(19)
(11)EP 3 643 788 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
29.04.2020 Bulletin 2020/18

(21)Application number: 17915186.5

(22)Date of filing:  20.06.2017
(51)Int. Cl.: 
C12Q 1/68  (2018.01)
(86)International application number:
PCT/CN2017/089195
(87)International publication number:
WO 2018/232594 (27.12.2018 Gazette  2018/52)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
MA MD

(71)Applicant: MGI Tech Co., Ltd.
Yantian District Shenzhen Guangdong 518083 (CN)

(72)Inventors:
  • YANG, Lin
    Shenzhen Guangdong 518083 (CN)
  • GAO, Ya
    Shenzhen Guangdong 518083 (CN)
  • HUANG, Guodong
    Shenzhen Guangdong 518083 (CN)
  • WANG, Yicong
    Shenzhen Guangdong 518083 (CN)
  • WANG, Yuqian
    Shenzhen Guangdong 518083 (CN)
  • ZHANG, Yanyan
    Shenzhen Guangdong 518083 (CN)
  • CHEN, Fang
    Shenzhen Guangdong 518083 (CN)
  • ZHONG, Na
    Shenzhen Guangdong 518083 (CN)
  • JIANG, Hui
    Shenzhen Guangdong 518083 (CN)
  • XU, Xun
    Shenzhen Guangdong 518083 (CN)

(74)Representative: Nemec, Harald 
Schwarz & Partner Patentanwälte Wipplingerstrasse 30
1010 Wien
1010 Wien (AT)

  


(54)PCR PRIMER PAIR AND APPLICATION THEREOF


(57) Provided is a PCR primer pair, comprising: a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random sequence; the first specific sequence is located on 3' end of the first primer, and the first random sequence is located on 5' end of the first primer; the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is located on 3' end of the second primer, and the second random sequence is located on 5' end of the second primer; moreover, the first specific sequence and the second specific sequence are an upstream primer and a downstream primer directed to a target sequence respectively, and the first random sequence and the second random sequence are inverse complementary.


Description

FIELD



[0001] The present disclosure relates to the field of biotechnology, in particular to PCR amplification, and more particular to PCR primer pair and application thereof.

BACKGROUND



[0002] Polymerase Chain Reaction (PCR) established by Mullis in 1983 has become a classical experimental method in molecular biology and related fields. Its application has been diversified, from gene amplification and gene detection, to gene cloning, genetic engineering, genetic analysis and the like, which even extends to non-biological areas. With the development in recent years, this technology itself has been improving, with continuously improved reliability. Meanwhile, a series of new concepts and experimental methods have been developed based on this basic principle of PCR, which has important application value in life science research.

[0003] Primers are one of the key factors in all PCR methods and technologies. Primer designs are currently available through computer and network. The advantages of online primer design include 1) no need to be proficient in software operation; and 2) simultaneous analysis of many variables. However, good primers designed by conventional primer design software do not definitely produce good results in practical working, especially for templates containing high GC regions and sequences similar to other regions, thus ultimately resulting in poor specificity for the product amplified in the presence of the primers, as well as poor PCR amplification efficiency. In addition, the primers with optimization for these regions usually cannot get satisfactory results.

[0004] Therefore, the existing conventional PCR primer design method still needs to be improved.

SUMMARY



[0005] The present disclosure aims to at least solve one of the technical problems existing in the prior art. For this purpose, an object of the present disclosure is to propose a PCR primer design strategy and a corresponding PCR primer pair which can effectively reduce GC bias during PCR amplification and improve amplification specificity.

[0006] First, it should be noted that the present disclosure has been completed based on the following findings of present inventors.

[0007] There are various websites and software available online for free services of online primer design, such as NetPrimer (www.premierbiosoft.com). Lots of different primer design stand-alone software has been commonly used, with their own advantages. For example, RightprimerTM (Bio2Disk), which has excellent proofreading function, can find out primers having highly strong specificity to sequences to be amplified in a short time by searching Genebank and aligning possible primers with background DNA. OligoTM (Molecular Biology Insights, Inc.) is suitable for designing primers for Multiplex PCR and Consensus PCR, and can provide suitable PCR conditions. PrimerPremier (or Premierbiosoft) can design primers according to protein sequences in the case of unknown nucleic acid sequences, which can be especially useful in cloning new genes when only part of protein sequences are known. PrimerDesigner210 (Scientific and Educational Software) is widely used due to its small size and complete functionality.

[0008] However, good primers designed by conventional primer design software do not definitely produce good results in practical working, especially for templates containing high GC regions and sequences similar to other target regions, thus ultimately resulting in poor specificity for the product amplified in the presence of the primers, as well as poor PCR amplification efficiency. In addition, the primers with optimization for these regions usually cannot get satisfactory results.

[0009] The present inventors after research have found that current PCR primers are required to be designed according to strict primer design conditions. PCR specificity and amplification efficiency greatly depend on the quality of primer designed, thus the primers generally cannot get good results in some repeat regions, high GC regions or regions with advanced structures, thereby plenty of energy and resources will cost for primer design and optimization. The present inventors have conducted a series of design and experimental explorations to solve the problems. Moreover, it is surprisingly discovered by the present inventors that such problems can be effectively addressed by addition of a pair of complementary sequences at the 5' ends of a conventional primer pair thus forming a primer pair with a stable primer-dimer structure which is reversely complement at the 5' end and overhanging at the 3' end.

[0010] Further, in a first aspect, the present disclosure in embodiments provides a PCR primer pair. In embodiments of the present disclosure, the PCR primer pair comprises a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random sequence, and the second primer comprises a second specific sequence and a second random sequence, wherein the first specific sequence is located at the 3' end of the first primer and the first random sequence is located at the 5' end of the first primer, the second specific sequence is located at the 3' end of the second primer and the second random sequence is located at the 5' end of the second primer, the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer for a target sequence, and the first random sequence and the second random sequence are reversely complementary. The present inventors have surprisingly found that the PCR primer pair of the present disclosure can effectively reduce the GC bias during PCR amplification, thus increasing amplification specificity. Specifically, use of conventional primers will result in GC bias to some extent during PCR amplification of the next-generation sequencing library, but the PCR primer pair of the present disclosure (sometimes referred to as "Padlock Primer") is capable of effectively reducing the GC bias during library PCR amplification.

[0011] In embodiments of the present disclosure, the first specific sequence and the second specific sequence each have a TM value of 55-65°C, and the first primer and the second primer each have a TM value of 65-75°C. Thus, the PCR reaction is subjected to a first round of linear amplification under a low annealing temperature of 55-65°C, followed by a second round of circular amplification under a high annealing temperature of 65-72°C in subsequent cycles. During the circular amplification, the specific sequence of primers cannot bind to the specific site of templates directly because the specific sequence has a TM value of 55-65°C which is lower than the high annealing temperature of circular amplification. Such a circular amplification can be effectively performed only when the 5' end and the 3' end of the padlock primer bind to the 5' end and the specific site of templates respectively, i.e. performing the circular amplification through two recognition-site binding.

[0012] The PCR primer pair of the present disclosure is suitable for PCR amplification and library construction for any form of DNA sample to be tested. It should be noted that the "DNA sample to be tested" described in the present disclosure is somewhat different from the conventional understanding which does not include treated DNA. However, in the present disclosure, the "DNA sample to be tested" may include both treated DNA and untreated DNA. Generally, during construction of sequencing library, the genomic DNA of sample will be fragmented and added with adaptors for sequencing, thus obtaining DNA fragments carrying sequencing adaptors corresponding to a sequencing platform, which will be subjected to subsequent amplification and other steps for obtaining sequencing products. Such a DNA fragment carrying sequencing adaptor corresponding to a sequencing platform is called as the "treated DNA". Correspondingly, DNA fragments which are not treated according to the method as described above are called the "untreated DNA". If the PCR primer pair of the present disclosure is for untreated DNA, specific target fragments can be amplified; but if the PCR primer pair of the present disclosure is for treated DNA, whole genomic DNA fragments can be amplified.

[0013] In some embodiments of the present disclosure, when the DNA sample to be tested is the treated DNA which carries a universal sequence, such as a sequencing adaptor, the first specific sequence and the second specific sequence have to specifically recognize a target sequence carrying the universal sequence accordingly, that is, the target sequence actually consists of a universal sequence and a target region sequence, in which the "universal sequence" herein means a sequence complement with the specific sequence of the PCR prime pair, including adaptor sequence for a sequencing platform, i.e. a sequencing adaptor. When the DNA sample to be tested is DNA fragments which do not carry a universal sequence, i.e. templates for PCR reaction, the first specific sequence and the second specific sequence have to specifically recognize the target sequence accordingly. Meanwhile, if a sequencing library is required, adaptor sequence for sequencing (i.e. a universal sequence) can be inserted into the first primer and the second primer respectively or between the specific sequence and the random sequence, so that PCR amplification products can be ligated with adaptors, thus can be effectively used in sequencing platforms.

[0014] In further embodiments of the present disclosure, at least one of the first primer and the second primer further comprises a tag sequence, by which a plurality of samples can be subjected to PCR amplification simultaneously, and the samples can be distinguished by corresponding tag sequences. The position of the tag sequence in the first primer and the second primer is not particularly limited as long as the tag sequence can distinguish different samples without affecting PCR amplification. According to some specific examples of the present disclosure, the tag sequence may be located between a specific sequence and a random sequence, by which a first tag sequence may be arranged between the first specific sequence and the first random sequence of the first primer, and/or a second tag sequence may be arranged between the second specific sequence and the second random sequence of the second primer. According to another embodiment of the present disclosure, the tag sequence may also be arranged within the random sequence, i.e. forming a part of the random sequence, by which, exhibiting the function of distinguishing different samples without affecting PCR amplification as well.

[0015] In embodiments of the present disclosure, the first random sequence and the second random sequence each have a length of 15-45 bp, and the first specific sequence and the second specific sequence each have a length of 15-30 bp.

[0016] In embodiments of the present disclosure, the 1-5th bases from each of the 5' end and the 3' end of the first primer are respectively subjected to modification, and the 1-5th bases from each of the 5' end and the 3' end of the second primer are respectively subjected to modification, so as to effectively prevent from cleavage by exonuclease. According to some specific examples of the present disclosure, the 1-5th bases from each of the 5' end and the 3' end of the first primer are respectively subjected to thio-modification, and the 1-5th bases from each of the 5' end and the 3' end of the second primer are respectively subjected to thio-modification.

[0017] In some embodiments of the present disclosure, the type of thio-modification is not particularly limited as long as the first primer and the second primer can be prevented from cleavage by exonuclease, such as degradation by 5-3' exonuclease or 3-5' exonuclease. According to some specific examples of the present disclosure, the thio-modification is any one selected from phosphorothioate modification, methyl-sulfate modification and peptide nucleic acid modification.

[0018] In an embodiment of the present disclosure, at least one of the first primer and the second primer is subjected to phosphorylation modification at the 5' end. Thus, the products obtained by using the PCR primer pair of the present disclosure, which is a loop-like substance with a nick (that is, the 5' end and the 3' end of the loop-like substance are not connected) can be ligated by a ligase so as to form complete circular DNA.

[0019] In a second aspect, the present disclosure in embodiments provides a PCR amplification kit. In embodiments of the present disclosure, the kit comprises the PCR primer pair as described above. In embodiments of the present disclosure, using the kit comprising the PCR primer pair of the present disclosure for PCR amplification, can bring low GC bias, high amplification specificity and excellent amplification effect during amplification, compared to conventional primers.

[0020] In a third aspect, the present disclosure in embodiments provides a method for PCR amplification. In embodiments of the present disclosure, the method performs the PCR amplification by using the PCR primer pair or the PCR amplification kit as described above. Thus, PCR amplification of templates can be effectively achieved through this method. Moreover, the method is capable of increasing specificity of PCR amplification, effectively reducing generation of non-specific products, and improving amplification efficiency.

[0021] In an embodiment of the present disclosure, the method comprises two rounds of amplification. In the first round of amplification, the PCR primer pair and a template are subjected to linear amplification under an annealing temperature of 55-65°C, and in the second round of amplification, a product of the linear amplification is subjected to circular amplification under an annealing temperature of 65-72°C. Thus, starting from the second PCR cycle (i.e. circular amplification in the second round), bases at the 5' end of the first primer or the second primer can reversely complement with bases at the 5' end of the newly-generated template, and the specific sequence at the 3' end of the first primer or the second primer can reversely complement with bases at the 3' end of the newly-generated template, that is, two recognition sites for binding between primer and template (referring to Figure 3), thus increasing specificity of PCR amplification, and effectively decreasing the generation of non-specific products.

[0022] In an embodiment of the present disclosure, the two rounds of amplification are performed as the following amplification reaction procedure:
step 1 preheating for 2 minutes at 98°C
step 2 denaturing for 10 seconds at 98°C
step 3 annealing for 2 minutes at 55-65°C
step 4 amplifying for 30 seconds at 72°C
step 5 denaturing for 10 seconds at 98°C
step 6 annealing for 1 minute at 65-72°C
step 7 repeating steps 5 and 6 for 5-35 cycles
step 8 extending for 5 minutes at 72°C


[0023] Therefore, the GC bias during PCR amplification is low, the amplification specificity is high and the amplification effect is excellent.

[0024] In a fourth aspect, the present disclosure in embodiments provides a method for preparing a circular DNA library. In embodiments of the present disclosure, the method comprises the steps of:
  1. (1) subjecting a DNA sample to be tested to PCR amplification according to the method for PCR amplification as described above, so as to obtain an amplification product comprising a loop-like substance, wherein
    the 5' end and the 3' end of the loop-like substance are not connected,
    at least one of the first primer and the second primer is subjected to phosphorylation modification at the 5' end,
    the 1-5th bases from each of the 5' end and the 3' end of the first primer are respectively subjected to thio-modification,
    the 1-5th bases from each of the 5' end and the 3' end of the second primer are respectively subjected to thio-modification, and
  2. (2) subjecting the amplification product comprising the loop-like substance to a ligation reaction by using a ligase, such that the 5' end and the 3' end of the loop-like substance are ligated, thus forming circular DNA, the circular DNA constituting the circular DNA library,
    wherein the circular DNA library is a single-stranded circular DNA library when either of the first primer and the second primer is subjected to phosphorylation modification at the 5' end; and the circular DNA library is a double-stranded circular DNA library when both the first primer and the second primer are subjected to phosphorylation modification at the 5' end.


[0025] In embodiments of the present disclosure, a single-stranded circular DNA library or a double-stranded circular DNA library can be efficiently prepared through the method. The obtained single-stranded circular DNA library or double-stranded circular DNA library is of good library quality, which exhibits good effect for DNA preservation or library sequencing.

[0026] In embodiments of the present disclosure, the method further comprises: (3) removing linear DNA. Thus, the obtained library is of good quality.

[0027] In some specific examples of the present disclosure, the linear DNA is removed through a linear-chain digestion reaction.

[0028] In embodiments of the present disclosure, the DNA sample to be tested in the step (1) is added with universal sequence at the 5' end and the 3' end respectively.

[0029] In embodiments of the present disclosure, the PCR primer pair of the present disclosure and use thereof have at least one of the following advantages:
  1. 1. The design strategy of the PCR primer pair of the present disclosure simplifies the primer design flow and optimizes the experimental steps. Each primer of the primer pair consists of a specific sequence at the 3' end and a random sequence (i.e. a complementary sequence) at the 5' end, thus the forward primer and the reverse primer form a stable dimer structure through complementary sequences, which does not need to meet strict conditions as conventional primers, thus greatly simplifying the design process. During conventional primer design, complementation of the 5' end and the 5' end of primers, generation of palindrome structure via primer itself and the like are necessarily to be avoided so as to ensure that no dimer structure is formed between primers and no self-extension of primers occurs for PCR. However, for designing the PCR primer pair of the present disclosure, such problems are not necessary to be considered, because the padlock primer is of a stable dimer structure, of which the 5' ends are complementary with each other and the 3' ends can complement with specific sequences and extend normally; in contrast, for a conventional primer pair, if its 5' ends are complementary with each other, its 3' ends will have no enough sequence to complement with specific sequences of templates. Moreover, the complementary sequences at the 5' end formed between two primers of the padlock primer pair of the present disclosure display potential energy which is greatly stronger than that of self-palindrome structure, thus the dimer structure at the 5' end is preferably formed even the 5' end and the 3' end have sequences complementary with each other.
  2. 2. The PCR amplification method of the present disclosure can increase specificity of PCR amplification, thus effectively reducing generation of non-specific products. Starting from the second PCR cycle, bases at the 5' end of the primer (i.e. the random sequence) can reversely complement with bases at the 5' end of the newly-generated template, and the specific sequence at the 3' end of the primer can reversely complement with bases at the 3' end of the newly-generated template, that is, two recognition sites for binding between primer and template (refer to Figure 3), thus significantly increasing binding ability between primer and template, and amplification specificity. Further, amplification efficiency is also effectively improved due to the increased binding ability.
  3. 3. Using the PCR primer pair of the present disclosure for PCR amplification can effectively reduce GC bias of different templates in amplification of sequencing libraries (especially, the next-generation sequencing library), because effective PCR amplification is only carried out after denaturation of template and binding of primer to template. The GC bias is generated because templates containing some high GC regions would have renatured rapidly before the primer binds to the template during PCR, thus these high GC regions cannot be efficiently amplified. For the padlock primer of the present disclosure, two recognition sites for binding primer to template are presented, which can greatly improve the binding ability between primer and template, thereby the padlock primer can be effectively paired with templates containing high GC regions, thus reducing the GC bias.
  4. 4. The products obtained by the PCR amplification method of the present disclosure are loop-like substances with a nick (that is, the 5' end and the 3' end of the loop-like substance are not connected), thus for experiments where the products have to be cyclized, the cyclization can be realized by addition of a ligase, without complex denaturation, quenching and the like steps, thereby effectively simplifying the experimental process.


[0030] Additional aspects and advantages of the present disclosure will be given in the following description partly, part of which will become apparent from the following description or be acknowledged through the practice of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS



[0031] The above and/or additional aspects and advantages of the present disclosure will become apparent and easily understood from the description of the embodiments in combination with the following drawings, in which

Figure 1 is a schematic diagram showing the structure of a PCR primer pair (i.e. a padlock primer) of the present disclosure according to an embodiment of the present disclosure;

Figure 2 is a schematic diagram showing the structure of a PCR primer pair (i.e. a padlock primer) of the present disclosure according to another embodiment of the present disclosure;

Figure 3 is a schematic diagram showing binding between primer and newly-generated strand in the circular amplification of the present disclosure according to an embodiment of the present disclosure;

Figure 4 is a schematic flow chart showing preparation of a BGISEQ-500 library by using a PCR primer pair of the present disclosure according to an embodiment of the present disclosure, in which markers respectively represent 1 - first round of linear PCR amplification; 2 - second round of circular PCR amplification; 3 - Ligation reaction; and 4 - Linear digestion;

Figure 5 is a graph showing detection results of agarose gel electrophoresis of PCR amplification products obtained by using padlock primers in Example 1;

Figures 6 to 9 are graphs showing 2100 electrophoresis detection results of different types of libraries obtained in Example 2, in which, Figures 6 to 9 respectively represent the 2100 electrophoresis detection results of cfDNA library of conventional primer, cfDNA library of padlock primer, E.coli genomic library of conventional primer and E.coli genomic library of padlock primer; and

Figures 10 and 11 are graphs showing detection results of GC bias of different types of libraries in Example 2.


DETAILED DESCRIPTION



[0032] Embodiments of the present disclosure are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are intended to be illustrative and for explanation of the present disclosure, which cannot be construed as limiting.

[0033] It should be noted, the terms "first" and "second" are used for purposes of description and are not intended to indicate or imply relative importance or significance or impliedly indicate quantity of the technical feature referred to. Thus, the feature defined with "first" and "second" may comprise one or more this feature either explicitly or implicitly. Further, in the description of the present disclosure, "a plurality of' means two or more than two, unless specified otherwise.

PCR primer pair



[0034] In a first aspect, the present disclosure in embodiments provides a PCR primer pair. According to embodiments of the present disclosure, the PCR primer pair comprises a first primer and a second primer, in which, the first primer comprises a first specific sequence and a first random sequence, and the second primer comprises a second specific sequence and a second random sequence, in which the first specific sequence is located at the 3' end of the first primer and the first random sequence is located at the 5' end of the first primer, the second specific sequence is located at the 3' end of the second primer and the second random sequence is located at the 5' end of the second primer, the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer for a target sequence, and the first random sequence and the second random sequence are reversely complementary. The present inventors have surprisingly found that the PCR primer pair of the present disclosure can effectively reduce the GC bias during PCR amplification, thus increasing amplification specificity. Specifically, use of conventional primers will result in GC bias to some extent during PCR amplification of the next-generation sequencing library, but the PCR primer pair of the present disclosure (sometimes referred to as "Padlock Primer") is capable of effectively reducing the GC bias during library PCR amplification.

[0035] It should be noted that the "first random sequence" and the "second random sequence" of the present disclosure may be unfixed or fixed sequences as long as they are reversely complementary to each other.

[0036] According to embodiments of the present disclosure, the first specific sequence and the second specific sequence each have a TM value of 55-65°C, and the first primer and the second primer each have a TM value of 65-75°C. Thus, the PCR reaction is subjected to a first round of linear amplification under a low annealing temperature of 55-65°C, followed by a second round of circular amplification under a high annealing temperature of 65-72°C in subsequent cycles. During the circular amplification, the specific sequence of primer cannot bind to the specific site of templates directly because the specific sequence has a TM value of 55-65°C which is lower than the high annealing temperature of circular amplification. Such a circular amplification can be effectively performed only when the 5' end and the 3' end of the padlock primer bind to the 5' end and the specific site of templates respectively, i.e. performing the circular amplification through two recognition-site binding.

[0037] The PCR primer pair of the present disclosure is suitable for PCR amplification and library construction for any form of DNA sample to be tested. It should be noted that the "DNA sample to be tested" described in the present disclosure is somewhat different from the conventional understanding which does not include treated DNA. However, in the present disclosure, the "DNA sample to be tested" may include both treated DNA and untreated DNA. Generally, during construction of sequencing library, the genomic DNA of sample will be fragmented and added with adaptor for sequencing, thus obtaining DNA fragments carrying sequencing adaptors corresponding to a sequencing platform, which will be subjected to subsequent amplification and other steps for obtaining sequencing products. Such a DNA fragment carrying sequencing adaptor corresponding to a sequencing platform is called as the "treated DNA". Correspondingly, DNA fragments which are not treated according to the method as described above are called the "untreated DNA". If the PCR primer pair of the present disclosure is for untreated DNA, specific target fragments can be amplified; but if the PCR primer pair of the present disclosure is for treated DNA, whole genomic DNA fragments can be amplified.

[0038] According to some embodiments of the present disclosure, when the DNA sample to be tested is the treated DNA which carries a universal sequence, such as a sequencing adaptor, the first specific sequence and the second specific sequence have to specifically recognize a target sequence carrying the universal sequence accordingly, that is, the target sequence actually consists of a universal sequence and a target region sequence, in which the "universal sequence" herein means a sequence complement with the specific sequence of the PCR prime pair, including adaptor sequence for a sequencing platform, i.e. a sequencing adaptor. When the DNA sample to be tested is DNA fragments which do not carry a universal sequence, i.e. templates for PCR reaction, the first specific sequence and the second specific sequence have to specifically recognize the target sequence accordingly. Meanwhile, if a sequencing library is required to be constructed, adaptor sequence for sequencing (i.e. a universal sequence) can be inserted into the random sequence or between the specific sequence and the random sequence for the first primer and the second primer respectively, so that PCR amplification products can be ligated with adaptors, thus can be effectively used in sequencing platform.

[0039] According to further embodiments of the present disclosure, at least one of the first primer and the second primer further comprises a tag sequence, by which a plurality of samples can be subjected to PCR amplification simultaneously, and the samples can be distinguished by corresponding tag sequences. The position of the tag sequence in the first primer and the second primer is not particularly limited as long as the tag sequence can distinguish different samples without affecting PCR amplification. According to some specific examples of the present disclosure, the tag sequence may be located between a specific sequence and a random sequence, by which a first tag sequence may be arranged between the first specific sequence and the first random sequence of the first primer, and/or a second tag sequence may be arranged between the second specific sequence and the second random sequence of the second primer. According to another embodiment of the present disclosure, the tag sequence may also be arranged within the random sequence, i.e. forming a part of the random sequence, by which, exhibiting the function of distinguishing different samples without affecting PCR amplification as well.

[0040] According to embodiments of the present disclosure, the first random sequence and the second random sequence each have a length of 15-45 bp, and the first specific sequence and the second specific sequence each have a length of 15-30 bp.

[0041] According to embodiments of the present disclosure, the 1-5th bases from each of the 5' end and the 3' end of the first primer are respectively subjected to thio-modification, and the 1-5th bases from each of the 5' end and the 3' end of the second primer are respectively subjected to thio-modification, so as to effectively prevent from cleavage by exonuclease.

[0042] According to some embodiments of the present disclosure, the type of thio-modification is not particularly limited as long as the first primer and the second primer can be prevented from cleavage by exonuclease, such as cleavage by 5-3' exonuclease or 3-5' exonuclease. According to some specific examples of the present disclosure, the thio-modification is any one selected from phosphorothioate modification, methyl-sulfate modification and peptide nucleic acid modification.

[0043] According to an embodiment of the present disclosure, at least one of the first primer and the second primer is subjected to phosphorylation modification at the 5' end. Thus, a loop-like substance with a nick which is obtained after two rounds of amplification (that is, the 5' end and the 3' end of the loop-like substance are not connected) can be ligated by a ligase so as to form complete circular DNA.

[0044] In addition, it should be noted, referring to Figure 1 which shows the schematic structure of the PCR primer pair of the present disclosure, the design strategy of the PCR primer pair of the present disclosure includes addition of a pair of complementary sequences at the 5'-ends of a conventional primer pair (including a forward primer and a reverse primer), thus forming a PCR primer pair (i.e. Padlock Primer, PP) which is reversely complementary at the 5' end and overhanging at the 3' end, in which two primers of the padlock primer pair form a stable primer-dimer structure, and the complementary sequences may be unfixed or fixed sequences. The first primer and the second primer of the PCR primer pair each have a length of 30-70 bp, and have a high TM value, generally of 65-75°C. The complementary sequences (i.e. the first random sequence and the second random sequence, being unfixed or fixed sequences) at the 5'-end of the padlock primer pair each have a length of 15-45 bp. The first specific sequence and the second specific sequence at the 3'-ends of the padlock primer pair, which are complementary to target sequences of a template respectively, each have a length of 15-30 bp, and have a low TM value, generally of 55-65°C.

[0045] Further, for the application of the PCR primer pair of the present disclosure, the PCR primer pair of the present disclosure is subjected to two different amplification processes (i.e. two rounds of amplification) to complete the PCR amplification, referring to Figure 3, in the first round of amplification, the annealing temperature is 55-65°C and the cycle number is 1; and in the second round of amplification, the annealing temperature is 65-72°C and the cycle number is 5-35. During the first round of amplification, only the specific sequence at the 3' end of the PCR primer pair can bind to template, thus the annealing temperature in this round is low. During the second round of amplification, the newly-generated template (i.e. the product of the first round of amplification) is firstly paired with the complementary sequence at the 5' end of the PCR primer pair (i.e. the first random sequence or the second random sequence), followed by pairing with the specific sequence at the 3' end (i.e. the first specific sequence or the second specific sequence), that is, two recognition sites for binding between primer and template, which greatly improved the annealing temperature of primer, resulting in a high annealing temperature.

[0046] Further, it should be noted, in the second round of amplification, the circular amplification can be effectively performed only when the 5' end and the 3' end of the primer bind to the newly-generated template simultaneously, thus both specificity of PCR amplification and binding between primer and template are greatly improved via two recognition sites, with improved PCR amplification efficiency. Therefore, using the PCR primer pair of the present disclosure for PCR amplification, is capable of significantly increasing the specificity of PCR amplification, effectively reducing the generation of non-specific products, and reducing the GC bias during amplification, compared to conventional PCR primers. Thus, use of such a primer pair in sequencing, especially in the next-generation sequencing library, can effectively reduce the genome-wide GC bias in library enrichment and amplification.

[0047] In addition, according to the embodiment of the present disclosure, the products obtained by PCR amplification in the presence of the PCR primer pair of the present disclosure can be directly cyclized by a ligation reaction, thus obtaining circular DNA. Specifically, the products can be directly subjected to cyclization in a ligation reaction system, without additional denaturation, quenching and other steps, thus cyclization process is simplified, and the process for preparing a circular DNA library is simplified accordingly.

Application



[0048] Further, in a second aspect, the present disclosure in embodiments also provides a PCR amplification kit. According to an embodiment of the present disclosure, the kit comprises the PCR primer pair as described above. According to an embodiment of the present disclosure, using the kit comprising the PCR primer pair of the present disclosure for PCR amplification, can bring low GC bias, high amplification specificity and excellent amplification effect during amplification, compared to conventional primers.

[0049] Further, the present disclosure in embodiments proposes use of the PCR primer pair and the kit comprising the PCR primer pair.

[0050] In a third aspect, the present disclosure in embodiments provides a method for PCR amplification. According to an embodiment of the present disclosure, the method performs PCR amplification by using the PCR primer pair or the PCR amplification kit as described above. Thus, PCR amplification of template can be effectively achieved by using this method. Moreover, this method can increase specificity of PCR amplification, effectively reduce generation of non-specific products, and improve amplification efficiency.

[0051] According to an embodiment of the present disclosure, the method comprises two rounds of amplification. In the first round of amplification, the PCR primer pair and a template are subjected to linear amplification under an annealing temperature of 55-65°C, and in the second round of amplification, a product of the linear amplification is subjected to circular amplification under an annealing temperature of 65-72°C. Thus, starting from the second PCR cycle (i.e. circular amplification in the second round), bases at the 5' end of the first primer or the second primer can reversely complement with bases at the 5' end of the newly-generated template, and the specific sequence at the 3' end of the first primer or the second primer can reversely complement with bases at the 3' end of the newly-generated template, that is, two recognition sites for binding between primer and template (referring to Figure 3), thus increasing specificity of PCR amplification, and effectively decreasing the generation of non-specific products.

[0052] According to an embodiment of the present disclosure, the two rounds of amplification are performed as the following amplification reaction procedure:
step 1 preheating for 2 minutes at 98°C
step 2 denaturing for 10 seconds at 98°C
step 3 annealing for 2 minutes at 55-65°C
step 4 amplifying for 30 seconds at 72°C
step 5 denaturing for 10 seconds at 98°C
step 6 annealing for 1 minute at 65-72°C
step 7 repeating steps 5 and 6 for 5-35 cycles
step 8 extending for 5 minutes at 72°C


[0053] Therefore, the GC bias during PCR amplification is low, the amplification specificity is high and the amplification effect is excellent.

[0054] In a fourth aspect, the present disclosure in embodiments provides a method for preparing a circular DNA library. According to embodiments of the present disclosure, the method comprises the steps of:
  1. (1) subjecting a DNA sample to be tested to PCR amplification according to the method for PCR amplification as described above, so as to obtain an amplification product comprising a loop-like substance, in which
    the 5' end and the 3' end of the loop-like substance are not connected,
    at least one of the first primer and the second primer is subjected to phosphorylation modification at the 5' end,
    the 1-5th bases from each of the 5' end and the 3' end of the first primer are respectively subjected to thio-modification,
    the 1-5th bases from each of the 5' end and the 3' end of the second primer are respectively subjected to thio-modification, and
  2. (2) subjecting the amplification product comprising the loop-like substance to a ligation reaction by using a ligase, such that the 5' end and the 3' end of the loop-like substance are ligated, thus forming circular DNA, the circular DNA constituting the circular DNA library,
    in which the circular DNA library is a single-stranded circular DNA library when either of the first primer and the second primer is subjected to phosphorylation modification at the 5' end; and the circular DNA library is a double-stranded circular DNA library when both the first primer and the second primer are subjected to phosphorylation modification at the 5' end.


[0055] According to embodiments of the present disclosure, a single-stranded circular DNA library or a double-stranded circular DNA library can be efficiently prepared through the method. The obtained single-stranded circular DNA library or double-stranded circular DNA library is of good library quality, which exhibits good effect for DNA preservation or library sequencing.

[0056] According to embodiments of the present disclosure, the method further comprises: (3) removing linear DNA. Thus, the obtained library is of good quality.

[0057] According to some specific examples of the present disclosure, the linear DNA is removed through a linear-chain digestion reaction.

[0058] According to embodiments of the present disclosure, the DNA sample to be tested in the step (1) is added with universal sequence at the 5' end and the 3' end respectively. As described above, the expression "universal sequence" used herein means a sequence paired with the specific sequence of the PCR prime pair, including an adaptor sequence for a sequencing platform, i.e. a sequencing adaptor. Thus, the obtained library can be directly used in on-line sequencing on a corresponding sequencing platform, when the DNA sample to be tested is added with the universal sequence such as a sequencing adaptor at two ends respectively.

[0059] Reference will be made in detail to examples of the present disclosure. It would be appreciated by those skilled in the art that the following examples are explanatory, and cannot be construed to limit the scope of the present disclosure. If the specific technology or conditions are not specified in the examples, a step will be performed in accordance with the techniques or conditions described in the literature in the art (for example, referring to J. Sambrook, et al. (translated by Huang PT), Molecular Cloning: A Laboratory Manual, 3rd Ed., Science Press) or in accordance with the product instructions. If the manufacturers of reagents or instruments are not specified, the reagents or instruments may be conventional products that are commercially available, for example, from Illumina Company.

Example 1: Padlock primer improves specificity and amplification efficiency relative to conventional primer


1.1 Primer design of padlock primer (i.e. PCR primer pair of the present disclosure, referred to as "PP primer" below)



[0060] 1.1.1 A conventional primer pair was designed through NC_000004.12 (Homo sapiens chromosome 4, GRCh38.p7 Primary Assembly), with an amplified product in a length of 277 bp. The conventional primer pair was added with complementary sequences of 24 bp, 19 bp and 11 bp at the 5' end respectively, thus obtaining three padlock primer pairs.

[0061] 1.1.2 Five conventional primer pairs (respectively having amplified products in a length of 88 bp, 235 bp, 318 bp, 404 bp and 498 bp) were designed through NC_000016.10 (Homo sapiens chromosome 16, GRCh38.p7 Primary Assembly), which were each added with a 18 bp of complementary sequence at the 5' end, thus obtaining five padlock primer pairs, referring to Table 1 and Figure 1 showing the schematic primer structure.

1.2 Experimental steps



[0062] For each of 6 conventional primer pairs and 6 padlock primer pairs (primer sequences referring to Table 1), 10 ng of genomic DNA was taken for PCR amplification by using the rTaq amplification kit from Takara (Catalog No: RR001B) according to the amplification system shown in the following table:
rTaq Buffer (10X) 2 µL
Genomic DNA 16.3 µL
Forward primer (10 µM) 0.5 µL
Reverse primer (10 µM) 0.5 µL
dNTP Mixture 0.5 µL
rTaqDNA Polymerase 0.2 µL
Total 20 µL
A) The amplification conditions for conventional primers were as follows:
Step 1 98°C, 2 minutes
Step 2 98°C, 10 seconds
Step 3 58°C, 2 minutes
Step 4 72°C, 30 seconds
Step 5 repeating steps 3 and 4 for 30 cycles
Step 8 72°C, 5 minutes
B) The amplification conditions for PP primers were as follows:
Step 1 98°C, 2 minutes
Step 2 98°C, 10 seconds
Step 3 58°C, 2 minutes
Step 4 72°C, 30 seconds
Step 5 98°C, 10 seconds
Step 6 68°C, 1 minute
Step 7 repeating steps 5 and 6 for 29 cycles
Step 8 72°C, 5 minutes


[0063] 1.2.1 The resulting products were subjected to purification and agarose gel electrophoresis, refer to Figure 5.
Table 1: Primer sequences
Primer Nos.Primer nameSequences (5'-3', SEQ ID NO:)Length
1 Control-F GCTCTGGGGGCTCGGCTT(1) 277bp
Control-R AACGCACGGGCACTGAGG(2)
2 PP-24BP-F

 
325bp (24bp of complementary sequence)
PP-24BP-R

 
3 PP-19BP-F

 
315bp (19bp of complementary sequence)
PP-19BP-F

 
4 PP-11BP-F

 
299bp (11bp of complementary sequence)
PP-11BP-R

 
5 Control-1F CCTTACGGATAGTATAACATAAAGTC(9) 88bp
Control-1R GACAGAGCGAGACTCCGT(10)
6 Control-2F TCCCGAGTAGCTGGGACT(11) 235bp
Control-2R TCTTTCTTATCTCCTCCAATGATCT(12)
7 Control-3F TACCACGCCCGGCTAA111111GTA(13) 318bp
Control-3R TATGGCTATCTGGTCCCAACATAATT(14)
8 Control-4F GATCTCCTGACCTCGTGATC(15) 404bp
Control-4R TGAAGGCTAATCCTAAGAGCTGAT( 16)
9 Control-5F AGAATTTAGCACGATAAAGAGAGCA(17) 498bp
Control-5R CTGGAGTGCAATGGCCTG(18)
10 PP-F1

 
124bp
PP-R1

 
11 PP-F2

 
271bp
PP-R2

 
12 PP-F3

 
354bp
PP-R3

 
13 PP-F4

 
440bp
PP-R4

 
14 PP-F5

 
534bp
PP-R5

 
Note: F: Forward primer; R: Reverse primer; Control: Conventional primer; PP: Padlock primer; and the complementary sequence of each PP primer is highlighted in box.


[0064] Figure 5 shows the detection results of agarose gel electrophoresis of PCR amplification products obtained by padlock primers. As shown in Figure 5, bands 1, 2, 3 and 4 respectively represent amplification products of a conventional primer pair, a padlock primer pair with 24 bp of complementary sequence, a padlock primer pair with 19 bp of complementary sequence, and a padlock primer pair with 11 bp of complementary sequence, with expected length of 277 bp, 325 bp, 315 bp and 299 bp respectively. Among them, band 4 was obtained in the presence of primers PP-11BP-F and PP-11BP-R, that is, representing the amplification product of the padlock primer pair with 11 bp of complementary sequence, however, showing a poor amplification effect, thus the present inventors limited the minimum length of the complementary sequence to be 15bp. Bands 5-9 represent amplification products of conventional primers, with expected length of 88bp, 235bp, 318bp, 404bp and 498bp. Bands 10-14 represent amplification products of the padlock primer pairs with 18 bp of complementary sequence, with expected length of 124 bp, 271 bp, 354 bp, 440 bp and 534 bp.

[0065] Conclusion: it can be seen from Figure 5 that conventional primers result in severe non-specific amplification; in contrast, the padlock primers of different lengths (within the length from 15 bp to 45 bp) exhibit significant improvement on amplification, in which longer the complementary sequence is, better specificity gets. Thus, the amplification specificity of primer is greatly improved through the padlock primer-related strategy.

Example 2: Application of padlock primers in enrichment of BGI-SEQ500 library



[0066] In this example, conventional primers and padlock primers of the present disclosure (refer to Figure 2, showing the schematic structure) were respectively used to prepare libraries for E. coli genomic DNA and cell-free DNA (i.e. cfDNA) as a template.

[0067] 10 ng of taken E.coli genomic DNA and 2 ng of extracted cfDNA were respectively fragmented into about 200 bp of fragments by Cavoris, followed by library construction by using the combined probe anchored polymerization sequencing kit from BGI (Catalog No. BOX3) through the following steps. Figure 4 shows the flow chat for library construction.

2.1 Terminal repairing and adenine (A) addition



[0068] Terminal repairing and adenine (A) addition for library construction were as follows:
DNA 40 µL
Buffer 9.4 µL
Enzyme 0.6 µL
Total 50 µL


[0069] Reaction conditions were 37°C for 30 minutes and 65°C for 15 minutes.
DNA: E.coli genomic DNA or cell-free DNA (cfDNA).

2.2 Adaptor ligation



[0070] The ligation system was as follows:
Terminal-repaired 50 µL
DNA ligase 1 µL
Ligation buffer 24 µL
Adaptor 5 µL
Total 80 µL


[0071] Reaction condition was 23°C for 30 minutes.

2.3 Library enrichment



[0072] The PCR amplification system was as follows:
2×kapa HIFI master mix 25 µL
DNA-ligated product 21 µL
Primer 1 (10µM) 2 µL
Primer 2 (10µM) 2 µL
Total 50 µL


[0073] The primer sequences are shown in Table 2:
Table 2
Primer nameSequences (5'-3', SEQ ID NO: )
Primer 1

 
Primer 2

 
Note: the bases underlined are tag sequence.


[0074] PCR reaction conditions were as follows:
Step 1 98°C, 2 minutes
Step 2 98°C, 10 seconds
Step 3 58°C, 1 minute
Step 4 72°C, 30 seconds
Step 5 98°C, 10 seconds
Step 6 68°C, 1 minute
Step 7 repeating steps 5 and 6 for 5-15cycles
Step 8 72°C, 5 minutes

2.4 Library cyclization



[0075] Cyclization reaction system was as follows:
PCR product 50 µL
10×TA buffer 6 µL
T4DNA ligase 4 µL
Total 60 µL


[0076] Reaction condition was 37°C for 30 minutes.

[0077] 2.5 The ligation products were subjected to purification followed by qubit quantification (refer to Table 3) and 2100 Bioanalyzer detection (refer to Figures 6- 9).

[0078] 2.6 The qualified library was subjected to on-machine sequencing by using BGISEQ-500 sequencing platform and the sequencing type of SE28+10. The basic parameters of off-machine data including GC bias, aligning rate, repetitive rate and the like were subjected to statistics analysis (refer to Table 4 and Figures 10-11).

[0079] Figures 6 to 9 show the 2100 electrophoresis detection results of different types of libraries obtained, including cfDNA library of conventional primer, cfDNA library of padlock primer, E.coli genomic library of conventional primer and E.coli genomic library of padlock primer.
Table 3: Library output
TypesInput (ng)Output (ng)
cfDNA library of conventional primer 2 430
cfDNA library of padlock primer 2 640
E.coli genomic library of conventional primer 10 1560
E.coli genomic library of padlock primer 10 2600
Table 4: Statistics of library off-machine data
TypesOriginal data (reads)Alignment rateUnique Hit RateRepetitive rateMismatch rate
cfDNA library of 6525963       0.5%
conventional primer   92.1% 97.5% 0.1%  
cfDNA library of 6635893       0.3%
padlock primer   93.4% 97.3% 0.2%  
E.coli genomic 2278185       0.4%
library of          
conventional primer   97.5% 97.5% 23.3%  
E.coli genomic 2242635       0.6%
library of padlock          
primer   97.7% 97.1% 20.6%  


[0080] Figures 10 and 11 show detection results of GC bias for different types of libraries, in which in Figure 10, the dotted line represents GC bias of conventional primer and the solid line represents GC bias of padlock primer, indicating less fluctuation with change of GC content for the library depth obtained by the padlock primer; and in Figure 11, the dotted line represents GC bias of conventional primer and the solid line represents GC bias of padlock primer, indicating less fluctuation with change of GC content for the library depth obtained by the padlock primer, thus the results demonstrate that the padlock primer can effectively improve GC bias.

[0081] Conclusion: it can be seen from Table 3 and Figures 6-9 that the library output of padlock primers has been improved significantly, indicating that padlock primers can improve the efficiency of PCR amplification; and Table 4 and Figures 10-11 show that the off-machine data (for example, basic parameters such as alignment rate) of padlock primers are not different from conventional primers apparently, however, the padlock primers can effectively improve the GC bias of library compared to conventional primers. The padlock primer can be applied for library construction not only on the BGISEQ-500 sequencing platform but only on other next-generation sequencing platforms such as illumina, proton and the like.

Industrial applicability



[0082] The PCR primer pair of the present disclosure can be effectively used for PCR amplification of DNA samples to be tested, effectively reduce GC bias during PCR amplification, and improve amplification specificity.

[0083] Although specific embodiments of the present disclosure have been described in detail, it would be appreciated by those skilled in the art that various modifications and alternatives of the details can be made according to teachings of the present disclosure, which are all within the scope of the present disclosure. The full scope of the present disclosure is given by the appended claims and any equivalents thereof.

[0084] Reference throughout this specification to terms "an embodiment", "some embodiments", "illustrative embodiment", "an example", "a specific example" or "some examples" means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the description with reference to the terms "an embodiment", "some embodiments", "illustrative embodiment", "an example", "a specific example" or "some examples" throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.














Claims

1. A PCR primer pair, comprising a first primer and a second primer, wherein
the first primer comprises a first specific sequence and a first random sequence, and
the second primer comprises a second specific sequence and a second random sequence, wherein
the first specific sequence is located at the 3' end of the first primer and the first random sequence is located at the 5' end of the first primer,
the second specific sequence is located at the 3' end of the second primer and the second random sequence is located at the 5' end of the second primer,
the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer for a target sequence, and
the first random sequence and the second random sequence are reversely complementary.
 
2. The PCR primer pair according to claim 1, wherein the first specific sequence and the second specific sequence each have a TM value of 55-65°C, and
the first primer and the second primer each have a TM value of 65-75°C.
 
3. The PCR primer pair according to claim 1, wherein
the first random sequence and the second random sequence each have a length of 15-45 bp, and
the first specific sequence and the second specific sequence each have a length of 15-30 bp.
 
4. The PCR primer pair according to claim 1, wherein the 1-5th bases from each of the 5' end and the 3' end of the first primer are respectively subjected to thio-modification, and
the 1-5th bases from each of the 5' end and the 3' end of the second primer are respectively subjected to thio-modification.
 
5. The PCR primer pair according to claim 4, wherein the thio-modification is selected from phosphorothioate modification, methyl-sulfate modification and peptide nucleic acid modification.
 
6. The PCR primer pair according to claim 1, wherein at least one of the first primer and the second primer is subjected to phosphorylation modification at the 5' end.
 
7. A PCR amplification kit, comprising the PCR primer pair according to any one of claims 1 to 6.
 
8. A method for PCR amplification, comprising:
performing the PCR amplification by using the PCR primer pair of any one of claims 1 to 6 or the PCR amplification kit of claim 7.
 
9. The method according to claim 8, comprising two rounds of amplification, wherein
in the first round of amplification, the PCR primer pair and a template are subjected to linear amplification under an annealing temperature of 55-65°C, and
in the second round of amplification, a product of the linear amplification is subjected to circular amplification under an annealing temperature of 65-72°C.
 
10. The method according to claim 9, wherein the two rounds of amplification are performed as the following amplification reaction procedure:
step 1 preheating for 2 minutes at 98°C
step 2 denaturing for 10 seconds at 98°C
step 3 annealing for 2 minutes at 55-65°C
step 4 amplifying for 30 seconds at 72°C
step 5 denaturing for 10 seconds at 98°C
step 6 annealing for 1 minute at 65-72°C
step 7 repeating steps 5 and 6 for 5-35 cycles
step 8 extending for 5 minutes at 72°C

 
11. A method for preparing a circular DNA library, comprising the steps of:

(1) subjecting a DNA sample to be tested to PCR amplification according to the method of any one of claims 8 to 10, so as to obtain an amplification product comprising a loop-like substance, wherein
the 5' end and the 3' end of the loop-like substance are not connected,
at least one of the first primer and the second primer is subjected to phosphorylation modification at the 5' end,
the 1-5th bases from each of the 5' end and the 3' end of the first primer are respectively subjected to thio-modification,
the 1-5th bases from each of the 5' end and the 3' end of the second primer are respectively subjected to thio-modification, and

(2) subjecting the amplification product comprising the loop-like substance to a ligation reaction by using a ligase, such that the 5' end and the 3' end of the loop-like substance are ligated, thus forming circular DNA, the circular DNA constituting the circular DNA library,
wherein the circular DNA library is a single-stranded circular DNA library when either of the first primer and the second primer is subjected to phosphorylation modification at the 5' end; and the circular DNA library is a double-stranded circular DNA library when both the first primer and the second primer are subjected to phosphorylation modification at the 5' end.


 
12. The method according to claim 11, further comprising:
(3) removing linear DNA.
 
13. The method according to claim 12, wherein the linear DNA is removed through a linear-chain digestion reaction.
 
14. The method according to claim 11, wherein in step (1), the DNA sample to be tested is added with universal sequence at the 5' end and the 3' end respectively.
 




Drawing

























REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description