNA staining, microarray, and flow cytometry.

For example, capillary electrophoresis may identify DNA fragments on the basis of both fluorescent moieties and fragment sizes. In some embodiments, capillary electrophoresis is performed by injecting the DNA fragments into a capillary, filled with polymer. The DNA is pulled through the tube by the application of an electric field, which separates the fragments such that the smaller fragments travel faster through the capillary. The fragments are then detected on the basis of fluorescent dyes that are attached to the primers used in PCR. This allows multiple fragments to be amplified and run simultaneously in a multiplexed manner. Sizes are assigned using labeled DNA size standards that are added to each sample. In capillary electrophoresis the intensity of signal of an amplification product is the number of relative fluorescence units (rfus) of its corresponding peak. The intensity value correlates with the amount of labeled amplification product.

In some embodiments, capillary electrophoresis is the preferred method for analyzing the amplification products of the present invention. Capillary electrophoresis devices are known in the art. Capillary electrophoresis devices useful according to the invention include, but are not limited to, ABI 3130XL Genetic Analyzer by Applied Biosystems (Foster City, California); MegaBACE 1000 Capillary Array Electrophoresis System by Amersham Pharmacia Biotech (Piscataway, N.J.); CEQ™ 8000 Genetic Analytic System by Beckman Coulter (Fullerton, Calif.); Agilent 2100 Bioanalyzer by Caliper Technologies (Mountain View, Calif.); and iCE280 System by Convergent Bioscience Ltd. (Toronto, Canada).

As is the case throughout this invention disclosure, including the background, the summary, the detailed description and the claims, the designation of the "left" probe and the "right" probe are arbitrary and are interchangeable. For example, the left probe have the features described for the right probe and the right probe may have the features for the left probe. In addition, the designation of the "first" primer and the "second" primer, or the "forward" primer and the "reverse" primer are also arbitrary and interchangeable. Further, when an oligo primer binding to a primer binding site on a template DNA in a PCR amplification reaction, the oligo primer is meant to bind either to the positive strand or to the negative strand of the template DNA. A primer is reversely complementary to a binding site on a single-stranded DNA when the primer is complementary to the opposite strand of the single-stranded DNA. As is well known in the art for a PCR reaction to proceed successfully, the forward primer and the reverse primer do not bind to the same strand of a DNA template. Instead, the forward primer and the reverse primer bind to different strands of a DNA template if an exponential amplification is desired.

Methods of multiplex nucleic acid analysis according to the present invention may be applied to detect various genetic aberrations, including, but not limited to, single nucleotide polymorphisms (SNPs), gene copy number variants (CNVs), chromosomal abnormalities (e.g., insertions, deletions and duplications), gene mutations (e.g., single nucleotide changes, insertions, and deletions), nucleic acid modifications (e.g., methylation, acetylation and phosphorylations), and abnormal gene expression. Some aberrations may be qualitative changes, e.g., the presence or absence of a SNP. Other aberrations may be quantitative, e.g., the change of gene copy number. The quantitative change may be big or small. For example, in a genomic DNA sample from a Down's syndrome patient, the copy number of chromosome 21 increases 50%. In contrast, in a blood sample of a pregnant woman conceiving a Down's syndrome fetus, the copy number of chromosome 21 may increase less than 10%.

The terms "analyzing", "determining," "measuring," "assessing," "assaying," "evaluating," and any grammatical equivalents are used interchangeably to refer to any form of quantitative or qualitative measurement, and include determining if a characteristic, trait, or feature is present or not. An analysis of nucleic acids may be relative or absolute. For example, an increase of copy number of a nucleic acid in a sample may be measured in relative to the copy number of the nucleic acid in a reference sample.

Recent human genomics research indicates that the genomic makeup between any two humans has over 99.9% similarity. The relatively small number of variations in DNA between individuals gives rise to differences in phenotypic traits, and may be related to many human diseases, susceptibility to various diseases, or response to treatment of disease. Variations in DNA between individuals occur in both coding and non-coding regions, and include changes of a single nucleotide, as well as insertions and deletions of nucleotides. Changes of a single nucleotide in the genome are referred to as single nucleotide polymorphisms, or "SNPs." The occurrences of SNPs in the genome are becoming correlated to the presence of and/or susceptibility to various diseases and conditions. As these correlations and other advances in human genetics are being made, medicine and personal health in general are moving toward a customized approach in which a patient will make appropriate medical and other choices in consideration of his or her genomic information, among other factors. Thus, there is a need to provide individuals and their care-givers with information specific to the individual's personal genome toward providing personalized medical and other decisions.

A multiplex SNP detection method according to the present invention is illustrated in Figure 4. In the example, 48 SNP sites are determined in a single assay simultaneously. For illustrative purposes, one SNP has a C or T nucleotide at the polymorphic position. A set of probes (here referred to as the right probe and the left probe as they appear in the figure) are designed. The left probe contains an allele-specific hybridization sequence (ASHS) with the SNP recognition nucleotide "C" or "T" on one end, a primer binding site X, a stuffer A sequence between the ASHS and the primer binding site, and a stuffer L1 sequence between the stuffer A sequence and the primer binding site. The stuffer A sequence helps distinguish the alleles for each SNP site. The right probe contains a locus-specific hybridization sequence (LSHS), a primer binding site Y, and a stuffer L2 sequence between the LSHS and the primer binding site. By varying the length of stuffer L1 and L2 sequences, a six-fold multiplexity may be achieved for the ligation products; ligation products for six SNP sites can be distinguished on the basis of fragment size. In some instances, a mismatch at a position 2, 3, or 4 nucleotides away from the SNP recognition site is introduced into the probe. These mismatches may help increase the specificity of the hybridization of the ASHS to the target site. See, e.g., Luo et al., Improving the fidelity of Thermus thermophilus DNA ligase, Nucleic Acids Res. 1996 Aug 1;24(15):3071-8.

In addition, by labeling the forward primers with FAM-blue, VIC-green, NED-yellow and PET-red and inserting a stuffer sequence in the reverse primer R2, the multiplexity for the SNP analysis is increased to 96 (=6 x2 x 4 x 2). As such, the multiplex SNP detection method may analyze 48 SNP sites simultaneously with each SNP site having two alleles. A person skilled in the art may increase the multiplexity for SNP detection by varying the parameters, e.g., inserting stuffer sequences in more probes, labeling the primers with more fluorescent tags, or inserting stuffer sequences in more primers. As such, the multiplexity may be increased to 192, 384, 768, or more if desirable.

As used herein, CNVs refer to variations in the number of copies of a nucleic acid sequence that contains 2 or more nucleotides in a test sample in comparison with the copy number of the nucleic acid sequence present in a reference sample. CNVs may include deletions, microdeletions, insertions, microinsertions, duplications, multiplications, inversions, translocations and complex multisite variants. CNVs may also encompass chromosomal aneuploidies and partial aneuploidies that may cause many genetic diseases, e.g., Down's syndrome, Turner's syndrome, diGeorge syndrome, Angelman syndrome, Cri-du-chat, Kallmann syndrome, Miller-Dieker syndrome, Prader-Willi syndrome (PWS), Smith-Magenis syndrome, Steroid sulfatase deficiency (X-linked ichthyosis), Williams syndrome, and Wolf-Hirschhorn syndrome.

An exemplary multiplex CNV detection method according to the present invention is illustrated in Figure 5. In the example, 96 CNVs are measured in a single assay simultaneously. For each CNV, a set of probes (here referred to as the right probe and the left probe as they appear in the figure) are designed. The left probe contains a locus-specific hybridization sequence (LSHS), a primer binding site X, and a stuffer L1 sequence between the LSHS and the primer binding site. The right probe contains a locus-specific hybridization sequence (LSHS), a primer binding site Y, and a stuffer L2 sequence between the LSHS and the primer binding site. By varying the length of stuffer L1 and stuffer L2 sequences, a 12-fold multiplexity may be achieved for the ligation products; ligation products for 12 CNVs can be distinguished on the basis of fragment size. In addition, by labeling the forward primers with FAM-blue, VIC-green, NED-yellow and PET-red and inserting a stuffer sequence in one of the reverse primers R2, the multiplexity for the CNVs analysis may be increased to 96 (=12 x 4 x 2). As such, the multiplex CNV detection method may analyze 96 CNVs simultaneously. A person skilled in the art may increase the multiplexity for CNV detection by varying the parameters, e.g., inserting stuffer sequences in more probes, labeling the primers with more fluorescent tags, or inserting stuffer sequences in more primers. As such, the multiplexity may be increased to 192, 384, 768, or more if desirable.

In some embodiments, the fluorescent intensity of each peak was compared to a standard value to determine the copy number of each CNV site. For example, if the intensity decreases in half compared to the standard value, the copy number may be considered as decreased in half. On the other hand, if the intensity increases 50% compared to the standard value, the copy number may be considered as increased 50%. A standard value may be a fluorescent intensity value specific to a testing system. A testing system means the whole experimental system including the reagents, primers, probes, procedures and devices used for the testing the CNVs. One test sample is considered to share the same testing system with another test sample if the whole experimental system is the same except that the initial DNA sample to be tested is different. As such a standard value may be obtained for a specific testing system based on prior testing results of DNA samples for which copy numbers of the CNV sites are known.

In other embodiments, a control sample is used to generate control fluorescent intensity values for the same CNV sites. The copy number for each CNV site is known for the control sample. When the peak intensity for a CNV site in a test sample is compared to the peak intensity for the same CNV site in the control sample, the copy number of the CNV site may be determined. For example, as shown in Figure 5 bottom chart, arrow 1 points to a CNV site for which the test sample has about 50% copy number of the number in the control sample because the peak intensity decreases about 50% when compared to the peak intensity for the same CNV site in the control sample. For another example, arrow 2 points to a CNV site for which the test sample has about 150% copy number of the number in the control sample because the peak fluorescent intensity in the test sample increases about 50% when compared to the peak for the same CNV site in the control sample.

In other preferred embodiments, the peak values for each CNV site are normalized against peak values for one or more reference target sites. A CNV copy number change may be detected by comparing the normalized peak value for a CNV site in a test sample to the normalized standard value for the same CNV site in the testing system or the normalized peak value for the same CNV site in a control sample. The normalization may correct the variations, e.g., the amount of DNA templates used for probe hybridization, the amount of ligation probes used for probe hybridization, the amount of PCR primers for amplifying ligation products, the ligation efficiency of each set of probes, and the amount of ligation products used for PCR amplification, in obtaining the peak values between different samples and/or between different CNV sites.

To obtain peak values of reference target sites, probes for the reference target sites may be designed in a similar manner and used simultaneously with the probes for the CNV sites in the hybridization and ligation reactions. In addition, probes for the reference target sites and probes for the CNV sites may share the same primer binding sites so that the same set of primers may be used to amplify the ligation products for both the reference sites and the CNV sites.

As such in some instances, peak values for a plurality of reference target sites are obtained in parallel with the peak values for a plurality of CNV sites so that the peak value for each of the plurality of the CNV sites may be normalized against the peak value for each of the plurality of the reference target sites. For example, peak values for 6 reference target sites may be obtained together with the peak values for 6 CNV sites. In this example, 6 sets of probes for the 6 reference target sites and 6 sets of probes for the 6 CNV sites are added to a sample and ligation products are obtained after hybridization and ligation reactions. A single set of primers may be used to amplify the ligation products and peak values are measured by analyzing the amplification products by capillary electrophoresis.

In some embodiments, the normalization of the peak value for a CNV site in a sample is to obtain a ratio (here referred to as R) of the peak value for the CNV site against the peak value for a reference target site. As such, the copy number of a CNV site in a test sample may be measured by comparing the ratio for the CNV site in the test sample (here referred to as R_test) with the standard ratio for the same CNV site in the testing system (here referred to as R_standard) or with the ratio for the same CNV site in a control sample (here referred to as R_control).

In some instances, the copy number of a CNV site in a test sample is measured by comparing the ratio for the CNV site in the test sample (R_test) with the standard ratio for the same CNV site in the testing system (R_standard). The copy number of the CNV site in the testing system is known (here referred to as C_standard). In this case, the copy number of the CNV site in the test sample (here referred to as C_test) may be calculated as follow: C_test= C_standard x R_test/R_stadard.

In other instances, the copy number of a CNV site in a test sample may be measured by comparing the ratio for the CNV site in the test sample (R_test) with the ratio for the same CNV site in a control sample (R_control). The copy number of the CNV site in the testing system is known (here referred to as C_control). In this case, the copy number of the CNV site in the test sample (C_test) may be calculated as follow: C_test = C_control x R_test/R_control.

As such, when 6 reference target sites are introduced to normalize the peak value for a CNV site, six copy number measurements (C_test) for the CNV site may be derived on the basis of the six peak values for the six reference target sites. Based on the six C_test measurements, the copy number of the CNV site is obtained according to certain statistical analysis. In one embodiment, the median value of the six C_test measurements is deemed the copy number of the CNV site. In another embodiment, the average value of the six C_test measurements is deemed the copy number of the CNV site.

In a multiplex CNV analysis method, multiple sets of primers may be used to amply ligation products in a single tube PCR reaction. In this case, each set of primers is used to amplify a group of ligation products from both CNV sites and reference target sites so that peak values of CNV sites maybe normalized against peak values of the reference target sites in the same group. Depending on probe designs, the number of CNV sites or reference target sites in each group may vary from 1 to 24. In some embodiments, there are 6 CNV sites and 6 reference target sites in each group. In other embodiments, there are 12 CNV sites and 12 reference target sites in each group. In still other embodiments, there are 9 CNV sites and 3 reference target sites in each group. In further other embodiments, there are 16 CNV sites and 8 reference target sites in each group.

The selection of a reference target site is sometimes based on criteria including, but not limited to, the copy number of each reference target site being stable in different samples and the sequences of the reference target sites being unique and not prone to interfering the reactions of the CNV sites of interest. In addition, when a plurality of reference target sites are used in a group, each reference target site is preferably from a different chromosome. In some embodiments, the quality of detecting copy number changes may be improved by increasing the number of reference target sites.

In some embodiments, when normalization is performed and normalized peak values for each CNV site are obtained for the test sample and the control sample, a comparison of the normalized peak values in the test sample with the values in the control sample may be carried out to determine any change of copy number of each CNV site. For example, if a normalized peak value for a CNV site in the test sample is 1.0 and the normalized peak value for the same CNV site in the control sample (the copy number for the CNV site is known to be 2) is 2.0, the copy number of the CNV site in the test sample is determined to be 1, about half of the copy number in the control sample. For another example, if a normalized peak value for a CNV site in the test sample is 1.0 and the normalized standard peak value for the testing system (the copy number for the CNV site is known to be 2) is 1.0, the copy number of the CNV site in the test sample is determined to be 2, about the same with the copy number in the control sample.

DNA mutations refer to nucleotide changes in the genome in comparison to a wild type genome. "Wild-type" refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, "mutant" refers to a gene or gene-product having at one or more sites a different nucleic acid sequence when compared to the wild-type gene or gene product.

In some embodiments, mutations in a gene may be screened by a method according to the present invention. An exemplary multiplex mutation screening method according to the present invention is illustrated in Figure 6. In the example, 96 target sites are screened in a single assay simultaneously. For each target site, a set of probes (here referred to as the right probe and the left probe as they appear in the figure) are designed. The left probe contains a locus-specific hybridization sequence (LSHS), a primer binding site X and a stuffer L1 sequence between the LSHS and the primer binding site X. The right probe contains a locus-specific hybridization sequence (LSHS), a primer binding site Y, and a stuffer L2 sequence between the LSHS and the primer binding site Y. By varying the length of stuffer sequences, a 12-fold multiplexity may be achieved for the ligation products; ligation products for 12 target sites can be distinguished on the basis of fragment size. In addition, by labeling the forward primers with FAM-blue, VIC-green, NED-yellow and PET-red and inserting a stuffer sequence in one of the reverse primers R2, the multiplexity for the mutation screening may be increased to 96 (=12 x 4 x 2). As such, the multiplex mutation screening method may analyze 96 target sites simultaneously. A person skilled in the art would consider increase the multiplexity for mutation screening by varying the parameters, e.g., inserting stuffer sequences in more probes, labeling the primers with more fluorescent tags, or inserting stuffer sequences in more primers. As such, the multiplexity may be increased to 192, 384, 768, or more if desirable.

As shown in Figure 6, sets of probes are designed to overlap with each other. The probes may target the positive stand, the negative stand, or both the positive and the negative stands. In all cases, similar to the rationale in Figure 5, the peak intensity for each target site is compared to a standard value or the peak intensity of the same target site in a control sample. A standard value may be derived in a similar way as described in Figure 5. As shown in Figure 6 bottom charts, the arrow points to a peak with about half the intensity of the same target site in the control sample. The decreased peak intensity suggests that the target site may contain a mutation. This is so because the two probes corresponding to the target site covers a mutation and therefore cannot hybridize to the target site to form a proper ligation product and finally result in no amplification product. This exemplary mutation screening method would provide a preliminary result as to the location of a mutation in a target nucleic acid. This may facilitate further sequencing analysis to identify the exact mutation.

In some embodiments, the target nucleic acids in a sample are RNA and the analysis of the target nucleic acids is to determine the presence, absence, or quantity of the RNA in the sample. In some instances according to the present invention, the RNA is directly used for probe hybridization, probe ligation, ligation product amplification, and amplification product analysis. In other instances, the RNA is reverse-transcribed into complementary DNA (cDNA) before probe hybridization and further steps. As is known in the art, reverse transcription of RNA into cDNA may be accomplished using reverse transcriptase. The analysis of RNA may help determine gene expression levels if the RNA is a transcription product of a gene.

As such, in some embodiments, target RNAs may be analyzed using a multiplex method according to the present invention. An exemplary multiplex target RNA analysis method according to the present invention is illustrated in Figure 7. In the example, 96 target RNAs or its reverse transcribed cDNAs are analyzed in a single assay simultaneously. For each target RNA or cDNA, a set of probes (here referred to as the right probe and the left probe as they appear in the figure) are designed. The left probe contains a locus-specific hybridization sequence (LSHS), a primer binding site X, and a stuffer L1 sequence between the LSHS and the primer binding site X. The right probe contains a locus-specific hybridization sequence (LSHS), a primer binding site Y, and a stuffer L2 sequence between the LSHS and the primer binding site Y. By varying the length of stuffer sequences, a 12-fold multiplexity may be achieved for the ligation products; ligation products for 12 target sites can be distinguished on the basis of fragment size. In addition, by labeling the forward primers with FAM-blue, VIC-green, NED-yellow and PET-red and inserting a stuffer sequence in one of the reverse primers R2, the multiplexity for the mutation screening is increased to 96 (=12 x 4 x 2). As such, the multiplex gene expression analysis may measure 96 target RNAs simultaneously. A person skilled in the art would consider increase the multiplexity for target RNA analysis by varying the parameters, e.g., inserting stuffer sequences in more probes, labeling the primers with more fluorescent tags, or inserting stuffer sequences in more primers. As such, the multiplexity may be increased to 192, 384, 768, or more if desirable.

In some embodiments, as shown in Figure 7 and similar to the rationale in Figure 5, the peak values for each RNA target is normalized against reference target sites before determining the presence, absence or amount of RNA copy number changes between different samples. Reference target sites may be any RNA, for example, RNA of housekeeping genes including, but not limited to, histone, β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or hypoxanthine-guanine phosphoribosyltransferase (HPRT) genes. In a multiplex RNA analysis, multiple groups of target RNA and reference target sites may be analyzed simultaneously. The same set of primers is used to amply ligation products for both the target RNA and the reference target sites within each group. The copy number of a target RNA is determined by comparing the normalized peak values for a target RNA in the test sample to the normalized peak values for the target RNA in the control sample. If the value increases 2 times, the copy number of the target RNA increase 2 times. Other aspects of the peak value normalization and copy number measurement methods are similar to those described for the normalization of peak values for CNV sites against reference target sites and therefore are not repeated here.

Multiplex nucleic acid analysis methods according to the present invention may be applied to detect nucleic acids from pathogens and transgenic organisms and thereby indentify the pathogens and transgenic organisms. Pathogens, including, but not limited to, a bacterium, a virus, a protozoan, a parasite, a mold, or a fungus, may cause diseases or other conditions in an animal. Traditionally, several methods including bacteriological analysis, virus isolation and culture, histopathology and an enzyme-linked immunosorbent assay (ELISA) (Adams and Thompson, 2008, Rev. Sci. Technol. 27, 197-209) have been developed for the phenotypic characterization and identification of pathogens. Alternatively, molecular diagnosis based on polymerase chain reaction (PCR), RT-PCR, (Dhar et al., 2002, J. Virol. Methods 104, 69-82; Nishizawa et al., 1995, J. Gen. Virol. 76, 1563-1569) or quantitative real-time PCR (DallaValle et al., 2005, Vet. Microbiol. 110, 167-179) using specific primer sets for nucleic acid amplification has been demonstrated for diagnosis of diseases. The development of a new multiplex, rapid, accurate, and sensitive diagnostic methods for the identification of pathogens may help in treating, controlling, or even eradicating many pathogen-related diseases. In addition, transgenic organisms, including transgenic plants, e.g., corn, rise, soybean and cotton and transgenic animals, e.g., cow, peg, sheep and dog may sometimes need to be identified.

One aspect of the present invention provides a multiplex detection of pathogens and transgenic organisms. In some embodiments, target nucleic acids specific to pathogens or transgenic organisms are obtained for multiplex analysis. In some instances, nucleic acids specific to pathogens or transgenic organisms may be DNA unique to the pathogens or the transgenic organisms or unique to the family of the pathogens or the transgenic organisms. The pathogens or transgenic organisms may be from many sources. For example, in screening blood and other bodily fluids and tissues for pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like, or transgenic genes, the sources may be blood and other bodily fluid or tissue samples, which may be obtained from living animals or deceased animals. In other instances, nucleic acids specific to a pathogen or transgenic organism may be RNA unique to the pathogens or transgenic organisms or unique to the family of the pathogens or transgenic organisms. For example, RNA viruses such as HIV, HBV, and HCV viruses contain unique RNA molecules and the detection of these unique RNA molecules helps determine the presence, absence or quantity of these RNA viruses.

In some embodiments, the target nucleic acids are amplified before the multiplex analysis. Such amplification is needed when the amount of target nucleic acids obtained from pathogens or transgenic organisms is limited. In other embodiments, when the target nucleic acids are RNA, a reverse transcription step may be optionally performed to convert the RNA into cDNA before the multiplex analysis.

An exemplary multiplex target nucleic acid analysis method for detecting pathogens and transgenic organisms according to the present invention is illustrated in Figure 8. In the example, 96 target nucleic acids are analyzed in a single assay simultaneously. For each target nucleic acid, a set of probes (here referred to as the right probe and the left probe as they appear in the figure) are designed. The left probe contains a locus-specific hybridization sequence (LSHS), a primer binding site X, and a stuffer L1 sequence between the LSHS and the primer binding site X. The right probe contains a locus-specific hybridization sequence (LSHS), a primer binding site Y, and a stuffer L2 sequence between the LSHS and the primer binding site Y. By varying the length of stuffer sequences, a 12-fold multiplexity may be achieved for the ligation products; ligation products for 12 target nucleic acids can be distinguished on the basis of fragment size. In addition, by labeling the forward primers with FAM-blue, VIC-green, NED-yellow and PET-red and inserting a stuffer sequence in one of the reverse primers R2, the multiplexity for the multiplex pathogen and transgenic organism detection is increased to 96 (=12 x 4 x 2). As such, the multiplex pathogen and transgenic organism detection analysis may assay 96 target nucleic acids simultaneously. A person skilled in the art would consider increase the multiplexity for target nucleic acid analysis for detecting pathogens and transgenic organisms by varying the parameters, e.g., inserting stuffer sequences in more probes, labeling the primers with more fluorescent tags, or inserting stuffer sequences in more primers. As such, the multiplexity may be increased to 192, 384, 768, or more if desirable.

As a person skilled in the art understands based on the disclosure of the present invention, the presence of peaks corresponding to target nucleic acids indicates the presence of the corresponding pathogens or transgenic organisms. As shown in Figure 8 bottom chart, the arrows point to two peaks corresponding to two target nucleic acids, indicating the presence of two corresponding pathogens. The absence of peaks corresponding to the remaining 86 target nucleic acids indicates the absence of the 86 corresponding pathogens. The encircled peaks are positive controls, whose presence indicates the testing system is functional.

In other embodiments, the method according to the present invention may be applied to measure quantitatively the amount of pathogens and transgenic organisms. The rationale behind this application procedure for this purpose are similar to those described for quantitatively measure RNA expression levels in the multiplex RNA analysis section supra except that exogenous nucleic acids may be used as reference target sites. In some embodiments, a certain amount of exogenous DNA or RNA fragments are added into samples and used as reference target sites. For example, in measuring the load of HIV virus in a plasma samples, the same amount of exogenous RNA fragments may be mixed with the same amount of plasma from each sample before RNA extraction. The quantitative evaluation of HIV load may be carried out by quantitatively measuring the HIV RNA level by normalizing the peak values for HIV RNA against the peak values for the exogenous reference RNA fragments.

Epigenetic modifications of genomic DNA, e.g., changes in DNA methylation patterns are related to many diseases or other health conditions. Abnormal methylation of normally unmethylated CpG-rich areas, also known as CpG-islands, have been associated with transcriptional inactivation of many disease genes, e.g., tumor suppressor genes, DNA repair genes, metastasis inhibitor genes. See, e.g., Jain PK, Epigenetics: the role of methylation in the mechanism of action of tumor suppressor genes, Ann N Y Acad Sci. 2003 Mar;983:71-83. There is a need for a multiplex, quick, and accurate method for detecting DNA methylation patterns, which may aid the diagnosis, prognosis, prediction, and evaluation of treatment plan for underlying diseases or other health conditions.

An exemplary multiplex DNA methylation detection method according to the present invention is illustrated in Figure 9. In the example, 96 target methylation sites are analyzed in a single assay simultaneously. In some instances, DNA bearing the target methylation sites in a test sample may be treated with methylation sensitive restriction endonucleases, e.g., HpaII or HhaI, so that unmethylated DNA are cleaved at a position near or close to the methylation site. The treatment of methylation sensitive restriction endonucleases may be carried out before, at the same time, or after the probe hybridization step. In some embodiments, the treatment is carried out before the probe hybridization step. In this case, if the DNA in the test sample is cleaved, the probes designed to hybridize near or close to the methylation site on the target DNA may not be ligated and no amplification products may be produced. In contrast, if the DNA in the test sample is methylated at the methylation site and therefore cannot be cleaved by the methylation sensitive restriction endonuclease, the probes hybridized near or close to the methylation site on the target DNA may be ligated and the corresponding amplification products may be produced. In other embodiments, the treatment is carried out at the same time or after the probe hybridization step. In this case, probes may hybridize to the target DNA near or close to the methylation site, the methylation sensitive endonuclease may cleave the hybridized probe/target DNA duplex if the target DNA is not methylated and therefore no amplification product is produced. In contrast, if the target DNA is methylated, the methylation sensitive endonuclease may not cleave the hybridized probe/target DNA duplex and therefore amplification product is produced.

In both situations, as shown in Figure 9, in an exemplary multiplex methylation detection method according to the present invention, for each target methylation site, a set of probes (here referred to as the right probe and the left probe as they appear in the figure) are designed. The left probe contains a locus-specific hybridization sequence (LSHS) and a primer binding site X, and a stuffer L1 sequence between the LSHS and the primer binding site X. The right probe contains a locus-specific hybridization sequence (LSHS), a primer binding site Y, and a stuffer L2 sequence between the LSHS and the primer binding site Y. By varying the length of stuffer L1 and stuffer L2 sequences, a 12-fold multiplexity may be achieved for the ligation products; ligation products for 12 target methylation sites can be distinguished on the basis of fragment size. In addition, by labeling the forward primers with FAM-blue, VIC-green, NED-yellow and PET-red and inserting a stuffer sequence in one of the reverse primers R2, the multiplexity for the methylation detection may be increased to 96 (=12 x 4 x 2). As such, the multiplex methylation detection may measure 96 target methylation sites simultaneously. A person skilled in the art may increase the multiplexity for methylation detection by varying the parameters, e.g., inserting stuffer sequences in more probes, labeling the primers with more fluorescent tags, or inserting stuffer sequences in more primers. As such, the multiplexity may be increased to 192, 384, 768, or more if desirable.

In other instances, the DNA in the test sample is treated with bisulfite so that unmethylated cytosines (here referred to as "C") are converted into uracils. The bisulfite treatment therefore makes nucleotide changes at unmethylation sites, converting an unmethylated "C" into "U" in the target DNA. In contrast, methylated "C" remains as "C" after bisulfite treatments. As such, specific probes are designed to bind either to the methylated allele or the unmethylated allele of the test DNA: the methylated allele with "Cs"at methylated C sites and "Us" at the unmethylated C sites, and the unmethylated allele with "Us" at all C sites. Depending on which allele needs to be detected, the design of these specific probes is briefly described below.

As shown in Figure 9, 96 methylation sites may be detected in a single assay simultaneously when a bisulfite treated is first performed on the target DNA. For illustrative purposes, a set of probes (here referred to as the right probe and the left probe as they appear in the figure) are designed. The left probe contains a methylation-specific hybridization sequence (MSHS) with the methylation site recognition nucleotides "G", a primer binding site X, a stuffer L1 sequence between the MSHS and the primer binding site X. The right probe contains a methylation-specific hybridization sequence (MSHS) with the methylation site recognition nucleotides "G", a primer binding site Y, and a stuffer L2 sequence between the MSHS and the primer binding site. By varying the length of stuffer L1 and stuffer L2 sequences, a 12-fold multiplexity may be achieved for the ligation products; ligation products for 12 methylation sites can be distinguished on the basis of fragment size. In addition, by labeling the forward primers with FAM-blue, VIC-green, NED-yellow and PET-red and inserting a stuffer sequence in one of the reverse primers, the multiplexity for the methylation detection may be increased to 96 (=12 x 4 x 2). As such, the multiplex methylation detection may measure 96 target methylation sites simultaneously. A person skilled in the art may increase the multiplexity for methylation detection by varying the parameters, e.g., inserting stuffer sequences in more probes, labeling the primers with more fluorescent tags, or inserting stuffer sequences in more primers. As such, the multiplexity may be increased to 192, 384, 768, or more if desirable. In some embodiments, when only unmethylated alleles need to be detected, the unmethylation specific probes are used instead of the methylation specific hybridization probes. In other embodiments, when both methylated and unmethylated alleles need to be detected, both methylated and unmethylated specific probes are used. The rationale for designing the methylated and unmethylated specific probes is similar to those described supra, and therefore is not repeated here.

In both the method using methylation sensitive restriction enzymes and the method using bisulfite treatment, the analysis of the amplification products may determine the presence or absence of methylated nucleotides in a target DNA. In the method using methylation sensitive restriction enzymes, amplification peaks appear only if the DNA is methylated. In the method using bisulfite treatment and MSHS probes which have the methylation site recognition nucleotides "G", amplification peaks appear only if the DNA is methylated. Therefore, in both methods, the presence of peaks indicates the presence of methylated nucleotides in the target DNA, and the absence of peaks indicates the absence of methylated nucleotides in the target DNA. As shown in Figure 9 bottom chart, the arrows point to the peaks in the amplification product, indicating that the corresponding methylation sites contain methylated nucleotides in the test sample. The absence of peaks corresponding to other methylation sites indicates that those other methylation sites in the test sample contain no methylated nucleotides. The encircled peaks are positive control, whose presence indicates the testing system is functional.

In some embodiments, the multiplex DNA methylation analysis may determine the relative amount of methylated DNA in a sample. To determine the relative amount of methylated DNA in a sample, the peak value for each target methylation site in a test sample is first normalized to the peak value of the reference target site, and then compared to the normalized peak value in a control sample. The selection of a reference target site is sometimes based on criteria including, but not limited to, the copy number of each reference target site being stable after methylation sensitive restriction enzyme digestion or bisulfite treatment in different samples and the sequences of the reference target sites being unique and not prone to interfering the reactions of the methylation sites of interest. In addition, when a plurality of reference target sites are used in a group, each reference target site is preferably from a different chromosome. In a multiplex DNA methylation analysis, multiple groups of target methylation sites and reference target sites may be analyzed simultaneously. The same set of primers is used to amply ligation products for both the target methylation sites and the reference target sites within each group. The relative amount of methylated DNA in the target methylation site is determined by comparing the normalized peak value for a target methylation site in the test sample to the normalized peak value for the target methylation site in the control sample. If the value increases 2 times, the relative amount of methylated DNA in the target methylation site increases 2 times. Other aspects of the normalization method are similar to those described for the normalization of peak values for CNV sites against reference target sites and therefore are not repeated here.

In another aspect, the present invention is a method for detecting a small quantitative variation of a nucleic acid between two samples. A small quantitative variation of a nucleic acid may be a small copy number change of a nucleic acid, e.g., a gene, a part of a chromosome, or a whole chromosome. As used herein, a small quantitative variation of a nucleic acid refers to any copy number changes that are less than 50%. Indeed, in some embodiments of the present invention, the method may detect small copy number changes of about 0.1% to about 30%. In other embodiments, the method may detect small copy number changes of about 8%, 6%, 4%, 2%, 1%, or 0.1 %.

For example, the copy number of human chromosome 21, human chromosome 18, human chromosome 13, human chromosome region 22q11.2, or the pseudoautosomal regions of human chromosomes X or Y in the maternal blood may change in a small scale if the fetus harbors a different copy number of those chromosome regions. In one instance, if a fetus has Down's syndrome, the copy number of chromosome 21 may increase in a small scale in maternal blood. The increase is usually less than 10%. As such, a method according to the present invention may be used to detect small copy number changes of human chromosome 21, human chromosome 18, human chromosome 13, human chromosome region 22q11.2, or the pseudoautosomal regions of human chromosomes X or Y in maternal blood. The pseudoautosomal regions (PAR1 and PAR2) of the human X and Y chromosomes are not inherited in a strictly sex-linked fashion and may be used to detect the copy number of the X and Y chromosome pairs. See, e.g., Mangs and Morris, The Human Pseudoautosomal Region (PAR): Origin, Function and Future, Curr Genomics. 2007 April; 8(2): 129-136.

In one embodiment, the method for detecting small copy number changes of a nucleic acid in a test sample comprises the steps of measuring the copy number of a plurality of target sites within the nucleic acid in the test sample, and determining the copy number of the nucleic acid by statistically analyzing the measured copy number for each of the plurality of target sites.

According to this present invention, to detect small copy number changes of a nucleic acid, a plurality of target sites in the nucleic acid are chosen for analysis so that a statistically significant result may be obtained to quantitatively determine a copy number change for the nucleic acid. As used herein, a plurality of targets sites refers to more than about 5 target sites, preferably more than about 10 target sites, more preferably more than 100 target sites. The number of target sites may increase to about 100-500 if a more sensitive detection is desirable. A person skilled in the art may decide the number of target sites based on the disclosure of the present invention or empirically according to prior testing results.

The measurement of copy numbers for each of the plurality of target sites may be accomplished by many techniques, including a multiplex nucleic acid analysis method similar to the CNV detection method as detailed supra, a multiplex nucleic acid analysis employing DNA sequencing techniques, and real-time PCR.

For one example, a multiplex nucleic acid analysis method similar to the CNV detection method as detailed supra is used. Similar to the scheme shown in Figure 5, the copy number of 96 target sites may be measured in a single assay simultaneously. The descriptions of the designs of probes, primers and testing procedures are not repeated here except that the statistical analysis of peak intensities is described below.

In some embodiments, the fluorescent peak intensity of each target site in a test sample is measured and compared to a standard peak value of the same target site in the testing system to determine the copy number of the target site. A standard value may be a fluorescent intensity value corresponding to the target site in the specific testing system. A testing system means the whole experimental system including the reagents, primers, probes, procedures and devices used for the testing the copy number changes. One test sample is considered to share the same testing system with another test sample if the whole experimental system is the same except the initial DNA sample to be tested. As such a standard value may be obtained for a specific testing system based on prior testing results of DNA samples. If the DNA samples are from normal or wild-type subject, the standard value is a normal or wild-type standard value. If the DNA samples are from an abnormal subject, the standard value is an abnormal standard value.

In other embodiments, a control sample is used to generate control fluorescent peak intensity values for the target sites. The copy number for each target site is known for the control sample. When the fluorescent peak value for a target site in a test sample is obtained and compared to the peak value for the same target site in the control sample, the copy number of the target site may be determined.

In some preferred embodiments, similar to the normalization and copy number measurement methods described for CNV copy number change detection supra, the peak values for each gene target site (i.e., target DNA of interest, which is not necessarily within a gene but can be within non-coding genomic DNA) are normalized against peak values for one or more reference target sites. The copy number changes are measured by comparing the normalized peak value for each gene target site in the test sample to the normalized standard value for the same gene target site in the testing system or the normalized peak value for the same gene target site in the control sample. The normalization may correct the variations, e.g., the amount of DNA templates used for probe hybridization, the amount of ligation probes and PCR primers, the ligation efficiency of each set of probes, and the amount of DNA used for PCR amplification, in obtaining the peak values between different samples and between different gene target sites. To obtain peak values of reference target sites, probes for the reference target sites may be designed in a similar manner and used simultaneously with the probes for the gene target sites in the hybridization and ligation reactions. In addition, probes for the reference target sites and probes for the gene target sites may share the same primer binding sites so that the same set of primers may be used to amplify the ligation products for both the reference sites and the gene target sites.

As such in some instances, peak values for a plurality of reference target sites are obtained in parallel with the peak values for a plurality of gene target sites so that the peak value for each of the plurality of the gene target sites may be normalized against the peak value for each of the plurality of the reference target sites. The number of reference target sites may be about 1 to about 100, and the number of gene target sites may be about 1 to about 100. For one example, peak values for 6 reference target sites may be obtained together with the peak values for 6 gene target sites. In this example, 6 sets of probes for the 6 reference target sites and 6 sets of probes for the 6 gene target sites are added to a sample and ligation products are obtained after hybridization and ligation reactions. For another example, peak values for 100 reference target sites may be obtained together with the peak values for 100 gene target sites. In this example, 100 sets of probes for the 100 reference target sites and 100 sets of probes for the 100 gene target sites are added to a sample and ligation products are obtained after hybridization and ligation reactions. In both examples, a single set of primers may be used to amplify the ligation products and peak values are measured by analyzing the amplification products by capillary electrophoresis.

In some embodiments, the number of reference target sites used for a gene target site may affect the sensitivity of detecting copy number changes of the gene target site. The more reference target sites are used, the smaller of the copy number change of the gene target site may be detected. Accordingly, when it is desirable to detect more copy number changes, for example a 0.1 % change of cancer cell-associated copy number variant, it is desirable to use relatively more reference target sites for each gene target site. A person skilled in the art may increase the number of reference target sites when it is apparent that more sensitive detection of copy number changes is desired.

In some embodiments, the normalization of the peak value for a gene target site in a sample is to obtain a ratio (here referred to as R) of the peak value for the gene target site against the peak value for a reference target site. As such, the copy number of a gene target site in a test sample may be measured by comparing the ratio for the gene target site in the test sample (here referred to as R_test) with the standard ratio for the same gene target site in the testing system (here referred to as R_standard) or with the ratio for the same gene target site in a control sample (here referred to as R_control).

In some instances, the copy number of a gene target site in a test sample is measured by comparing the ratio for the gene target site in the test sample (R_test) with the standard ratio for the same gene target site in the testing system (R_standard). The copy number of the gene target site in the testing system is known (here referred to as C_standard). In this case, the copy number of the gene target site in the test sample (here referred to as C_test) may be calculated as follow: C_test= C_standard x R_test/R_standard.

In other instances, the copy number of a gene target site in a test sample may be measured by comparing the ratio for the gene target site in the test sample (R_test) with the ratio for the same gene target site in a control sample (R_control). The copy number of the gene target site in the testing system is known (here referred to as C_control). In this case, the copy number of the gene target site in the test sample (C_test) may be calculated as follow: C_test = C_control x R_{test /} R_control.

As such, when 6 reference target sites are introduced to normalize the peak value for a gene target site, six copy number measurements (C_test) for the gene target site may be derived on the basis of the six peak values for the six reference target sites. Based on the six C_test measurements, the copy number of the gene target site is obtained according to certain statistical analysis. In one embodiment, the median value of the six C_test measurements is deemed the copy number of the gene target site. In another embodiment, the average value of the six C_test measurements is deemed the copy number of the gene target site.

Based on the copy number for each of the plurality of target sites, the copy number for the nucleic acid may be determined by methods including, but not limited to, taking the average of the copy numbers of all target sites or the median value of the copy numbers for all target sites, or taking the average of the copy numbers of all target sites or the median value of the copy numbers of all target sites after abandoning some egregious values if desirable.

In some embodiments, the testing for each gene target site in the nucleic acid may be repeated so that multiple copy number calculation results may be obtained. For example, the testing for each gene target site within a nucleic acid may be repeated three times and three copy number calculation results for the nucleic acid may be obtained. The average or median value of the three copy number calculation results may be deemed as the copy number of the nucleic acid. As such, the number of repeats of the testing may also affect the sensitivity of the method. If a more sensitive detection of copy number changes is desired, the testing may be repeated more times. A person skilled in the art may increase the number of repeated testing if he or she desires to detect smaller copy number changes, e.g., 0.1%.

As such, the sensitivity of detecting small copy number changes of a nucleic acid in a sample may be influenced by many factors including, but not limited to, the number of gene target sites within the nucleic acid, the number of reference target sites used for each gene target site, and the number of repeated testing for each gene target site. The increase of the number of gene target sites within the nucleic acid, the number of reference target sites used for each gene target site, and/or the number of repeated testing for each gene target site may enhance the sensitivity of the detection. A person skilled in the art may adjust the numbers according to the present invention if a more sensitive detection is needed in measuring very small copy number changes of a nucleic acid of interest in a sample.

Various statistical methods may be applied to calculate the copy number of each target site based on experimental results and determine the copy number of the nucleic acid based on the calculated copy number of each target site. A specific example of statistical analysis of small copy number changes is detailed below in Example 4: "Detection of chromosome 21 copy number changes."

For another example, real-time PCR may be used to detect the copy number of each of the plurality of the selected target sites as is known in the art. Real-time PCR may determine the copy number of a target site in a monoplex or multiplex manner. Based on the copy numbers for each of the plurality of target sites, the copy number for the nucleic acid may be determined by methods including, but not limited to, taking the average of the copy numbers of all target sites or the median value of the copy numbers for all target sites, or taking the average of the copy numbers of all target sites or the median value of the copy numbers of all target sites after abandoning some egregious values if desirable.

In yet another aspect of the present invention, a kit is provided for multiplex nucleic acid analysis and small copy number change detection. In one embodiment, the kit for assaying nucleic acids in a sample include one or more sets of probes corresponding to a target nucleic acid so that the probes in each set, when hybridized to the target nucleic acid may be ligated to form a third probe. In another embodiment, the kit further includes one or more sets of primers for amplifying the third probe.

Each set of probes may include a first probe having a first portion at least partially complementary to a first region of the target nucleic acid and a second portion as a first primer binding site, and a second probe having a first portion at least partially complementary to a second region of the target nucleic acid and a second portion as a second primer binding site. In some instances, the 5' end of the first probe is essentially adjacent to the 3' end of the second probe and the first and the second probes may be ligated to form a third probe. In other instances, the 5' end of the first probe is not adjacent to the 3' end of the second probe and the first and the second probes may not be ligated to form a third probe without filling the gaps. Gap filling may be achieved by another probe that can hybridize to the gap on the target nucleic acid or by extending one of the two probes in a polymerase reaction. For example, the 3' end of the second probe may be extended to fill the gap until the extended 3' end is substantially adjacent to the 5' end of the first probe.

In some embodiments, the kit may also contain reagents including, but not limited to, a ligase, e.g., a Taq DNA ligase or a T4 DNA ligase, a buffer for a ligation reaction, a DNA polymerase, e.g., a Taq DNA polymerase, a buffer for polymerase chain reaction, or a combination thereof.

In some embodiments, the set of primers in a kit may include a first primer at least partially complementary to the first primer binding site and a second primer at least partially complementary to the second primer binding site. In some instances, at least one primer of the set of primers is labeled with a detectable moiety. The moiety may be an oligonucleotide tag or a fluorescent dye such as a fluorescein fluorophore. A fluorophore may be FAM (5-or 6-carboxyfluorescein), VIC, NED, PET, Fluorescein, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, and Yakima Yellow.

In some embodiments, an oligonucleotide GTTTCTT was included in the 5' portion of at least one of the primers. In some preferred embodiments, the reverse primer used for amplifying the ligation products includes oligonucleotide GTTTCTT or its functional equivalents, e.g., GTTTCTTG.

In some embodiments, at least one primer of the set of primers includes a stuffer sequence with a length of about 10-500 nucleotides. The stuffer sequence in some primers may have about 10 to about 500 nucleotides. The stuffer sequence in other primers may be about 10 to about 60 nucleotides. In some preferred embodiments, no primer has more than about 125 nucleotides. In other preferred embodiments, no primer has more than about 75 nucleotides.

In other embodiments, at least one probe of the set of probes includes a stuffer sequence with a length of about 1-200 nucleotides. In other instances, the stuffer sequence has about 1 to about 55 nucleotides. In some preferred embodiments, the third probe has no more than about 250 nucleotides. In other preferred embodiments, the third probe has no more than about 140 nucleotides. In still some preferred embodiments, no probe has more than about 125 nucleotides. In still some other preferred embodiments, no probe has more than about 70 nucleotides. In some further preferred embodiments, no probe has more than about 60 nucleotides.

In some embodiments, the target nucleic acid is a dystrophin gene and the kit is for detecting Duchenne muscular dystrophy. The sets of probes in the kit comprise one or more probe pairs selected from SEQ ID NOs: 158-541.

In other embodiments, the target nucleic acid is on human chromosome 21 and the kit is for detecting fetal Down's syndrome in maternal blood. The sets of probes in the kit comprise one or more probe pairs selected from SEQ ID NOs: 559-942.

It should be understood that this invention is not limited to the particular methodologies, protocols and reagents, described herein. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

In addition, the reactions outlined below may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents which may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc., which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may be used, depending on the sample preparation methods and purity of the target.

While the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the full scope of the invention, as described in the appended specification and claims. Other features, objects, and advantages of the disclosed subject matter will be apparent from the detailed description, figures, examples and claims. Methods and materials substantially similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter.

This example demonstrates a multiplex SNP detection method according to the present invention. In this example, 48 SNPs were detected simultaneously in a blood sample by employing fluorescent dye labeled forward primers and varying the length of the reverse primer by adding a stuffer sequence according to the scheme in Figure 4.

The 48 SNPs were rs1056893, rs1058588, rs10790286, rs10791649, rs11107, rs11155787, rs11161732, rsl249950, rs12719860, rs1359185, rs1572983, rs2161916, rs2231926, rs2241280, rs2241571, rs2241802, rs2279072, rs2294092, rs2297129, rs2304035, rs2304102, rs2305150, rs2306331, rs2401751, rs2779500, rs2986014, rs3182535, rs3731631, rs3736582, rs3749877, rs3809806, rs3816800, rs4141253, rs4362, rs4371677, rs469783, rs4829830, rs4920098, rs624821, rs625372, rs639225, rs6784322, rs6892205, rs894344, rs934472, rs938883, rs9389034, and rs9791113. All SNP names and other information may be found in the National Center for Biotechnology Information dbSNP database. For each SNP, three probes were designed: the 3' probe (the right probe), and two 5' probes (the left probe) corresponding to two alleles. The 3' and 5' probes were designed in a manner so that when both are hybridized to the target sequence under a suitable condition, there is no gap between the two probes. The probes were synthesized by Life Technologies Corporation. The names, sequence ID numbers, primer binding sequences, and stuffer sequences for each of the 144 (3 x 48) probes are shown in Table 1.

For the 3' probe, the 5' end nucleotide was phosphorylated to provide a phosphate which would be connected to the hydroxyl group in the 3' end nucleotide of the 5' probe. Each 3' probe included a locus specific hybridization sequence (LSHS) in the 5' portion followed by a stuffer L2 sequence, and a primer binding sequence Y in the 3' portion. In some 3' probes, SNP_Y1 sequence, SEQ ID NO: 155 was used. In other 3' probes, SNP-Y2 sequence, SEQ ID NO: 156 was used.

Two different 5' probes were designed for each SNP with each 5' probe corresponding to a different allele. The 3' portion of each 5' probe was an allele-specific hybridization sequence (ASHS) and the 3' end nucleotide corresponded to the specific nucleotide in each individual allele. The 5' portion of each 5' probe had a primer binding sequence X. In this example, four primer binding sequence X: SNP_X1, SEQ ID NO: 151; SNP_X2, SEQ ID NO: 152; SNP_X3, SEQ ID NO: 153; SNP_X4, SEQ ID NO: 154 were used in the 5' probes. In some 5' probes, a stuffer A sequence was inserted between the 3' portion and the 5' portion of the 5' probes. In addition, in some 5' probes, a stuffer L1 sequence was inserted between the 5' portion and the stuffer A sequence. The position of the stuffer A and stuffer L1 sequences are interchangeable.

In addition, four forward primers and two reverse primers were designed for amplifying the ligation products. The four forward primers (SNP_F1, SEQ ID NO: 145; SNP_F2, SEQ ID NO: 146; SNP_F3, SEQ ID NO: 147; and SNP_F4, SEQ ID NO: 148) had unique sequences that were consistent with the four primer binding sequence X (SNP_X1, SEQ ID NO: 151; SNP_X2, SEQ ID NO: 152; SNP_X3, SEQ ID NO: 153; SNP_X4, SEQ ID NO: 154), respectively. The four forward primers (SNP_F1, SNP_F2, SNP_F3, and SNP_F4) were labeled on the 5' ends with four different fluorescent dyes: FAM-blue, VIC-green, NED-yellow, and PET-red, respectively. The two reverse primers (SNP_R1, SEQ ID NO: 149; and SNP_R2, SEQ ID NO: 150) had unique sequences that were reversely complementary to the pimer binding sequence Y (SNP_Y1 sequence, SEQ ID NO: 155 and SNP-Y2 sequence, SEQ ID NO: 156), respectively. The SNP_R primer also had a stuffer sequence SNP_R_Stuffer, SEQ ID NO:157 in the 5' portion. All primers and probes were synthesized by Life Technologies Corporation.

To perform the 48 multiplex SNP detection assay, a ligation product was first generated. Briefly, genomic DNA was extracted from a 2ml whole blood sample using the classic phenol:chloroform method. The blood sample was collected from a healthy volunteer at Shanghai Ruijin Hospital, Shanghai, China. From the extracted genomic DNA, 100-200 microgram (µg) DNA was dissolved in 10 microliter (µl) 1xTE buffer (10mM Tris.Cl, pH8.0, 1mM EDTA from Sigma-Aldrich). The dissolved genomic DNA was denatured at 98°C for 5 minutes and then immediately cooled down on ice. At the same time, a 2x ligation premix solution was prepared according to the following formula: a 10µl 2xligation premix was made of 2µl 10xTaq ligase buffer, 1µl 40U/µl Taq Ligase from NewEngland Biolabs, Inc., 1µl ProbeMix (each probe with a final concentration of 0.005 micromolar in 1xTE), and 6µl ddH₂O (Distilled Milli-Q water from Milli-Q Advantage A10, Millipore). 10 µl 2X ligation premix was mixed with the denatured 10 µl genomic DNA and the mixture was allowed to undergo 4 cycles of denaturation, hybridization and ligation under the following conditions: 95 °C for 30 seconds, and then 58 °C for 4 hours. The ligation product thus obtained could be stored on ice for same day use or freezed in -20 °C for future use.

With the ligation product, an amplification step was then performed to obtain an amplification product. Briefly, a PCR reaction was performed using the amplification product as the template. The PCR reaction mixture was prepared as follows: a 20 µl reaction system was made by mixing 2µl 10x PCR buffer (Qiagen, Germany), 2µl 2.5mM dNTP mix (2.5mM each of dATP, dTTP, dCTP and dGTP from Takara Bio Inc.), 2µl primer mix (SNP_F1, SNP_F2, SNP_F3, SNP_F4, SNP_R1 and SNP_R2 at final concentrations of 1µM, 1µM, 1µM, 1µM, 2µM and 2µM, respectively), 1µl Ligation product, 0.2µl 5U/µl HotStarTaq Plus Taq DNA polymerase (Qiagen, Germany), and 12.8µl ddH₂O. The PCR mixure was allowed to undergo a polymerase chain reaction under the following conditions: 95 °C for 2 minutes, followed by 35 cycles of 94 °C for 20 second, 57°C for 40 second, and 72 °C for 1.5 minutes, and after the 35th cycle, the reaction mixture was kept at 60 °C for 1 hour. To analyze the amplifcation product, 1 µl of the amplification product was first diluted with ddH₂O 10 times into 10 µl. Then 1 µl was taken out of the 10 µl diluted amplification product and mixed with 0.1 µl GeneScan™ 500 LIZ® size standard (Life Technologies, Inc.) and 8.9 µl Hi-Di formamide (Life Technologies, Inc.). The mixture was detaured at 95 °C for 5 minutes and analyzed with capillary electrophoresis by ABI3130XL according to manufacturer's manual. The capillary electrophoresis data was processed using Genemapper 4.0. SEQ ID NO. Probe Name Sequence X Stuffer L1 Stuffer A Stuffer L2 Sequence Y 1 rs1056893_3 ATTA Y2 2 rs1056893_C X3 3 rs1056893_T X3 TT 4 rs1058588_3 Y2 5 rs1058588_C X2 6 rs1058588_T X2 TT 7 rs10790286_3 ATT Y2 8 rs10790286_C X4 9 rs10790286_T X4 TT 10 rs10791649_3 ATTACGCGATTAC Y2 11 rs10791649_A X4 TT 12 rs10791649_G X4 13 rs11107_3 ATTACGCGATTACG Y1 14 rs11107_A X2 A TT 15 rs11107_G X2 A 16 rs11155787_3 ATTACGCGATTAC Y1 17 rs11155787_C X3 A 18 rs11155787_T X3 A TT 19 rs11161732_3 ATTACGCGA Y1 20 rs11161732_A X1 ATTA TT 21 rs11161732_G X1 ATTA 22 rs1249950_3 ATTAC Y1 23 rs1249950_C X1 24 rs1249950_T X1 TT 25 rs12719860_3 ATTACGCGATTAC Y2 26 rs12719860_A X1 ATTAC TT 27 rs12719860_C X1 ATTAC 28 rs1359185_3 ATTACGCGATTA Y2 29 rs1359185_A X4 ATT TT 30 rs1359185_G X4 ATT 31 rs1572983_3 ATTACGCGA Y2 32 rs1572983_C X2 33 rs1572983_T X2 TT 34 rs2161916_3 A Y2 35 rs2161916_A X4 TT 36 rs2161916_G X4 37 rs2231926_3 ATTACGC Y1 38 rs2231926_A X4 TT 39 rs2231926_G X4 40 rs2241280_3 ATTACGCGAT Y2 41 rs2241280_A X1 ATTA TT 42 rs2241280_G X1 ATTA 43 rs2241571_3 AT Y1 44 rs2241571_C X2 45 rs2241571_T X2 TT 46 rs2241802_3 ATTACGCGAT Y2 47 rs2241802_A X4 TT 48 rs2241802_G X4 49 rs2279072_3 ATTACGCGATT Y2 50 rs2279072_C X2 51 rs2279072_T X2 TT 52 rs2294092_3 ATTACGCGATT Y1 53 rs2294092_C X1 ATTAC TT 54 rs2294092_G X1 ATTAC 55 rs2297129_3 Y2 56 rs2297129_A X3 TT 57 rs2297129_G X3 58 rs2304035_3 A Y2 59 rs2304035_A X1 TT 60 rs2304035_G X1 61 rs2304102_3 ATTACGCGATTA Y2 62 rs2304102_A X1 TT 63 rs2304102_G X1 64 rs2305150_3 ATTACGCGATTA Y1 65 rs2305150_C X2 ATT 66 rs2305150_T X2 ATT TT 67 rs2306331_3 ATTACGCG Y1 68 rs2306331_C X2 69 rs2306331_T X2 TT 70 rs2401751_3 ATTA Y1 71 rs2401751_A X2 TT 72 rs2401751_G X2 73 rs2779500_3 ATTACGCGA Y1 74 rs2779500_C X1 TT 75 rs2779500_G X1 76 rs2986014_3 ATT Y1 77 rs2986014_C X3 78 rs2986014_T X3 TT 79 rs3182535_3 Y1 80 rs3182535_A X3 TT 81 rs3182535_G X3 82 rs3731631_3 ATTACGCGATTAC Y2 83 rs3731631_A X2 AT TT 84 rs3731631_G X2 AT 85 rs3736582_3 ATTA Y2 86 rs3736582_C X2 TT 87 rs3736582_G X2 88 rs3749877_3 ATTACGC Y2 89 rs3749877_A X3 TT 90 rs3749877_G X3 91 rs3809806_3 ATTACGCG Y1 92 rs3809806_C X4 93 rs3809806_T X4 TT 94 rs3816800_3 AT Y1 95 rs3816800_C X1 TT 96 rs3816800_G X1 97 rs4141253_3 ATTACGCGATT Y1 98 rs4141253_C X3 99 rs4141253_T X3 TT 100 rs4362_3 ATTACGC Y2 101 rs4362_C X4 102 rs4362_T X4 TT 103 rs4371677_3 ATTACGCGATT Y2 104 rs4371677_A X3 TT 105 rs4371677_G X3 106 rs469783_3 ATTACGCGATT Y2 107 rs469783_C X3 ATTACGCGAT 108 rs469783_T X3 ATTACGCGAT TT 109 rs4829830_3 ATTACGCGAT Y1 110 rs4829830_A X2 TT 111 rs4829830_C X2 112 rs4920098_3 ATTACG Y2 113 rs4920098_C X1 114 rs4920098 T X1 TT 115 rs624821_3 ATTACGCGATTA Y1 116 rs624821_A X4 117 rs624821_T X4 TT 118 rs625372_3 Y1 119 rs625372_C X4 120 rs625372_T X4 TT 121 rs639225_3 ATTACGCGATTA Y2 122 rs639225_A X2 ATTA TT 123 rs639225_G X2 ATTA 124 rs6784322_3 ATTACGCGATTACG Y1 125 rs6784322_A X4 A 126 rs6784322_T X4 A TT 127 rs6892205_3 ATTACGCGA Y1 128 rs6892205_A X3 AT TT 129 rs6892205_G X3 AT 130 rs894344_3 ATTACGCGAT Y1 131 rs894344_A X1 TT 132 rs894344_G X1 133 rs934472_3 AT Y1 134 rs934472_A X4 TT 135 rs934472_C X4 136 rs938883_3 ATTACGCGATTACGC Y2 137 rs938883_C X3 A 138 rs938883_T X3 A TT 139 rs9389034_3 ATTACGCG Y1 140 rs9389034_C X3 141 rs9389034_T X3 TT 142 rs9791113_3 ATTACGCG Y2 143 rs9791113_C X1 TT 144 rs9791113_G X1

As shown in Figures 10A-E, the amplification products obtained in the assay could be separated and the peaks corresponding to each SNP allele could be individually identified by capillary electrophoresis. All the amplification products were from the same PCR reaction. Figure 10A showed the chromatograms of all amplification products which were labeled with four different fluorescent dyes. Each peak represented one amplification product corresponding to an individual SNP allele. Figures 10B, 10C, 10C, and 10E, which were individually derived from Figure 10A, showed the chromatograms for amplification products labeled with blue, green, yellow and red, respectively. As seen in Figures 10B, 10C, 10D, and 10E, the peaks from the amplification products labeled with the same fluorescent dye could be individually identified on the basis of different fragment sizes. The two alleles for each SNP were separated on the basis of a fragment size difference of about 2 nucleotides.

For example, SNP rs3816800 had two alleles with G and C, respectively. The ligated probe size for A1 allele was 95 base pair (bp) and the size for A2 97 bp; and the CE reference size for A1 allele was 92.46 and the size for A2 was 94.56. As shown in Figure 10B, there were two peaks one with the fragment size of about 93 bp and the other with the fragment size of about 95 bp. Therefore, the genotype for SNP rs3816800 was determined to be heterozygous G/C.

A CE reference size for a SNP allele was obtained as follows: first, the corresponding set of probes were used to perform hybridization, ligations and amplification in a DNA sample harboring the SNP allele, and then the amplification products were analyzed by capillary electrophoresis to obtain the CE reference size for the SNP allele with the presence of a size standard. For example, the set of probes rs11161732_3, SEQ ID NO: 19 and rs11161732_A SEQ ID NO: 20 were used to obtain the CE reference size for the rs11161732_A allele and the CE reference size was determined to be 114.64.

For another example, SNP rs1249950 had two alleles with C and T, respectively. The ligated probe size for A1 allele was 100 bp and the size for A2 102 bp; and the CE reference size for A1 allele was 97.82 and the size for A2 was 100.11. As shown in Figure 10B, there was only one peak with the fragment size of about 100 bp. Therefore, the genotype for SNP rs1249950 was determined to be homozygous T/T.

As such, the genotypes of all the 48 SNP were determined according to the capillary electrophoresis results (see Table 2). These results were all consistent with the results obtained by direct DNA sequencing. SNP rs# A1 A2 PCR Primers A1LP SIZE A2LP SIZE A1LP CE REF SIZE A2LP CE REF SIZE GENOTYPE rs11161732 G A SNP_F1/SNP_R1 115 117 112.56 114.64 G/A rs1249950 C T SNP_F1/SNP_R1 100 102 97.82 100.11 T/T rs2294092 G C SNP_F1/SNP_R1 120 122 118.23 120.55 C/C rs2779500 G C SNP_F1/SNP_R1 105 107 103.2 105.41 G/C rs3816800 G C SNP_F1/SNP_R1 95 97 92.46 94.56 G/C rs894344 G A SNP_F1/SNP_R1 110 112 107.75 109.69 G/G rs12719860 C A SNP_F1/SNP_R2 154 156 153.51 155.7 C/C rs2241280 G A SNP_F1/SNP_R2 149 151 147.53 149.88 A/A rs2304035 G A SNP_F1/SNP_R2 128 130 126.12 128.23 G/G rs2304102 G A SNP_F1/SNP_R2 144 146 143.77 146.15 G/A rs4920098 C T SNP_F1/SNP_R2 134 136 130.98 133.25 T/T rs9791113 G C SNP_F1/SNP_R2 139 141 135.06 137.18 G/C rs11107 G A SNP_F2/SNP_R1 115 117 113.01 114.86 G/A rs2241571 C T SNP_F2/SNP_R1 95 97 93.51 95.49 C/T rs2305150 C T SNP_F2/SNP_R1 120 122 118.24 120.33 C/T rs2306331 C T SNP_F2/SNP_R1 105 107 103.44 105.59 C/T rs2401751 G A SNP_F2/SNP_R1 100 102 98.97 101.18 G/A rs4829830 C A SNP_F2/SNP_R1 110 112 109.48 111.44 C/C rs1058588 C T SNP_F2/SNP_R2 128 130 126.65 128.78 C/C rs1572983 C T SNP_F2/SNP_R2 139 141 136.39 138.43 C/T rs2279072 C T SNP_F2/SNP_R2 144 146 142.77 145.27 C/C rs3731631 G A SNP_F2/SNP_R2 149 151 149.5 151.89 G/A rs3736582 G C SNP_F2/SNP_R2 134 136 132.01 134.03 G/C rs639225 G A SNP_F2/SNP_R2 154 156 154.65 156.72 G/A rs11155787 C T SNP_F3/SNP_R1 115 117 112.56 114.86 C/T rs2986014 C T SNP_F3/SNP_R1 100 102 97.59 100.11 C/T rs3182535 G A SNP_F3/SNP_R1 95 97 92.3 94.93 G/A rs4141253 C T SNP_F3/SNP_R1 110 112 107.53 109.38 C/C rs6892205 G A SNP_F3/SNP_R1 120 122 117.01 119.3 G/G rs9389034 C T SNP_F3/SNP_R1 105 107 102.45 104.59 C/C rs1056893 C T SNP_F3/SNP_R2 134 136 131.68 133.7 T/T rs2297129 G A SNP_F3/SNP_R2 129 131 126.95 128.85 G/A rs3749877 G A SNP_F3/SNP_R2 139 141 137.29 139.63 A/A rs4371677 G A SNP_F3/SNP_R2 144 146 142.65 145.15 G/A rs469783 C T SNP_F3/SNP_R2 154 156 153.63 155.58 C/C rs938883 C T SNP_F3/SNP_R2 149 151 148.29 150.59 C/T rs2231926 G A SNP_F4/SNP_R1 102 104 104.62 106.56 G/A rs3809806 C T SNP_F4/SNP_R1 107 109 109.25 111.22 C/C rs624821 A T SNP_F4/SNP_R1 112 114 114.09 116.13 A/A rs625372 C T SNP_F4/SNP_R1 92 94 93.83 94.87 T/T rs6784322 A T SNP_F4/SNP_R1 117 119 118.09 120.18 A/T rs934472 C A SNP_F4/SNP_R1 97 99 98.51 100.64 C/C rs10790286 C T SNP_F4/SNP_R2 131 133 133 135.14 C/T rs10791649 G A SNP_F4/SNP_R2 146 148 148.51 151.06 G/A rs1359185 G A SNP_F4/SNP_R2 151 153 153.16 155.24 A/A rs2161916 G A SNP_F4/SNP_R2 125 127 126.73 128.78 A/A rs2241802 G A SNP_F4/SNP_R2 141 143 141.98 144.4 G/G rs4362 C T SNP_F4/SNP_R2 136 138 137.52 139.63 C/T

This example demonstrates a multiplex CNV detection method according to the present invention. In this example, there were 192 target sequences including 129 target sites in the Duchenne muscular dystrophy (DMD) gene, and 63 target reference sites on chromosomes X, Y, 2-12, 14, 16-20. DMD is a recessive X-linked form of muscular dystrophy, which results in muscle degeneration. The disorder is caused by mutations in the dystrophin gene, located on the human X chromosome, which codes for the protein dystrophin, an important structural component within muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane. Approximately two-thirds of the mutations in DMD are copy number variants of one or more exons in the dystrophin gene. The exemplary multiplex CNV detection method employed fluorescent dye labeled forward primers and reverse primers with stuffer sequences according to the scheme in Figure 5.

Specifically, probes were designed to cover 192 target sites with at least one target in each of the 79 exons of the DMD gene. Exons 22-42, 58-60, and 62-79 contained one target site. Exons 1, 3-15, 18-20, 47, 49, 54-57, and 61 contained two target sites. Exons 2, 16-17, 21, 43-46, 48, and 50-53 contain three target sites. For each target site, a probe pair was designed including a 3' probe (the left probe) and a 5' probe (the right probe). The 3' and 5' probes were designed in a manner so that when both are hybridized to the target sequence under a suitable condition, there is no gap between the two probes. The probes were synthesized by Life Technologies Corporation. The names, sequence ID numbers, primer binding sites and stuffer sequences of the 384 probes (192 probe pairs) for the 192 target sites are shown in Table 3.

For each 3' probe, the 5' end nucleotide was phosphorylated to provide a phosphate which would be connected to the hydroxyl group in the 3' end nucleotide of the 5' probe. Each 3' probe included a locus specific hybridization sequence (LSHS) in the 5' portion followed by a stuffer L2 sequence, and a primer binding sequence Y in the 3' portion. In this example, four primer binding sequence Y (MDM_Y1, SEQ ID NO: 554; MDM_Y2, SEQ ID NO: 555; MDM_Y3, SEQ ID NO: 556; MDM_Y4, SEQ ID NO: 557) were used in the 3' probes.

Each 5' probe included a locus specific hybridization sequence (LSHS) in the 3' portion followed by a stuffer L1 sequence and a primer binding sequence X in the 5' portion. In this example, four primer binding sequence X (MDM_X1, SEQ ID NO: 550; MDM_x2, SEQ ID NO: 551; MDM_X3, SEQ ID NO: 552; MDM_X4, SEQ ID NO: 553) were used in the 5' probes.

In addition, four forward primers and four reverse primers were designed for amplifying the ligation products. The four forward primers (DMD_F1, SEQ ID NO: 542; DMD_F2, SEQ ID NO: 543; DMD_F3, SEQ ID NO: 544; and DMD_F4 SEQ ID NO: 545) had unique sequences that were consistent with the four primer binding sequence X (MDM_X1, SEQ ID NO: 550; MDM_x2, SEQ ID NO: 551; MDM_X3, SEQ ID NO: 552; MDM_X4, SEQ ID NO: 553), respectively. The four forward primers: DMD_F1, DMD_F2, DMD_F3, and DMD_F4 were labeled on the 5' ends with four different fluorescnent dyes: FAM-blue, VIC-green, NED-yellow, and PET-red, respectively. The four reverse primers (DMD_R1, SEQ ID No: 546; DMD_R2, SEQ ID No: 547; DMD_R3, SEQ ID No: 548; and DMD_R4, SEQ ID No: 549) had unique sequences there were reversely complementary to the four primer binding sequence Y (MDM_Y1, SEQ ID NO: 554; MDM_Y2, SEQ ID NO: 555; MDM_Y3, SEQ ID NO: 556; MDM_Y4, SEQ ID NO: 557), respectively. The DMD_R2 and DMD_R4 reverse primers also had a stuffer sequence DMD_R_Stuffer, SEQ ID No: 558 in the 5' portion. All primers and probes were synthesized by Life Technologies Corporation. SEQ ID NO. Probe NAME Sequenc e X Stuffer L1 Stuffer L2 Sequen ce Y 158 ChrX_A_3 ATTACGCGA Y4 159 ChrX_A_5 X2 ATTACG 160 ChrX_B_3 ATTACG Y3 161 ChrX_B_5 X1 162 ChrX_C_3 ATTAC Y3 163 ChrX_C_5 X3 ATTAC 164 ChrY_A_3 Y3 165 ChrY_A_5 X3 166 ChrY_B_3 ATTACGCGA Y3 167 ChrY_B_5 X3 ATTACGCGATT 168 ChrY_C_3 Y4 169 ChrY_C_5 X3 A 170 DMD_E01A_3 ATTACGCGATT Y4 171 DMD_E01A_5 X4 AT 172 DMD_E01B_3 ATTAC Y4 173 DMD_E01B_5 X4 ATTA 174 DMD_E02A_3 ATTACGCGATT Y4 175 DMD_E02A_5 X4 ATTACGC 176 DMD_E02B_3 ATTACGC Y4 177 DMD_E02B_5 X4 ATTACGC 178 DMD_E02C_3 AT Y4 179 DMD_E02C_5 X4 180 DMD_E03A_3 ATTACGC Y1 181 DMD_E03A_5 X4 182 DMD_E03B_3 AT Y2 183 DMD_E03B_5 X4 184 DMD_E04A_3 ATT Y1 185 DMD_E04A_5 X1 186 DMD_E04B_3 ATTACGC Y2 187 DMD_E04B_5 X4 188 DMD_E05A_3 ATTACGCG Y1 189 DMD_E05A_5 X3 190 DMD_E05B_3 ATTACG Y2 191 DMD_E05B_5 X1 ATTACG 192 DMD_E06A_3 A Y2 193 DMD_E06A_5 X3 194 DMD_E06B_3 ATTACGCG Y2 195 DMD_E06B_5 X1 196 DMD_E07A_3 ATT Y2 197 DMD_E07A_5 X1 198 DMD_E07B_3 ATTACGCGA Y1 199 DMD_E07B_5 X3 ATT 200 DMD_E08A_3 ATT Y1 201 DMD_E08A_5 X2 202 DMD_E08B_3 ATTAC Y2 203 DMD_E08B_5 X2 ATT 204 DMD_E09A_3 ATTACGCGA Y2 205 DMD_E09A_5 X1 206 DMD_E09B_3 ATT Y2 207 DMD_E09B_5 X3 208 DMD_E10A_3 ATTACGCG Y1 209 DMD_E10A_5 X3 210 DMD_E10B_3 ATTACG Y1 211 DMD_E10B_5 X2 ATT 212 DMD_E11A_3 ATTA Y2 213 DMD_E11A_5 X1 214 DMD_E11B_3 ATTAC Y2 215 DMD_E11B_5 X2 ATTA 216 DMD_E12A_3 ATTACGC Y1 217 DMD_E12A_5 X4 A 218 DMD_E12B_3 ATTACGCGAT Y2 219 DMD_E12B_5 X4 220 DMD_E13A_3 ATTACGCG Y2 221 DMD_E13A_5 X3 222 DMD_E13B_3 ATTACGCGA Y1 223 DMD_E13B_5 X2 224 DMD_E14A_3 AT Y2 225 DMD_E14A_5 X4 226 DMD_E14B_3 ATTAC Y1 227 DMD_E14B_5 X1 ATTACGCG 228 DMD_E15A_3 ATTACGC Y1 229 DMD_E15A_5 X4 ATTA 230 DMD_E15B_3 ATTACGCG Y1 231 DMD_E15B_5 X2 A 232 DMD_E16A_3 ATT Y4 233 DMD_E16A_5 X2 ATTAC 234 DMD_E16B_3 ATTA Y4 235 DMD_E16B_5 X2 236 DMD_E16C_3 ATTAC Y4 237 DMD_E16C_5 X1 ATTACGCG 238 DMD_E17A_3 A Y3 239 DMD_E17A_5 X2 A 240 DMD_E17B_3 Y4 241 DMD_E17B_5 X1 242 DMD_E17C_3 ATTAC Y3 243 DMD_E17C_5 X2 244 DMD_E18A_3 ATTACGCGATTA Y1 245 DMD_E18A_5 X2 A 246 DMD_E18B_3 ATTACGC Y2 247 DMD_E18B_5 X4 248 DMD_E19A_3 ATTA Y2 249 DMD_E19A_5 X1 250 DMD_E19B_3 ATTACGCGATT Y2 251 DMD_E19B_5 X4 252 DMD_E20A_3 ATT Y2 253 DMD_E20A_5 X2 254 DMD_E20B_3 ATTACG Y1 255 DMD_E20B_5 X4 256 DMD_E21A_3 A Y4 257 DMD_E21A_5 X1 258 DMD_E21B_3 Y3 259 DMD_E21B_5 X4 260 DMD_E21C_3 ATTA Y3 261 DMD_E21C_5 X2 ATT 262 DMD_E22A_3 Y3 263 DMD_E22A_5 X1 264 DMD_E23A_3 ATT Y3 265 DMD_E23A_5 X4 266 DMD_E24A_3 A Y3 267 DMD_E24A_5 X4 268 DMD_E25A_3 Y4 269 DMD_E25A_5 X2 270 DMD_E26A_3 ATT Y3 271 DMD_E26A_5 X3 272 DMD_E27A_3 Y3 273 DMD_E27A_5 X3 274 DMD_E28A_3 ATT Y3 275 DMD_E28A_5 X4 A 276 DMD_E29A_3 AT Y3 277 DMD_E29A_5 X1 ATTACGC 278 DMD_E30A_3 A Y4 279 DMD_E30A_5 X2 280 DMD_E31A_3 ATTAC Y3 281 DMD_E31A_5 X4 ATTACGCGATTACGC 282 DMD_E32A_3 ATTACGCGAT Y4 283 DMD_E32A_5 X3 ATT 284 DMD_E33A_3 ATTACG Y4 285 DMD_E33A_5 X3 286 DMD_E34A_3 AT Y4 287 DMD_E34A_5 X1 ATTACGCGATT 288 DMD_E35A_3 ATTA Y4 289 DMD_E35A_5 X3 290 DMD_E36A_3 ATTA Y3 291 DMD_E36A_5 X4 ATTACG 292 DMD_E37A_3 ATTACGC Y4 293 DMD_E37A_5 X2 ATTACGC 294 DMD_E38A_3 ATTA Y4 295 DMD_E38A_5 X3 AT 296 DMD_E39A_3 ATTACG Y4 297 DMD_E39A_5 X3 ATTACGC 298 DMD_E40A_3 ATT Y3 299 DMD_E40A_5 X1 ATTACGCG 300 DMD_E41A_3 ATTA Y3 301 DMD_E41A_5 X1 302 DMD_E42A_3 ATTACG Y3 303 DMD_E42A_5 X1 A 304 DMD_E43A_3 ATT Y3 305 DMD_E43A_5 X4 306 DMD_E43B_3 Y3 307 DMD_E43B_5 X3 308 DMD_E43C_3 ATTA Y4 309 DMD_E43C_5 X2 ATTACGCGA 310 DMD_E44A_3 ATTA Y1 311 DMD_E44A_5 X2 312 DMD_E44B_3 ATTACGCG Y2 313 DMD_E44B_5 X3 ATT 314 DMD_E44C_3 AT Y2 315 DMD_E44C_5 X3 ATTA 316 DMD_E45A_3 ATT Y1 317 DMD_E45A_5 X4 A 318 DMD_E45B_3 ATTACGCGA Y1 319 DMD_E45B_5 X1 A 320 DMD_E45C_3 ATTACGCG Y1 321 DMD_E45C_5 X1 ATTACG 322 DMD_E46A_3 ATTACGCGA Y4 323 DMD_E46A_5 X3 ATTACG 324 DMD_E46B_3 ATTACG Y4 325 DMD_E46B_5 X1 326 DMD_E46C_3 ATT Y4 327 DMD_E46C_5 X2 ATT 328 DMD_E47A_3 A Y1 329 DMD_E47A_5 X4 330 DMD_E47B_3 ATTACGCGATTA Y2 331 DMD_E47B_5 X1 A 332 DMD_E48A_3 ATTACG Y3 333 DMD_E48A_5 X3 AT 334 DMD_E48B_3 Y3 335 DMD_E48B_5 X2 336 DMD_E48C_3 ATTACGCGAT Y3 337 DMD_E48C_5 X3 ATTACGC 338 DMD_E49A_3 ATTACGCGAT Y1 339 DMD_E49A_5 X1 340 DMD_E49B_3 ATTACGCG Y1 341 DMD_E49B_5 X2 342 DMD_E50A_3 ATTA Y4 343 DMD_E50A_5 X4 344 DMD_E50B_3 Y4 345 DMD_E50B_5 X4 346 DMD_E50C_3 ATTACG Y4 347 DMD_E50C_5 X4 ATTAC 348 DMD_E51A_3 ATTACGCG Y1 349 DMD_E51A_5 X3 ATTACGCGA 350 DMD_E51B_3 ATT Y1 351 DMD_E51B_5 X3 352 DMD_E51C_3 ATTACGCGAT Y2 353 DMD_E51C_5 X1 354 DMD_E52A_3 ATTA Y2 355 DMD_E52A_5 X2 356 DMD_E52B_3 ATTAC Y1 357 DMD_E52B_5 X1 358 DMD_E52C_3 ATTACGCGATTAC Y2 359 DMD_E52C_5 X2 360 DMD_E53A_3 ATTAC Y3 361 DMD_E53A_5 X1 ATTACG 362 DMD_E53B_3 A Y3 363 DMD_E53B_5 X1 364 DMD_E53C_3 Y3 365 DMD_E53C_5 X2 366 DMD_E54A_3 ATTACGCG Y2 367 DMD_E54A_5 X2 368 DMD_E54B_3 A Y2 369 DMD_E54B_5 X4 370 DMD_E55A_3 ATTACGCG Y1 371 DMD_E55A_5 X3 372 DMD_E55B_3 ATTA Y1 373 DMD_E55B_5 X3 374 DMD_E56A_3 ATTACGCGA Y2 375 DMD_E56A_5 X2 376 DMD_E56B_3 Y2 377 DMD_E56B_5 X4 ATTA 378 DMD_E57A_3 ATT Y2 379 DMD_E57A_5 X3 380 DMD_E57B_3 ATTACGCG Y2 381 DMD_E57B_5 X3 382 DMD_E58A_3 ATTACGC Y3 383 DMD_E58A_5 X2 ATT 384 DMD_E59A_3 ATTA Y3 385 DMD_E59A_5 X2 386 DMD_E60A_3 ATT Y1 387 DMD_E60A_5 X4 388 DMD_E61A_3 ATT Y1 389 DMD_E61A_5 X3 390 DMD_E61B_3 AT Y1 391 DMD_E61B_5 X4 ATTACGCGA 392 DMD_E62A_3 ATTA Y1 393 DMD_E62A_5 X2 394 DMD_E63A_3 ATTACGC Y4 395 DMD_E63A_5 X2 396 DMD_E64A_3 ATTACGCG Y2 397 DMD_E64A_5 X3 398 DMD_E65A_3 ATTA Y2 399 DMD_E65A_5 X2 400 DMD_E66A_3 ATTACGCGA Y1 401 DMD_E66A_5 X1 402 DMD_E67A_3 ATTAC Y1 403 DMD_E67A_5 X1 404 DMD_E68A_3 ATTACGCG Y4 405 DMD_E68A_5 X3 ATT 406 DMD_E69A_3 AT Y3 407 DMD_E69A_5 X1 408 DMD_E70A_3 ATTAC Y3 409 DMD_E70A_5 X3 AT 410 DMD_E71A_3 ATTAC Y4 411 DMD_E71A_5 X1 412 DMD_E72A_3 ATTAC Y3 413 DMD_E72A_5 X2 AT 414 DMD_E73A_3 ATTA Y3 415 DMD_E73A_5 X2 ATT 416 DMD_E74A_3 ATTAC Y3 417 DMD_E74A_5 X4 ATT 418 DMD_E75A_3 Y3 419 DMD_E75A_5 X4 420 DMD_E76A_3 AT Y4 421 DMD_E76A_5 X1 422 DMD_E77A_3 ATTACGC Y4 423 DMD_E77A_5 X1 ATTACGCGA 424 DMD_E78A_3 Y4 425 DMD_E78A_5 X3 426 DMD_E79A_3 ATTACGCGA Y4 427 DMD_E79A_5 X1 ATTAC 428 REF10p_A_3 ATTACGCG Y2 429 REF10p_A_5 X1 430 REF10p_B_3 ATTAC Y4 431 REF10p_B_5 X4 432 REF10p_C_3 ATTACGCGATT Y3 433 REF10p_C_5 X4 434 REF10q_A_3 Y3 435 REF10q_A_5 X2 436 REF10q_B_3 ATTACGCGATTA Y1 437 REF10q_B_5 X3 ATTACGCGATTACGCG 438 REF11p_A_3 ATTACGC Y1 439 REF11p_A_5 X4 440 REF11p_B_3 ATTACGCGATTAC Y3 441 REF11p_B_5 X2 AT 442 REF11q_A_3 ATTACGCGATTAC Y2 443 REF11q_A_5 X4 ATTACGCGATTA 444 REF11q_B_3 ATTACG Y3 445 REF11q_B_5 X4 446 REF12p_A_3 ATTACGCGATTACG Y2 447 REF12p_A_5 X4 ATT 448 REF12q_A_3 ATTACGC Y1 449 REF12q_A_5 X3 450 REF12q_B_3 ATTACGCGA Y4 451 REF12q_B_5 X1 452 REF14q_A_3 Y2 453 REF14q_A_5 X2 454 REF16p_A_3 ATTACGCGATTAC Y2 455 REF16p_A_5 X2 ATTACG 456 REF16p_B_3 ATTACGCGATTACG Y4 457 REF16p_B_5 X2 ATTACG 458 REF16q_A_3 ATTACGCGAT Y3 459 REF16q_A_5 X1 ATTAC 460 REF16q_B_3 Y2 461 REF16q_B_5 X3 462 REF17q_A_3 ATTACGCGATTA Y2 463 REF17q_A_5 X1 ATTACGCGATTACG 464 REF18p_A_3 ATTACGCGATTA Y1 465 REF18p_A_5 X1 AT 466 REF18p_B_3 ATTACGC Y3 467 REF18p_B_5 X2 468 REF19q_A_3 ATTACGCGATTAC Y2 469 REF19q_A_5 X3 ATTACGC 470 REF19q_B_3 Y4 471 REF19q_B_5 X1 472 REF19q_C_3 Y3 473 REF19q_C_5 X4 474 REF20p_A_3 ATTACGCGATT Y1 475 REF20p_A_5 X2 ATTA 476 REF20p_B_3 ATTACGCGA Y4 477 REF20p_B_5 X2 478 REF20q_A_3 ATTACGCGATTA Y2 479 REF20q_A_5 X1 ATTACGC 480 REF20q_B_3 ATTACGCGATTACGCGA Y4 481 REF20q_B_5 X4 482 REF20q_C_3 ATT Y3 483 REF20q_C_5 X3 484 REF2p_A_3 ATTACGCGAT Y1 485 REF2p_A_5 X3 ATTACGCGA 486 REF2p_B_3 Y4 487 REF2p_B_5 X2 488 REF2q_A_3 ATTACGCGATTACG Y4 489 REF2q_A_5 X3 ATTACG 490 REF2q_B_3 ATTACGCGATTA Y2 491 REF2q_B_5 X2 ATTACGCGAT 492 REF3p_A_3 ATTACG Y3 493 REF3p_A_5 X1 494 REF3p_B_3 ATTACGCGATTAC Y4 495 REF3p_B_5 X4 ATTACGCGATTAC 496 REF3p_C_3 Y2 497 REF3p_C_5 X4 498 REF3p_D_3 ATTACGCGA Y1 499 REF3p_D_5 X1 500 REF3q_A_3 ATTACGCGATTACG Y1 501 REF3q_A_5 X4 ATTACGCGATTAC 502 REF3q_B_3 ATTACGCGA Y4 503 REF3q_B_5 X3 A 504 REF3q_C_3 A Y2 505 REF3q_C_5 X3 506 REF4q_A_3 Y4 507 REF4q_A_5 X3 508 REF4q_B_3 ATTACGC Y1 509 REF4q_B_5 X2 510 REF5p_A_3 A Y1 511 REF5p_A_5 X2 512 REF5q_A_3 ATTACGCGATTACGCG Y1 513 REF5q_A_5 X1 ATTACGCGATTACG 514 REF6p_A_3 Y1 515 REF6p_A_5 X3 516 REF6p_B_3 Y3 517 REF6p_B_5 X1 518 REF6q_A_3 ATTACGCGATTACG Y1 519 REF6q_A_5 X2 ATTACGCGATT 520 REF6q_B_3 ATTACGCGATTACGCG Y4 521 REF6q_B_5 X1 ATTAC 522 REF7p_A_3 ATTACGC Y2 523 REF7p_A_5 X4 524 REF7p_B_3 ATTACGCGATT Y3 525 REF7p_B_5 X3 ATTAC 526 REF8p_A_3 ATTACGCGA Y1 527 REF8p_A_5 X4 ATTACG 528 REF8p_B_3 ATTAC Y2 529 REF8p_B_5 X1 530 REF8q_A_3 A Y3 531 REF8q_A_5 X3 532 REF8q_B_3 A Y1 533 REF8q_B_5 X1 ATTACGC 534 REF9p_A_3 ATTACGCGATTAC Y2 535 REF9p_A_5 X3 ATTACGCGATT 536 REF9p_B_3 Y1 537 REF9p_B_5 X4 538 REF9q_A_3 Y4 539 REF9q_A_5 X4 540 REF9q_B_3 ATTACGCGA Y2 541 REF9q_B_5 X2

To perform the 192 multiplex CNV detection assay, a ligation product was first generated. Briefly, genomic DNA was extracted from 2ml whole blood samples using the classic phenol:chloroform method. The blood samples were collected from a male DMD patient and a healthy male volunteer at Shanghai Ruijin Hospital, Shanghai, China. The genomic DNA from the male DMD patient was the test sample. The genomic DNA from the healthy volunteer was the control sample. From the extracted genomic DNA, 100-200 microgram (µg) DNA was dissolved in 10 microliter (µl) 1xTE buffer (10mM Tris.Cl, pH8.0, 1mM EDTA from Sigma-Aldrich). The dissolved genomic DNA was denatured at 98 °C for 5 minutes and then immediately cooled down on ice. At the same time, a 2x ligation premix solution was prepared according to the following formula: a 10µl 2x ligation premix was made of 2µl 10xTaq ligase buffer, 1µl 40U/µl Taq Ligase from NewEngland Biolabs, Inc., 1µl ProbeMix (the 384 probes with a final concentration of 0.005 micromolar for each probe in 1xTE), and 6µl ddH₂O (Distilled Milli-Q water from Milli-Q Advantage A10, Millipore). 10 µl 2xligation premix was mixed with the denatured 10 µl genomic DNA and the mixture was allowed to undergo 4 cycles of denaturation, hybridization, and ligation under the following conditions: 95 °C for 30 seconds, and then 58 °C for 4 hours. The ligation product thus obtained could be stored on ice for same day use or freezed in -20 °C for future use.

With the ligation product, an amplification step was then performed to obtain an amplification product. Briefly, two PCR reactions were performed using the same amplification product as template DNA. One PCR reaction had DMD_F1, DMD_F2, DMD_F3, DMD_F4, DMD_R1 and DMD_R2 as primers. The other PCR reaction had DMD_F1, DMD_F2, DMD_F3, DMD_F4, DMD_R3 and DMD_R4 as primers. The PCR reaction mixture was prepared as follows: a 20 µl reaction system was made by mixing 2µl 10x PCR buffer (Qiagen, Germany), 2µl 2.5mM dNTP mix (2.5mM each of dATP, dTTP, dCTP and dGTP from Takara Bio Inc.), 2µl primer mix (DMD_F1, DMD_F2, DMD_F3, DMD_F4, DMD_R1 and DMD_R2 at final concentrations of 1µM, 1µM, 1µM, 1µM, 2µM and 2µM, respectively; or DMD_F1, DMD_F2, DMD_F3, DMD_F4, DMD_R3 and DMD_R4 at final concentrations of 1µM, 1µM, 1µM, 1µM, 2µM and 2µM, respectively), 1µl Ligation product, 0.2µl 5U/µl HotStarTaq Plus Taq DNA polymerase (Qiagen, Germany), and 12.8µl ddH₂O. The PCR mixure was allowed to undergo a polymerase chain reaction under the following conditions: 95 °C for 2 minutes; followed by 35 cycles of 94 °C for 20 second, 57 °C for 40 second, and 72 °C for 1.5 minutes; and after the 35th cycle, the reaction mixture was kept in 60 °C for 1 hour. To analyze the amplifcation product, 1 µl of the amplification product was first diluted with ddH₂O 10 times into 10 µl. Then 1 µl was taken out of the 10 µl diluted amplification product and mixed with 0.1 µl GeneScan™ 500 LIZ® size standard (Life Technologies, Inc.) and 8.9 µl Hi-Di formamide (Life Technologies, Inc.). The mixture was detaured at 95 °C for 5 minutes and run through capillary electrophoresis by ABI3130XL according to manufacturer's manual. The chromatograms from the two PCR reactions were designated as Panel A and Panel B, respectively. The capillary electrophoresis data was processed using Genemapper 4.0 to obtain peak intensity values for each amplification product.

As shown in Figures 11A-H, the amplification products obtained in the assay could be separated by capillary electrophoresis and the peaks could be individually identified on the chromatograms. Figures 11A and 11B showed the chromatograms of all amplification products for the control sample panel A and panel B, respectively. Each panel represented the analysis of amplification products from one PCR reaction. Figures 11C and 11D showed the chromatograms of all amplification products for the patient sample panel A and panel B, respectively. Each peak represented one amplification product corresponding to an individual target site.

Figures 11E and 11F showed the chromatograms for amplification products labeled with blue fluorescent dyes for the control sample and the patient sample, respectively. Figures 11G and 11H showed the chromatograms for amplification products labeled with green fluorescent dyes for the control sample and the patient sample, respectively. As seen in Figures 11E-11H, the peaks from the amplification products labeled with the same fluorescent dye could be identified on the basis of different fragment sizes. By comparing the chromatograms from the control and the patient samples, the presence or absence of peaks of similar fragment sizes may be determined. For example, when comparing the chromatograms in Figures 11E and 11F, the peaks for the target sites E22A, E41A, E40A, E29A, E21A and E34A were missing in the chromatogram from the patient sample, indicating the deletion of the sequences in the DMD gene corresponding to these target sites. For another example, when comparing the chromatograms in Figures 11G and 11H, the peaks for the target sites E18A, and E20A are missing in the chromatogram from the patient sample, indicating the deletion of the sequences in the DMD gene corresponding to these target sites.

Alternatively, in another method to analyze the electrophoresis data, a ratio for the test sample (R_test) was obtained for each DMD, chromosome X, and chromosome Y target sites (herein also referred to as gene target sites) by dividing the peak intensity value of the gene target site with the peak intensity value of each of the reference target sites in the same group. The same PCR primer pair was used for each gene target site and the reference target site in the same group. For example, As shown in Table 4, in one group which shared the same primer pair DMD_F1/DMD_R1, there were amplification products for 12 probe loci (herein also referred to as probe target sites) including 8 gene target loci: DMD_E04A, DMD_E14B, DMD_E45B, DMD_E45C, DMD_E49A, DMD_E52B, DMD_E66A, and DMD_E67A; and 4 reference target loci: REF18p_A, REF3p_D, REF5q_A, and REF8q_B. To obtain the ratio for the locus DMD_E04A in the test sample (R_test), the peak intensity for the locus DMD_E04A was divided by the peak intensity for each of the reference target sites REF18p_A, REF3p_D, REF5q_A, and REF8q_B so that four ratios (R_test) were derived.

Similarly, a ratio (R_control) in the control sample was obtained for each gene target site. Assuming the copy number of the target nucleic acid in the control sample (C_control) is 1, the copy number of each gene target site (C_test) was then calculated according to the formula: C_test = C_control X R_test / R_control.

The copy number for a gene target site in the test sample equals to the ratio between the R_test and the R_control, assuming the copy number of the target nucleic acid in the control sample is 1 (e.g., there is one X chromosome in a male patient). Because four reference target sites were introduced for each gene target site, four R_test and therefore four C_test were obtained for each gene target site. The median value the four C_test was deemed as the copy number for that gene target site in the test sample. The copy numbers thus calculated for each gene target site are shown in Table 4. Copy numbers for gene target sites corresponding to exons 18-41 were zero, indicating that these exons were deleted in the DMD patient sample. PANEL PROBE LOCUS TARGET REGION PCR PRIMERS LP SIZE CE REF SIZE COPY NUMBER MEASUREMENT A DMD_E04A DMD EXON04 DMD_F1/DMD_R1 101 97.95 1.02 A DMD_E14B DMD EXON14 DMD_F1/DMD_R1 125 122.35 1.02 A DMD_E45B DMD EXON45 DMD_F1/DMD_R1 116 113.84 1.08 A DMD_E45C DMD EXON45 DMD_F1/DMD_R1 122 120.03 1.07 A DMD_E49A DMD EXON49 DMD_F1/DMD_R1 113 110.3 1.04 A DMD_E52B DMD EXON52 DMD_F1/DMD_R1 104 102.87 1.03 A DMD_E66A DMD EXON66 DMD_F1/DMD_R1 110 108.1 1 A DMD_E67A DMD EXON67 DMD_F1/DMD_R1 98 94.86 1.07 A REF18p_A REFERENCE DMD_F1/DMD_R1 119 117.75 / A REF3p_D REFERENCE DMD_F1/DMD_R1 107 104.93 / A REF5q_A REFERENCE DMD_F1/DMD_R1 128 124.92 / A REF8q_B REFERENCE DMD_F1/DMD_R1 102 100.21 / A DMD_E05B DMD EXON05 DMD_F1/DMD_R2 164 163.68 0.98 A DMD_E06B DMD EXON06 DMD_F1/DMD_R2 148 147.96 0.99 A DMD_E07A DMD EXON07 DMD_F1/DMD_R2 139 137.04 1.04 A DMD_E09A DMD EXON09 DMD_F1/DMD_R2 151 150.45 0.97 A DMD_E11A DMD EXON11 DMD_F1/DMD_R2 136 134.22 1.06 A DMD_E19A DMD EXON19 DMD_F1/DMD_R2 142 140.22 0 A DMD_E47B DMD EXON47 DMD_F1/DMD_R2 159 160.53 1.08 A DMD E51C DMD EXON51 DMD_F1/DMD_R2 154 154.36 0.96 A REF10p_A REFERENCE DMD_F1/DMD_R2 133 130.34 / A REF17q_A REFERENCE DMD_F1/DMD_R2 166 166.35 / A REF20q_A REFERENCE DMD_F1/DMD_R2 157 157.72 / A REF8p_B REFERENCE DMD_F1/DMD_R2 145 144.59 / B DMD_E22A DMD EXON22 DMD_F1/DMD_R3 104 102.07 0 B DMD_E29A DMD EXON29 DMD_F1/DMD_R3 125 122.73 0 B DMD_E40A DMD EXON40 DMD_F1/DMD_R3 122 119.3 0 B DMD_E41A DMD EXON41 DMD_F1/DMD_R3 110 108.32 0 B DMD_E42A DMD EXON42 DMD_F1/DMD_R3 116 113.83 1.12 B DMD_E53A DMD EXON53 DMD_F1/DMD_R3 128 124.34 1.1 B DMD_E53B DMD EXON53 DMD_F1/DMD_R3 101 99.46 1.01 B DMD_E69A DMD EXON69 DMD_F1/DMD_R3 98 96.33 1.05 B ChrX_B ChrX Non-PAR DMD_F1/DMD_R3 113 110.95 0.96 B REF16q_A REFERENCE DMD_F1/DMD_R3 119 116.64 / B REF3p_A REFERENCE DMD_F1/DMD_R3 107 105.37 / B REF6p_B REFERENCE DMD_F1/DMD_R3 95 92.77 / B DMD_E16C DMD EXON16 DMD_F1/DMD_R4 162 163.37 1.01 B DMD_E17B DMD EXON17 DMD_F1/DMD_R4 139 138.03 0.99 B DMD_E21A DMD EXON21 DMD_F1/DMD_R4 141 140.14 0 B DMD_E34A DMD EXON34 DMD_F1/DMD_R4 160 160.74 0 B DMD_E46B DMD EXON46 DMD_F1/DMD_R4 150 150.99 0.98 B DMD_E71A DMD EXON71 DMD_F1/DMD_R4 148 148.7 0.99 B DMD_E76A DMD EXON76 DMD_F1/DMD_R4 136 134.94 0.98 B DMD_E77A DMD EXON77 DMD_F1/DMD_R4 166 165.44 0.94 B DMD_E79A DMD EXON79 DMD_F1/DMD_R4 159 157.44 0.99 B REF12q_B REFERENCE DMD_F1/DMD_R4 145 142.57 / B REF19q_B REFERENCE DMD_F1/DMD_R4 133 131.12 / B REF6q_B REFERENCE DMD_F1/DMD_R4 157 154.42 / A DMD_E08A DMD EXON08 DMD_F2/DMD_R1 101 99.78 0.97 A DMD_E10B DMD EXON10 DMD_F2/DMD_R1 116 114.26 0.97 A DMD_E13B DMD EXON13 DMD_F2/DMD_R1 113 111.65 0.96 A DMD_E15B DMD EXON15 DMD_F2/DMD_R1 125 123.2 0.91 A DMD_E18A DMD EXON18 DMD_F2/DMD_R1 122 120.8 0 A DMD_E44A DMD EXON44 DMD_F2/DMD_R1 104 103.18 1.02 A DMD_E49B DMD EXON49 DMD_F2/DMD_R1 110 108.73 1.04 A DMD_E62A DMD EXON62 DMD_F2/DMD_R1 98 96.8 0.96 A REF20p_A REFERENCE DMD_F2/DMD_R1 119 118.73 / A REF4q_B REFERENCE DMD_F2/DMD_R1 107 105.96 / A REF5p_A REFERENCE DMD_F2/DMD_R1 95 92.81 / A REF6q_A REFERENCE DMD_F2/DMD_R1 128 126.84 / A DMD_E08B DMD EXON08 DMD_F2/DMD_R2 163 164.24 0.95 A DMD_E11B DMD EXON11 DMD_F2/DMD_R2 154 155.4 0.97 A DMD_E20A DMD EXON20 DMD_F2/DMD_R2 139 138.23 0 A DMD_E52A DMD EXON52 DMD_F2/DMD_R2 142 141.19 0.94 A DMD_E52C DMD EXON52 DMD_F2/DMD_R2 160 161.38 0.93 A DMD_E54A DMD EXON54 DMD_F2/DMD_R2 148 147.35 0.97 A DMD_E56A DMD EXON56 DMD_F2/DMD_R2 151 151.48 0.99 A DMD_E65A DMD EXON65 DMD_F2/DMD_R2 136 134.87 0.94 A REF14q_A REFERENCE DMD_F2/DMD_R2 133 131.43 / A REF16p_A REFERENCE DMD_F2/DMD_R2 157 158.24 / A REF2q_B REFERENCE DMD_F2/DMD_R2 166 167.39 / A REF9q_B REFERENCE DMD_F2/DMD_R2 145 145.43 / B DMD_E17A DMD EXON17 DMD_F2/DMD_R3 99 96.61 1.03 B DMD_E17C DMD EXON17 DMD_F2/DMD_R3 113 111.55 1.03 B DMD_E21C DMD EXON21 DMD_F2/DMD_R3 125 123.15 0 B DMD_E48B DMD EXON48 DMD_F2/DMD_R3 104 102.95 0.97 B DMD_E53C DMD EXON53 DMD_F2/DMD_R3 101 99.67 1.03 B DMD_E58A DMD EXON58 DMD_F2/DMD_R3 122 120.59 1 B DMD_E59A DMD EXON59 DMD_F2/DMD_R3 110 108.73 0.96 B DMD_E72A DMD EXON72 DMD_F2/DMD_R3 116 115.09 1.02 B DMD_E73A DMD EXON73 DMD_F2/DMD_R3 128 126.65 0.98 B REF10q_A REFERENCE DMD_F2/DMD_R3 93 92.06 / B REF11p_B REFERENCE DMD_F2/DMD_R3 119 118.29 / B REF18p_B REFERENCE DMD_F2/DMD_R3 107 106.2 / B DMD_E16A DMD EXON16 DMD_F2/DMD_R4 154 155.18 0.96 B DMD_E16B DMD EXON16 DMD_F2/DMD_R4 148 149.05 1.18 B DMD_E25A DMD EXON25 DMD_F2/DMD_R4 139 138.79 0 B DMD_E30A DMD EXON30 DMD_F2/DMD_R4 136 134.72 0 B DMD_E37A DMD EXON37 DMD_F2/DMD_R4 163 163.48 0 B DMD_E43C DMD EXON43 DMD_F2/DMD_R4 160 160.74 0.87 B DMD_E46C DMD EXON46 DMD_F2/DMD_R4 166 166.08 1.18 B DMD_E63A DMD EXON63 DMD_F2/DMD_R4 151 151.38 1.06 B ChrX_A ChrX Non-PAR DMD_F2/DMD_R4 148 146.02 0.9 B REF16p_B REFERENCE DMD_F2/DMD_R4 157 157.23 / B REF20p_B REFERENCE DMD_F2/DMD_R4 145 142.32 / B REF2p_B REFERENCE DMD_F2/DMD_R4 133 131.1 / A DMD_E05A DMD EXON05 DMD_F3/DMD_R1 116 113.74 0.99 A DMD_E07B DMD EXON07 DMD_F3/DMD_R1 122 119.71 0.99 A DMD_E10A DMD EXON10 DMD_F3/DMD_R1 113 110.62 1.03 A DMD_E51A DMD EXON51 DMD_F3/DMD_R1 125 122.06 0.96 A DMD_E51B DMD EXON51 DMD_F3/DMD_R1 101 97.89 0.95 A DMD_E55A DMD EXON55 DMD_F3/DMD_R1 110 107.27 0.99 A DMD_E55B DMD EXON55 DMD_F3/DMD_R1 104 101.74 0.93 A DMD_E61A DMD EXON61 DMD_F3/DMD_R1 98 94.88 1.1 A REF10q_B REFERENCE DMD_F3/DMD_R1 128 125.01 / A REF12q_A REFERENCE DMD_F3/DMD_R1 107 104.7 / A REF2p_A REFERENCE DMD_F3/DMD_R1 119 115.98 / A REF6p_A REFERENCE DMD_F3/DMD_R1 95 92.7 / A DMD_E06A DMD EXON06 DMD_F3/DMD_R2 136 134.22 1 A DMD_E09B DMD EXON09 DMD_F3/DMD_R2 142 140.95 0.94 A DMD_E13A DMD EXON13 DMD_F3/DMD_R2 154 153.36 1.02 A DMD_E44B DMD EXON44 DMD_F3/DMD_R2 160 160.42 0.88 A DMD_E44C DMD EXON44 DMD_F3/DMD_R2 163 163.71 0.95 A DMD_E57A DMD EXON57 DMD_F3/DMD_R2 139 137.9 0.96 A DMD_E57B DMD EXON57 DMD_F3/DMD_R2 148 147.35 0.93 A DMD_E64A DMD EXON64 DMD_F3/DMD_R2 151 150.11 0.97 A REF16q_B REFERENCE DMD_F3/DMD_R2 133 130.13 / A REF19q_A REFERENCE DMD_F3/DMD_R2 157 157.24 / A REF3q_C REFERENCE DMD_F3/DMD_R2 145 143.98 / A REF9p_A REFERENCE DMD_F3/DMD_R2 166 166.34 / B DMD_E26A DMD EXON26 DMD_F3/DMD_R3 110 107.5 0 B DMD_E27A DMD EXON27 DMD_F3/DMD_R3 101 99.78 0 B DMD_E43B DMD EXON43 DMD_F3/DMD_R3 98 95.66 0.96 B DMD_E48A DMD EXON48 DMD_F3/DMD_R3 118 115.51 1 B DMD_E48C DMD EXON48 DMD_F3/DMD_R3 128 125.33 1.2 B DMD_E70A DMD EXON70 DMD_F3/DMD_R3 115 112.29 1.01 B ChrX_C ChrX Non-PAR DMD_F3/DMD_R3 122 119.94 1.07 B ChrY_A ChrY Non-PAR DMD_F3/DMD_R3 104 101.87 1.01 B ChrY_B ChrY Non-PAR DMD_F3/DMD_R3 130 126.33 1.06 B REF20q_C REFERENCE DMD_F3/DMD_R3 107 104.85 / B REF7p_B REFERENCE DMD_F3/DMD_R3 119 118.09 / B REF8q_A REFERENCE DMD_F3/DMD_R3 95 93.55 / B DMD_E32A DMD EXON32 DMD_F3/DMD_R4 163 162.74 0 B DMD_E33A DMD EXON33 DMD_F3/DMD_R4 142 140.58 0 B DMD_E35A DMD EXON35 DMD_F3/DMD_R4 148 147.63 0 B DMD_E38A DMD EXON38 DMD_F3/DMD_R4 151 150.99 0 B DMD_E39A DMD EXON39 DMD_F3/DMD_R4 166 164.62 0 B DMD_E46A DMD EXON46 DMD_F3/DMD_R4 163 160.1 0.96 B DMD_E68A DMD EXON68 DMD_F3/DMD_R4 157 154.02 0.95 B DMD_E78A DMD EXON78 DMD_F3/DMD_R4 136 133.08 0.98 B ChrY_C ChrY Non-PAR DMD_F3/DMD_R4 139 136.74 1.05 B REF2q_A REFERENCE DMD_F3/DMD_R4 157 157.2 / B REF3q_B REFERENCE DMD_F3/DMD_R4 146 143.15 / B REF4q_A REFERENCE DMD_F3/DMD_R4 133 131.02 / A DMD_E03A DMD EXON03 DMD_F4/DMD_R1 110 111.76 1.03 A DMD_E12A DMD EXON12 DMD_F4/DMD_R1 114 113.87 0.95 A DMD_E15A DMD EXON15 DMD_F4/DMD_R1 119 119.22 0.98 A DMD_E20B DMD EXON20 DMD_F4/DMD_R1 107 109.13 0 A DMD_E45A DMD EXON45 DMD_F4/DMD_R1 96 98.31 0.95 A DMD_E47A DMD EXON47 DMD_F4/DMD_R1 98 100 0.98 A DMD_E60A DMD EXON60 DMD_F4/DMD_R1 101 102.36 0.98 A DMD_E61B DMD EXON61 DMD_F4/DMD_R1 122 122.48 0.96 A REF11p_A REFERENCE DMD_F4/DMD_R1 104 105.44 / A REF3q_A REFERENCE DMD_F4/DMD_R1 125 126.72 / A REF8p_A REFERENCE DMD_F4/DMD_R1 116 117.23 / A REF9p_B REFERENCE DMD_F4/DMD_R1 92 94.43 / A DMD_E03B DMD EXON03 DMD_F4/DMD_R2 130 132.06 0.97 A DMD_E04B DMD EXON04 DMD_F4/DMD_R2 148 151.02 1.07 A DMD_E12B DMD EXON12 DMD_F4/DMD_R2 160 164.03 1 A DMD_E14A DMD EXON14 DMD_F4/DMD_R2 139 141.8 1.02 A DMD_E18B DMD EXON18 DMD_F4/DMD_R2 151 154.36 0 A DMD_E19B DMD EXON19 DMD_F4/DMD_R2 157 160.11 0 A DMD_E54B DMD EXON54 DMD_F4/DMD_R2 136 137.26 1.01 A DMD_E56B DMD EXON56 DMD_F4/DMD_R2 145 148.68 1.14 A REF11q_A REFERENCE DMD_F4/DMD_R2 163 167.72 / A REF12p_A REFERENCE DMD_F4/DMD_R2 154 158.37 / A REF3p_C REFERENCE DMD_F4/DMD_R2 133 134.98 / A REF7p_A REFERENCE DMD_F4/DMD_R2 142 144.34 / B DMD_E21B DMD EXON21 DMD_F4/DMD_R3 98 100.62 0 B DMD_E23A DMD EXON23 DMD_F4/DMD_R3 95 97.78 0 B DMD_E24A DMD EXON24 DMD_F4/DMD_R3 107 107.99 0 B DMD_E28A DMD EXON28 DMD_F4/DMD_R3 113 114.88 0 B DMD_E31A DMD EXON31 DMD_F4/DMD_R3 128 126.18 0 B DMD_E36A DMD EXON36 DMD_F4/DMD_R3 121 120.6 0 B DMD_E43A DMD EXON43 DMD_F4/DMD_R3 110 110.95 1.05 B DMD_E74A DMD EXON74 DMD_F4/DMD_R3 122 124.12 1.01 B DMD_E75A DMD EXON75 DMD_F4/DMD_R3 101 102.59 1 B REF10p_C REFERENCE DMD_F4/DMD_R3 116 118.4 / B REF11q_B REFERENCE DMD_F4/DMD_R3 104 106.31 / B REF19q_C REFERENCE DMD_F4/DMD_R3 92 94.78 / B DMD_E01A DMD EXON01 DMD_F4/DMD_R4 160 163.69 0.9 B DMD_E01B DMD EXON01 DMD_F4/DMD_R4 155 158.11 1.08 B DMD_E02A DMD EXON02 DMD_F4/DMD_R4 167 168.9 0.9 B DMD_E02B DMD EXON02 DMD_F4/DMD_R4 169 171.8 0.99 B DMD_E02C DMD EXON02 DMD_F4/DMD_R4 145 147.83 1.02 B DMD_E50A DMD EXON50 DMD_F4/DMD_R4 148 152.18 0.97 B DMD_E50B DMD EXON50 DMD_F4/DMD_R4 133 135.04 1.01 B DMD_E50C DMD EXON50 DMD_F4/DMD_R4 157 161.27 0.98 B REF10p_B REFERENCE DMD_F4/DMD_R4 142 144.28 / B REF20q_B REFERENCE DMD_F4/DMD_R4 154 155.12 / B REF3p_B REFERENCE DMD_F4/DMD_R4 163 166.76 / B REF9q A REFERENCE DMD_F4/DMD_R4 130 131.56 /

This example provides an exemplary method of multiplex point mutation screening according to the present invention. In this example, as in Example 2, there were 192 target sequences including 129 sites in the Duchenne muscular dystrophy (DMD) gene, and 63 reference sites on chromosomes X, Y, 2-12, 14, 16-20. Some mutations causing DMD are point mutations in the dystrophin gene. The multiplex point mutation screening in the DMD gene employed fluorescent dye labeled forward primers and reverse primers with stuffer sequences according to the scheme in Figure 6.

In this exemplary mutation screening method, the same probes and primers as those designed and used in Example 2 were used for the hybridization, ligation, and amplification reactions except that the test genomic DNA was obtained from another male DMD patient. The amplification products were analyzed through capillary electrophoresis, the peak intensities were obtained, and the copy numbers for each target site were calculated as detailed in Example 2. For sake of brevity, the descriptions of the probes, primers, ligation reaction, amplification reaction, data procurement, and copy number calculation, are not repeated in this example.

The copy number thus calculated for all gene target sites are shown in Figure 12A and partially shown in Figure 12B. The copy number for E07B (exon 7B gene target site in the dystrophin gene) was zero, suggesting that the probes for this site could not match perfectly with the target site. On the other hand, the copy number for E07A was about 1, meaning that part of exon7 had a normal copy number and there was no whole exon 7 deletion in the test DNA sample. The lack of amplification product for the E07B gene target site could also be seen in the chromatograms (Figure 12C), which showed that the peak corresponding to E07B gene target site was missing in the DMD test panel (lower panel) when compared to the peak in the control panel (upper panel). The E07B gene target site was then sequenced to find that there was an adenosine ("A") insertion in the E07B probe region. See the adenosine position as indicated by the arrow in Figure 12D. Because of the adenosine insertion in exon 7 of the dystrophin gene, the DMD_07B_5 probe SEQ ID NO: 294 was unable to anneal to the target site, leading to the disappearance of the peak corresponding to the Exon07B target site.

Therefore, the multiplex mutation screening method can identify gene regions where mutations may exist. The screening may be carried out simultaneously with the detection for CNVs, as demonstrated in Examples 2 and 3 where the same set of probes and primers were used and the same procedure was followed to obtain electrophoresis data.

This example demonstrates a method of detecting human chromosome 21 copy number changes according to the present invention. In this example, a mere 2% increase of the copy number of human chromosome 21 in a sample could be detected. The method employed fluorescent dye labeled forward primers and reverse primers with stuffer sequences according to the scheme in Figure 5.

In the exemplary method, probes were designed to cover 192 target sites including 96 sites on human chromosome 21, and 96 reference sites on human chromosomes 2-12, 14, and 16-20. For each target site, a probe pair was designed including a 3' probe (the right probe) and a 5' probe (the left probe). The 3' and 5' probes were designed in a manner so that when both are hybridized to the target sequence under a suitable condition, there is no gap between the two probes. The probes were synthesized by Life Technologies Corporation. The names, sequence ID numbers, primer binding sequences, and stuffer sequences of the 384 probes (192 probe pairs) are shown in Table 5. The probe pairs for the target sites on Chromosome 21 were SEQ ID NOs: 559-750. The probe pairs for the reference sites were SEQ ID NOs: 751-942.

Similar to the design in Example 2, for each 3' probe, the 5' end nucleotide was phosphorylated to provide a phosphate which would be connected to the hydroxyl group in the 3' end nucleotide of the 5' probe. Each 3' probe included a locus specific hybridization sequence (LSHS) in the 5' portion followed by a stuffer L2 sequence, and a primer binding sequence Y in the 3' portion. In this example, four primer binding sequence Y: Chr21_Y1, SEQ ID NO: 955; Chr21_Y2, SEQ ID NO: 956; Chr21_Y3, SEQ ID NO: 957; and Chr21_Y4, SEQ ID NO: 958 were used in the 3' probes.

Similar to the design in Example 2, each 5' probe included a locus specific hybridization sequence (LSHS) in the 3' portion followed by a stuffer L1 sequence and a primer binding sequence X in the 5' portion. In this example, four primer binding sequence X: Chr21_X1, SEQ ID NO: 951; Chr21_X2, SEQ ID NO: 952; Chr21_X3, SEQ ID NO: 953; and Chr21_X4, SEQ ID NO: 954 were used in the 5' probes.

In addition, similar to the design in Example 2, four forward primers and four reverse primers were designed for amplifying the ligation products. The four forward primers (Chr21_F1, SEQ ID NO: 943; Chr21_F2, SEQ ID NO: 944; Chr21_F3, SEQ ID NO: 945; and Chr21_F4, SEQ ID NO: 946) had unique sequences that were consistent with the four primer binding sequence X (Chr21_X1, SEQ ID NO: 951; Chr21_X2, SEQ ID NO: 952; Chr21_X3, SEQ ID NO: 953; and Chr21_X4, SEQ ID NO: 954), respectively. The four forward primers: Chr21_F1, Chr21_F2, Chr21_F3 and Chr21_F4 were labeled on the 5' ends with four different fluorescnent dyes: FAM-blue, VIC-green, NED-yellow, and PET-red, respectively. The four reverse primers (Chr21_R1, SEQ ID NO: 947; Chr21_R2, SEQ ID NO: 948; Chr21_R3, SEQ ID NO: 949; and Chr21_R4, SEQ ID NO: 950) had unique sequences that were reversely complementary to the four primer binding sequence Y (Chr21_Y1, SEQ ID NO: 955; Chr21_Y2, SEQ ID NO: 956; Chr21_Y3, SEQ ID NO: 957; and Chr21_Y4, SEQ ID NO: 958), respectively. The Chr21_R2 and Chr21_R4 reverse primers also had a stuffer sequence Chr21 _R_Stuffer, SEQ ID NO: 959 in the 5' portion. All primers were synthesized by Life Technologies Corporation. SEQ ID NO. Probe Name Sequence X Stuffer L1 Stuffer L2 Sequence Y 559 Chr21_01_3 ATT Y1 560 Chr21_01_5 X2 561 Chr21_02_3 ATTACGCGAT Y1 562 Chr21_02_5 X3 563 Chr21_03_3 ATTACGCG Y1 564 Chr21_03_5 X3 ATTACGCGA 565 Chr21_04_3 ATTACGCGATTACG Y2 566 Chr21_04_5 X1 ATTAC 567 Chr21_05_3 ATTACGCGATTA Y1 568 Chr21_05_5 X2 ATT 569 Chr21_06_3 ATTACGCGATTA Y2 570 Chr21_06_5 X1 ATTACG 571 Chr21_07_3 ATTACG Y2 572 Chr21_07_5 X3 573 Chr21_08_3 ATTACGCGATT Y2 574 Chr21_08_5 X1 AT 575 Chr21_09_3 ATTACGCGAT Y2 576 Chr21_09_5 X3 577 Chr21_10_3 ATTACG Y3 578 Chr21_10_5 X3 579 Chr21_11_3 ATTACGCGAT Y3 580 Chr21_11_5 X3 581 Chr21_12_3 ATTACGCGATTA Y3 582 Chr21_12_5 X1 ATTACGCG 583 Chr21_13_3 A Y4 584 Chr21_13_5 X4 585 Chr21_14_3 ATTACGCGAT Y4 586 Chr21_14_5 X2 587 Chr21_15_3 ATTACGCGAT Y2 588 Chr21_15_5 X2 ATTAC 589 Chr21_16_3 ATTACGCGA Y2 590 Chr21_16_5 X3 ATTACGCG 591 Chr21_17_3 ATTACGCGATTAC Y2 592 Chr21_17_5 X2 ATTAC 593 Chr21_18_3 ATTACG Y4 594 Chr21_18_5 X1 595 Chr21_19_3 ATTACGCGATT Y3 596 Chr21_19_5 X1 597 Chr21_20_3 ATTACGCGAT Y1 598 Chr21_20_5 X4 599 Chr21_21_3 ATTACGCGATTA Y3 600 Chr21_21_5 X3 ATT 601 Chr21_22_3 ATTACGCGA Y4 602 Chr21_22_5 X2 ATTACGC 603 Chr21_23_3 ATTACGCGATTA Y3 604 Chr21_23_5 X3 ATTACGC 605 Chr21_24_3 ATTACGCGA Y1 606 Chr21_24_5 X4 607 Chr21_25_3 ATTA Y1 608 Chr21_25_5 X4 609 Chr21_26_3 ATTA Y2 610 Chr21_26_5 X4 611 Chr21_27_3 ATTACGCGATTA Y1 612 Chr21_27_5 X4 ATTAC 613 Chr21_28_3 AT Y2 614 Chr21_28_5 X4 615 Chr21_29_3 ATT Y2 616 Chr21_29_5 X2 617 Chr21_30_3 ATTACGCGA Y2 618 Chr21_30_5 X4 619 Chr21_31_3 ATTACGCGATT Y3 620 Chr21_31_5 X2 ATTACGCG 621 Chr21_32_3 ATTA Y3 622 Chr21_32_5 X4 623 Chr21_33_3 ATTA Y4 624 Chr21_33_5 X2 625 Chr21_34_3 ATTACGCG Y4 626 Chr21_34_5 X4 AT 627 Chr21_35_3 ATTACGCG Y3 628 Chr21_35_5 X2 ATTACGCGAT 629 Chr21_36_3 ATTACGCGAT Y4 630 Chr21_36_5 X3 AT 631 Chr21_37_3 ATTACGCGATT Y1 632 Chr21_37_5 X2 633 Chr21_38_3 ATTACGC Y1 634 Chr21_38_5 X1 635 Chr21_39_3 ATTACGCGATT Y2 636 Chr21_39_5 X2 637 Chr21_40_3 ATTACGCGATTAC Y1 638 Chr21_40_5 X3 AT 639 Chr21_41_3 ATTACGC Y2 640 Chr21_41_5 X1 641 Chr21_42_3 ATTACGCGA Y3 642 Chr21_42_5 X4 643 Chr21_43_3 ATTACGC Y3 644 Chr21_43_5 X1 645 Chr21_44_3 ATTACGCGAT Y2 646 Chr21_44_5 X4 647 Chr21_45_3 ATTACGCGATTAC Y3 648 Chr21_45_5 X1 649 Chr21_46_3 AT Y4 650 Chr21_46_5 X2 651 Chr21_47_3 ATTACGCGATTACGC Y4 652 Chr21_47_5 X3 ATTACGCGATTACGCGATT 653 Chr21_48_3 ATTACGCGATTA Y1 654 Chr21_48_5 X3 ATTACGCGATTAC 655 Chr21_49_3 ATTACGCGATTACG Y2 656 Chr21_49_5 X3 ATTACGCGAT 657 Chr21_50_3 ATTACGCGATTAC Y4 658 Chr21_50_5 X1 ATTAC 659 Chr21_51_3 ATTACGCGATT Y3 660 Chr21_51_5 X2 661 Chr21_52_3 ATTACGCGATTA Y4 662 Chr21_52_5 X4 ATTA 663 Chr21_53_3 ATTACGCGATTA Y1 664 Chr21_53_5 X2 ATTAC 665 Chr21_54_3 ATTA Y1 666 Chr21_54_5 X1 667 Chr21_55_3 ATTACGCGATT Y2 668 Chr21_55_5 X4 ATTAC 669 Chr21_56_3 ATTACGCGATTAC Y3 670 Chr21_56_5 X4 ATTAC 671 Chr21_57_3 ATTA Y4 672 Chr21_57_5 X3 673 Chr21_58_3 ATTACGCGAT Y3 674 Chr21_58_5 X1 ATTACGCGA 675 Chr21_59_3 ATTACGCGATTAC Y2 676 Chr21_59_5 X3 677 Chr21_60_3 AT Y1 678 Chr21_60_5 X3 679 Chr21_61_3 ATTA Y2 680 Chr21_61_5 X1 681 Chr21_62_3 ATTACGCGA Y2 682 Chr21_62_5 X2 ATTACGCG 683 Chr21_63_3 ATTACGCGATTAC Y3 684 Chr21_63_5 X2 685 Chr21_64_3 AT Y2 686 Chr21_64_5 X3 687 Chr21_65_3 ATTACGCGATTA Y4 688 Chr21_65_5 X4 689 Chr21_66_3 ATTACGCGATT Y3 690 Chr21_66_5 X4 ATTACG 691 Chr21_67_3 ATTACGCGATTAC Y4 692 Chr21_67_5 X1 693 Chr21_68_3 ATTA Y3 694 Chr21_68_5 X1 695 Chr21_69_3 ATTACGCGATTACG Y4 696 Chr21_69_5 X2 ATTACGCGATTAC 697 Chr21_70_3 ATTACGCGATTAC Y4 698 Chr21_70_5 X3 ATT 699 Chr21_71_3 ATTACGC Y1 700 Chr21_71_5 X2 701 Chr21_72_3 ATTACGC Y2 702 Chr21_72_5 X2 703 Chr21_73_3 ATTACGCGATTA Y4 704 Chr21_73_5 X3 ATTACGCGATT 705 Chr21_74_3 ATTACGC Y3 706 Chr21_74_5 X2 707 Chr21_75_3 ATTACGCGATTAC Y1 708 Chr21_75_5 X2 ATTACGCG 709 Chr21_76_3 ATTACGC Y3 710 Chr21_76_5 X3 ATTACGCGAT 711 Chr21_77_3 ATTACGCGATTAC Y1 712 Chr21_77_5 X1 ATTACG 713 Chr21_78_3 ATT Y3 714 Chr21_78_5 X2 715 Chr21_79_3 ATTACGCGATTA Y1 716 Chr21_79_5 X1 717 Chr21_80_3 ATTACGCGATTA Y4 718 Chr21_80_5 X4 ATT 719 Chr21_81_3 ATTACGCGATTAC Y1 720 Chr21_81_5 X1 ATT 721 Chr21_82_3 ATTACGCGATTACGC Y2 722 Chr21_82_5 X1 A 723 Chr21_83_3 ATTA Y4 724 Chr21_83_5 X1 725 Chr21_84_3 ATTACGCGATTAC Y3 726 Chr21_84_5 X4 727 Chr21_85_3 AT Y3 728 Chr21_85_5 X3 729 Chr21_86_3 AT Y4 730 Chr21_86_5 X3 731 Chr21_87_3 ATTACG Y4 732 Chr21_87_5 X1 ATTACGCGATT 733 Chr21_88_3 AT Y3 734 Chr21_88_5 X4 A 735 Chr21_89_3 ATTA Y4 736 Chr21_89_5 X4 737 Chr21_90_3 ATTACGCGATT Y1 738 Chr21_90_5 X4 ATTACGC 739 Chr21_91_3 ATTACGC Y1 740 Chr21_91_5 X3 741 Chr21_92_3 ATTACGCGATTAC Y4 742 Chr21_92_5 X2 743 Chr21_93_3 ATT Y1 744 Chr21_93_5 X4 745 Chr21_94_3 ATTACGCGATTAC Y1 746 Chr21_94_5 X1 ATTACGC 747 Chr21_95_3 ATTACGCGATT Y4 748 Chr21_95_5 X1 749 Chr21_96_3 ATTACGCGATTACGC Y2 750 Chr21_96_5 X4 ATTA 751 REF10p_A_3 Y2 752 REF10p_A_5 X1 753 REF10p_B_3 ATTAC Y1 754 REF10p_B_5 X4 755 REF10p_C_3 ATTAC Y4 756 REF10p_C_5 X4 757 REF10p_D_3 ATTACGCGATT Y3 758 REF10p_D_5 X4 759 REF10q_A_3 ATTACGCG Y1 760 REF10q_A_5 X2 ATTAC 761 REF10q_B_3 Y3 762 REF10q_B_5 X2 763 REF10q_C_3 ATTACGCGATTA Y1 764 REF10q_C_5 X3 ATTACGCGATTACGCG 765 REF11p_A_3 ATTACGC Y1 766 REF11p_A_5 X4 767 REF11p_B_3 ATTACGCGA Y3 768 REF11p_B_5 X1 ATTAC 769 REF11p_C_3 ATTACGCGATTAC Y3 770 REF11p_C_5 X2 AT 771 REF11p_D_3 ATTA Y1 772 REF11p_D_5 X1 773 REF11q_A_3 Y4 774 REF11q_A_5 X3 ATTACGCGATTA 775 REF11q_B_3 ATTACGCGATTAC Y2 776 REF11q_B_5 X4 ATTACGCGATTA 777 REF11q_C_3 ATTACG Y3 778 REF11q_C_5 X4 A 779 REF12p_A_3 AT Y4 780 REF12p_A_5 X4 ATTACGCGAT 781 REF12p_B_3 ATTACGCGATTAC Y4 782 REF12p_B_5 X3 ATTACGCGATTACGC 783 REF12p_C_3 ATTACGCGATTACG Y2 784 REF12p_C_5 X4 ATT 785 REF12q_A_3 ATTACGC Y1 786 REF12q_A_5 X3 787 REF12q_B_3 ATTACGCGAT Y4 788 REF12q_B_5 X2 ATTAC 789 REF12q_C_3 ATTACGCGA Y4 790 REF12q_C_5 X1 791 REF14q_A_3 Y2 792 REF14q_A_5 X2 793 REF14q_B_3 Y3 794 REF14q_B_5 X1 795 REF16p_A_3 Y2 796 REF16p_A_5 X4 797 REF16p_B_3 Y3 798 REF16p_B_5 X2 ATTACGCGATTACGCGA 799 REF16p_C_3 ATTACGCGATTAC Y2 800 REF16p_C_5 X2 ATTACG 801 REF16p_D_3 ATTACGCGATTACG Y4 802 REF16p_D_5 X2 ATTACG 803 REF16q_A_3 ATTACGCGAT Y3 804 REF16q_A_5 X1 ATTACG 805 REF16q_B_3 ATTACGCGATTA Y3 806 REF16q_B_5 X4 ATTACGCGA 807 REF16q_C_3 ATTAC Y1 808 REF16q_C_5 X3 ATTACGCG 809 REF16q_D_3 Y2 810 REF16q_D_5 X3 811 REF17q_A_3 Y4 812 REF17q_A_5 X3 AT 813 REF17q_B_3 ATTACGCGATTA Y2 814 REF17q_B_5 X1 ATTACGCGATTACG 815 REF18p_A_3 ATTACGCGATTA Y1 816 REF18p_A_3 X1 AT 817 REF18p_B_3 ATTACGC Y3 818 REF18p_B_3 X2 819 REF19p_A_3 Y4 820 REF19p_A_3 X4 821 REF19p_B_3 AT Y4 822 REF19p_B_5 X2 823 REF19q_A_3 ATT Y2 824 REF19q_A_5 X1 825 REF19q_B_3 ATTACGCGATTAC Y2 826 REF19q_B_5 X3 ATTACGC 827 REF19q_C_3 Y4 828 REF19q_C_5 X1 829 REF19q_D_3 Y3 830 REF19q_D_5 X4 831 REF20p_A_3 ATTACGCGATT Y1 832 REF20p_A_3 X2 ATTA 833 REF20p_B_3 A Y2 834 REF20p_B_3 X4 ATTACGCGAT 835 REF20p_C_3 ATTACGCGA Y4 836 REF20p_C_3 X2 A 837 REF20q_A_3 ATTACGCGATTAC Y2 838 REF20q_A_5 X3 839 REF20q_B_3 ATTACGCGATTA Y2 840 REF20q_B_5 X1 ATTACGC 841 REF20q_C_3 ATTACGCGATTACGCGA Y4 842 REF20q_C_3 X4 ATTAC 843 REF20q_D_3 ATTAC Y2 844 REF20q_D_5 X2 845 REF20q_E_3 ATT Y3 846 REF20q_E_3 X3 847 REF2p_A_3 ATTACGCGAT Y1 848 REF2p_A_5 X3 ATTACGCGA 849 REF2p_B_3 ATTACGCGATTAC Y3 850 REF2p_B_5 X3 ATTACGCGATT 851 REF2p_C_3 Y4 852 REF2p_C_5 X2 853 REF2p_D_3 ATT Y1 854 REF2p_D_5 X2 855 REF2q_A_3 ATTACGCGATTACG Y4 856 REF2q_A_3 X3 ATTACG 857 REF2q_B_3 ATTACGCGATTA Y2 858 REF2q_B_3 X2 ATTACGCGAT 859 REF3p_A_3 ATTACG Y3 860 REF3p_A_5 X1 861 REF3p_B_3 ATTACGCGATTAC Y4 862 REF3p_B_5 X4 ATTACGCGATTAC 863 REF3p_C_3 ATTA Y2 864 REF3p_C_5 X4 865 REF3p_D_3 ATTAC Y1 866 REF3p_D_5 X3 867 REF3p_E_3 ATTACGCGA Y1 868 REF3p_E_5 X1 869 REF3q_A_3 ATTACGCGATTACG Y1 870 REF3q_A_5 X4 ATTACGCGATTAC 871 REF3q_B_3 ATTACGCGA Y4 872 REF3q_B_5 X3 ATT 873 REF3q_C_3 A Y2 874 REF3q_C_5 X3 875 REF3q_D_3 ATTAC Y4 876 REF3q_D_5 X1 877 REF4q_A_3 Y4 878 REF4q_A_5 X3 879 REF4q_B_3 Y2 880 REF4q_B_5 X1 ATTACGCGATTAC 881 REF4q_C_3 ATTACGC Y1 882 REF4q_C_5 X2 883 REF5p_A_3 ATTACGCGATTAC Y3 884 REF5p_A_5 X1 ATTACGCGATTACGC 885 REF5p_B_3 ATTACGCGA Y1 886 REF5p_B_5 X4 ATT 887 REF5p_C_3 A Y1 888 REF5p_C_5 X2 889 REF5q_A_3 ATTAC Y3 890 REF5q_A_5 X2 891 REF5q_B_3 ATTACGCGATTA Y4 892 REF5q_B_5 X1 ATTACGCGATTACGCGA 893 REF5q_C_3 ATTACGCGATTACGCG Y1 894 REF5q_C_5 X1 ATTACGCGATTACG 895 REF6p_A_3 ATT Y3 896 REF6p_A_5 X3 897 REF6p_B_3 Y1 898 REF6p_B_5 X3 899 REF6p_C_3 Y3 900 REF6p_C_5 X1 901 REF6q_A_3 ATTACGCGATTACG Y1 902 REF6q_A_5 X2 ATTACGCGATT 903 REF6q_B_3 ATTACGCGATTACGCG Y4 904 REF6q_B_5 X1 ATTACGC 905 REF6q_C_3 ATTACGCG Y3 906 REF6q_C_5 X4 907 REF7p_A_3 ATTACGC Y2 908 REF7p_A_5 X4 909 REF7p_B_3 ATTACGCGATT Y3 910 REF7p_B_5 X3 ATTA 911 REF7p_C_3 ATTAC Y2 912 REF7p_C_5 X3 913 REF7p_D_3 AT Y4 914 REF7p_D_5 X1 ATTACGCGATTACGCG 915 REF8p_A_3 ATTACGCGA Y1 916 REF8p_A_5 X4 ATTACG 917 REF8p_B_3 ATTACGCGATTACG Y3 918 REF8p_B_5 X2 ATTACGCGATTACG 919 REF8p_C_3 ATTAC Y2 920 REF8p_C_5 X1 921 REF8p_D_3 AT Y2 922 REF8p_D_5 X2 ATTACGCGATT 923 REF8q_A_3 A Y3 924 REF8q_A_5 X3 925 REF8q_B_3 A Y1 926 REF8q_B_5 X1 927 REF8q_C_3 ATTACGC Y3 928 REF8q_C_5 X4 ATTAC 929 REF9p_A_3 ATTACGCGATTACGCG Y4 930 REF9p_A_5 X2 ATTACGCGATTACGC 931 REF9p_B_3 ATTACGCGATTAC Y2 932 REF9p_B_5 X3 ATTACGCGATT 933 REF9p_C_3 ATTACGC Y1 934 REF9p_C_5 X1 ATTACGC 935 REF9p_D_3 Y1 936 REF9p_D_5 X4 937 REF9q_A_3 A Y3 938 REF9q_A_5 X3 ATTACGCGATTA 939 REF9q_B_3 Y4 940 REF9q_B_5 X4 941 REF9q_C_3 ATTACGCGA Y2 942 REF9q C 5 X2