(19)
(11)EP 3 133 170 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
18.03.2020 Bulletin 2020/12

(21)Application number: 16180033.9

(22)Date of filing:  10.09.2009
(51)Int. Cl.: 
C12Q 1/6827  (2018.01)
C12Q 1/6841  (2018.01)

(54)

IMAGING INDIVIDUAL MRNA MOLECULES USING MULTIPLE SINGLY LABELED PROBES

ABBILDUNG EINZELNER MRNA-MOLEKÜLE MITHILFE MEHRERER INDIVIDUELL MARKIERTER SONDEN

IMAGERIE DE MOLÉCULES D'ARNM INDIVIDUELLES UTILISANT DES SONDES MULTIPLES À MARQUAGE UNIQUE


(84)Designated Contracting States:
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 SE SI SK SM TR

(30)Priority: 10.09.2008 US 191724 P

(43)Date of publication of application:
22.02.2017 Bulletin 2017/08

(62)Application number of the earlier application in accordance with Art. 76 EPC:
09813630.2 / 2324123

(73)Proprietor: Rutgers, the State University of New Jersey
New Brunswick, NJ 08909 (US)

(72)Inventors:
  • RAJ, Arjun
    Wynnewood, PA 19096 (US)
  • TYAGI, Sanjay
    New York, NY 10038 (US)

(74)Representative: Rees, Kerry 
WP Thompson 138 Fetter Lane
London EC4A 1BT
London EC4A 1BT (GB)


(56)References cited: : 
WO-A1-97/14816
WO-A2-02/22874
US-B1- 6 329 152
WO-A1-2006/132710
WO-A2-2007/001986
  
  • FEMINO A M ET AL: "Visualization of single RNA transcripts in situ", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, WASHINGTON, DC; US, vol. 280, no. 5363, 24 April 1998 (1998-04-24), pages 585-590, XP002306891, ISSN: 0036-8075, DOI: 10.1126/SCIENCE.280.5363.585
  • LEE SANGHOON ET AL.: "Use of multiple 16S rRNA-targeted fluorescent probes to increase signal strength and measure cellular RNA from natural planktonic bacteria", MARINE ECOLOGY PROGRESS SERIES, vol. 101, 4 November 1993 (1993-11-04), pages 193-201, XP002685594, ISSN: 0171-8630
  • ARJUN RAJ ET AL: "Imaging individual mRNA molecules using multiple singly labeled probes", NATURE METHODS, vol. 5, no. 10, 1 October 2008 (2008-10-01), pages 877-879, XP055041223, ISSN: 1548-7091, DOI: 10.1038/nmeth.1253
  • KOSMAN D ET AL: "Multiplex detection of RNA expression in Drosophila embryos", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, vol. 305, no. 5685, 6 August 2004 (2004-08-06), page 846, XP002394668, ISSN: 0036-8075, DOI: 10.1126/SCIENCE.1099247
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

Field of the invention



[0001] This invention is defined by the appended claims.

Background



[0002] As it has become increasingly apparent that gene expression in individual cells deviates significantly from the average behavior of cell populations, new methods that provide accurate integer counts of mRNA copy numbers in individual cells are needed. Ideally, such methods should also reveal the intracellular locations of the mRNAs, as mRNA localization is often used by cells to spatially restrict the activity gene.

[0003] In situ hybridization, followed by microscopic analysis, is a well-established means of studying gene expression. The first generation of in situ hybridizations was performed with radioactive probes. Early improvements involved linking the probes to enzymes that catalyze chromogenic or fluorogenic reactions. However, because the products of these reactions were small molecules or precipitates that diffuse away from the probe, the location of the target molecules could not be precisely determined. Conversely, probes labeled directly with a few fluorophores maintained spatial resolution, but the sensitivity that can be achieved is relatively poor.

[0004] Robert Singer and colleagues developed an in situ hybridization procedure that was not only sensitive enough to permit the detection of single mRNA molecules, but also restricted the signals to close proximity of the targets. They hybridized five oligonucleotide probes simultaneously to each mRNA target, each of which was about 50-nucleotides in length and each of which was labeled with five fluorophore moieties. Although the authors convincingly demonstrated single molecule sensitivity and other groups have successfully used these probes, the system has not been widely adopted. One reason for this is difficulty in the synthesis and purification of heavily labeled oligonucleotides. Usually, flurophore moieties are introduced via primary amino groups that are incorporated into oligonucleotides during their synthesis. When multiple amino groups are introduced into the same oligonucleotide some are lost due to side reactions such as transamidation. Coupling of fluorophores to the remaining amino groups is inefficient and requires several consecutive coupling reactions and it is difficult to purify oligonucleotides in which all designed sites are coupled to fluorophores from those that are partially coupled. Also, when some fluorophores are present in multiple copies on the same oligonucleotide they interact with each other altering the hybridization characteristics of the oligonucleotides and exhibiting severe self-quenching. These problems are obviated if each probe had just a single terminal amino group to serve as the site of attachment.

[0005] Another issue with the use of small numbers of heavily labeled probes is that a significant portion of the fluorescence is lost for every probe that does not bind to the target, whereas every non-specific binding event increases the background. This leads to a widened distribution of number of probes bound to each target mRNA. For instance, when using 5 fluorescent probes targeted to a single mRNA, Femino et al estimated that the majority of the fluorescent spots observed had intensities indicating the presence of only 1 or 2 probes. Science 280, 585-590 (1998). This makes it difficult to unambiguously identify those fluorescent spots as mRNA molecules, since it is impossible to determine whether the detection of an individual probe arises from legitimate binding to the target mRNA or non-specific binding. These "thresholding" problems limit the ability of such methods to provide reliable counts of mRNA numbers in individual cells. Further documents describe probes for in-situ hybridization methods: WO97/14816 A1, US6329152 B1, WO2007/001986 A2, WO02/22874 A2, Lee Sanghoon et al., Marine Ecology Progress Series, 101, pp.193. 04-11-1993, Kosman D. et al., Science,305, pp. 846, 06.08.2004.

[0006] Thus there remains a need for improved methods to provide reliable counts of mRNA numbers in individual cells and a need for probes that are easily synthesized and purified.

Summary of the invention



[0007] This disclosure provides a method for detecting individual nucleic acid molecules, such as, for example, RNA molecules, e.g., mRNA molecules in fixed, permeabilized cells using a plurality of nucleic acid hybridization probes that are singly fluorescently labeled, as with the same fluorophore. The inventors have surprisingly discovered that if at least 30, preferably 40-60, and very preferably 48 different probes, all labeled with the same fluorophore, are hybridized simultaneously to a target sequence of an mRNA molecule, a fluorescent spot is created that can be detected from the combined fluorescences of the multiple probes. The probes are non-overlapping; that is, the region of the target sequence to which each probe hybridizes is unique (or non-overlapping). Probes in a set of 30 or more for a selected target sequence can be designed to hybridize adjacently to one another or to hybridize non-adjacently, with stretches of the target sequence, from one nucleotide to a hundred nucleotides or more, not complementary to any of the probes. Accordingly, in one aspect, the disclosure provides a method for probing a target sequence of nucleic acid molecules such as, for example, mRNAs in a fixed, permeabilized cell, said target sequence including at least 30 non-overlapping probe binding regions of 15-100 nucleotides, comprising immersing said cell in an excess of at least 30 nucleic acid hybridization probes, each singly labeled with the same fluorescent label and each containing a nucleic acid sequence that is complementary to a different probe binding region of said target sequence; washing said fixed cell to remove unbound probes; and detecting fluorescence from said probes.

[0008] Probes useful in this invention may be DNA, RNA or mixtures of DNA and RNA. They may include non-natural nucleotides, and they may include non-natural internucleotide linkages. Non-natural nucleotides that increase the binding affinity of probes include 2'-O-methyl ribonucleotides. The lengths of probes useful in this disclosure are 15-40 nucleotides for typical DNA or RNA probes of average binding affinity. Preferred lengths of DNA probes and RNA probes are in the range of 15-20 nucleotides, more preferably 17-25 nucleotides and even more preferably 17-22 nucleotides. The inventors have constructed the probes to be about 20 nucleotides long. If means are included to increase a probe's binding affinity, the probe can be shorter, as short as seven nucleotides, as persons in the art will appreciate. A fluorophore can be attached to a probe at any position, including, without limitation, attaching a fluorophore to one end of a probe, preferably to the 3' end. The probes may be included in a hybridization solution that contains the multiple probes in excess, commonly in the range of 0.2-1 nanograms per microliter. Sufficient solution is added to cover and wet the cell so that the cell is immersed in the probe-containing solution.

[0009] A single cell can be probed simultaneously for multiple mRNA target sequences, either more than one target sequence of one mRNA molecule, or one or more sequences of different mRNA molecules. Additionally, one target sequence of an mRNA molecule can be probed with more than one set of probes, wherein each set is labeled with a distinguishable fluorophore, and the fluorophores are distinguishable. For example, in probing a gene sequence, at least 30 green-labeled probes can be used to probe one portion of the gene sequence as its target sequence, and at least 30 red-labeled probes can be used to probe a different portion of the gene sequence as its target sequence. Using more than one color for each of multiple targets permits use of color-coding schemes in highly multiplexed probing methods according to this disclosure.

[0010] Methods of this disclosure may include simply looking to see if one or more spots representing a target sequence are present. Methods according to this disclosure also include counting spots of a given color corresponding to a given mRNA species. When it is desired to detect more than one species of mRNA, different sets of probes labeled with distinct fluorophores can be used in the same hybridization mixture. A gene expression profile for each species of mRNA is constructed by counting spots of different colors.

[0011] Spots can be detected utilizing microscopic methods. It is not necessary to use a confocal microscope, as a wide-field fluorescence microscope is sufficient. To distinguish spots that positively reflect a target sequence from dim spots that may reflect background fluorescence or nonspecific binding, methods according to this disclosure include detection. In one embodiment, the detection comprises filtering images with a three-dimensional linear Laplacian of Gaussian filter and applying a detection threshold. If one plots the number of spots in three dimensions for all thresholds ranging from zero to the maximum pixel intensity in the filtered image, there is a wide plateau, indicative of a region in which the number of spots detected is insensitive to threshold. Thus, the method further comprises plotting the number of spots, determining the boundaries of a plateau region, and selecting the threshold preferably within that region.

[0012] In another aspect, this invention includes sets of probes for in situ hybridization that enable detection of individual mRNA molecules in cells. The probes render each molecule so intensely fluorescent that it can be seen as a fine fluorescent spot in fluorescence microscopy.

[0013] A computer program can be used to identify and count all the mRNA molecules in the cell from the microscopic image. In situ hybridizations performed with the sets of probes described above allow accurate and simple gene expression analysis, detection of pathogens and pathogenic states such as cancer.

[0014] Accordingly, in another aspect, provided is a method of screening for compounds which alter the amount of a subcellular distribution of the target sequence. The method includes incubating a cell with a test compound for a period of time sufficient to elicit a response, detecting the amount of distribution pattern of the target sequence, and comparing this amount or distribution with an amount or distribution of the target mRNA in a control cell which was treated identically, but not incubated with the test compound.

[0015] In yet another aspect, the disclosure provides a computer readable medium, comprising instructions for: obtaining a 3-D stack of 2-D fluorescent images; filtering said 3-D stack using a 3-D filter; counting a total number of 3-D spots in said filtered 3-D stack for each of a plurality of intensity thresholds; obtaining an optimum intensity threshold representative of a plateau region in a plot of said total number of 3-D spots verses the intensity threshold at which said total number was counted; and using the total number of 3-D spots obtained at said optimum threshold as representative of a number of fluorescing particles detected in said 3-D stack.

[0016] The invention also provides a kit, generally comprising the set of probes and the computer-readable media as described above.

Brief description of the drawings



[0017] 

Figure 1 shows simultaneous detection of a unique sequence and a repeated sequence in individual mRNA molecules. FIG. 1A is a schematic depiction of the construct used. The 48 probes used to detect the GFP coding sequence were labeled with Alexa-594 and the four different probes used to detect the tandem repeat in the 3'-UTR were labeled with TMR. FIG. 1B illustrates maximum intensity merges of a pair of z-stack of fluorescent images of CHO cells taken in the Alexa-594 channel (left) and the TMR channel (right) corresponding to GFP coding region probes and UTR probes, respectively. FIG. 1C illustrates false color merge of the images in FIG. 1B enclosed by the red (GFP) and green (UTR) squares, with red circles representing computationally identified GFP mRNA particles, green circles representing UTR particles, and yellow circles representing co-localized particles. All scale bars are 5 µm long.

Figure 2 shows intensity analysis of colocalized spots. Spot intensities corresponding to the GFP-targeted probes (Alexa 594 channel, y axis) and multimeric UTR-targeted probes (TMR channel, x axis) were computed by taking the maximum intensity in the computationally identified spot region and subtracting the mean intensity of an annular region surrounding the spot. Marginal histograms show the distributions of GFP spot intensities (right) and UTR spot intensities (top).

Figure 3 shows sensitivity of method when using different numbers of probes. FIG. 3A illustrates spot intensity (defined as maximum intensity within the spot minus the mean background taken in an annular region surrounding the spot) as a function of the number of probes chosen. Intensities for 12 and 24 probes are artifactual in that spots were not readily identifiable in those cases, so spots identified were biased towards being brighter. FIG. 3B illustrates the number of spots (i.e., connected components) found upon thresholding the filtered image plotted as a function of the threshold value, ranging from 0 to the maximum intensity of the filtered image (normalized to 1) for different numbers of probes. The grey bar indicates the threshold used for the analysis in FIG 3A.

Figure 4 shows comparison with the mRNA detection method of Femino et al. (Science 1998). FIG. 4A is a schematic depicting the method described in this manuscript with 48 singly labeled probes (left) and the method of Femino et al. in which each 45 bp probe contains five fluorophores each and is targeted to a sequence element that is repeated 32 times in the 3'UTR of the target mRNA expressed from a transgene in Chinese hamster ovary cells. FIG. 3B illustrates a comparison of spot intensities when using 48 singly labeled probes or using a 45 bp probe labeled with five fluorophores. Error bars represent one standard deviation.

Figure 5 shows computational identification of mRNA spots. FIG. 5A illustrates raw image data (maximum intensity merge) obtained from imaging FKBP5 mRNA particles in A549 cells induced with dexamethasone. FIG. 5B illustrates mage (maximum merge) obtained by running raw data through Laplacian of a Gaussian filter to enhance spots. FIG. 5C illustrates the number of spots (i.e., connected components) found upon thresholding the filtered image from FIG. 5B is plotted as a function of the threshold value, ranging from 0 to the maximum intensity of the filtered image (normalized to 1). FIG. 5D is an image showing the results of using the threshold represented by the grey line in FIG. 5C with each distinct spot assigned a random color. All scale bars are 5
µm long.

Figure 6 shows simultaneously imaging single molecules of three different mRNAs in mammalian cells. FIG. 6A- FIG. 6C illustrate images showing FLJ11127, Cox-2 and FKBP5 mRNA particles in the same set of A549 cells not treated with dexamethasone. FIG. 6D- FIG. 6F illustrate images showing FLJ11127, Cox-2 and FKBP5 particles in cells treated for 8 hours with 24nM dexamethasone. FIG. 6G illustrates fold induction for all three genes as measured by FISH and realtime RT-PCR; error bars for FISH were obtained by bootstrapping and those for RTPCR were obtained by repetition as described in the supplementary information. All images are maximum merges of a z-stack of fluorescent images spanning the extent of the cells with nuclear DAPI counterstaining in purple, and all scale bars are 5 µm long.

Figure 7 shows examination of fluorescent spot bleedthrough. FIG. 7A is an illustration of images of an FLJ11127 mRNA spot labeled with TMR as seen through the TMR, Alexa 594 and Cy5 filter channels. Linescans of fluorescent intensity corresponding to the line through the image are given below, with the different linescans corresponding to measurements taken at increasing z (0.25 µm spacing). The green linescan corresponds to the z-slice shown in the image itself. A similar analysis was performed for a Cox-2 mRNA spot labeled with Alexa 594 (FIG. 7B) and an FKBP5 mRNA particle labeled with Cy5 (FIG. 7C). All linescan intensity measurements had the camera background subtracted but range between 0 and 200 arbitrary fluorescence units.

Figure 8 shows demonstration that the oxygen-scavenger increases photostability of Cy5. FIG. 8A illustrates the mean of the maximum spot fluorescence for a number of FLJ11127 mRNAs labeled using TMR conjugated probes was plotted as a function of the number of 2 second exposures using a filter specific for TMR. Curves were generated for images taken both with (blue) and without (red) the oxygen scavenging system. A similar analysis was performed for Cox-2 mRNAs labeled using Alexa-594 conjugated probes with 2 second exposures (FIG. 8B) and FKBP5 mRNAs labeled using Cy5 conjugated probes with 2.5 second exposures (FIG. 8C). FIG. 8D illustrates the bleach rate per exposure (in units of fraction of fluorescence lost per exposure) for the TMR, Alexa-594 and Cy5 conjugates probes in (FIG. 8A-FIG. 8C) both with and without the oxygen-scavenging anti-bleach system. The bleach rate was calculated by fitting each individual particle's decay curve to an exponential and taking the mean of the fitted decay constants. The error bars correspond to one standard deviation. A minimum of 6 particles were chosen in each condition.

Figure 9 shows imaging localized mRNAs in C. elegans and D. melanogaster. FIG. 9A is an illustration of elt-2 mRNA molecules (red) in an early stage embryo (∼100 cell stage) from C. elegans; the nuclei have been counterstained with DAPI (blue). FIG. 9B is an illustration of elt-2 mRNA molecules in an L1 larva from C. elegans. Inside the blue box, a single focal plane is shown in which the intestinal track is visible. FIG. 9C illustrates a schematic depiction of dpp and engrailed expression in the imaginal wing discs of third instar larvae from D. melanogaster. FIG. 9D is an image showing the locations of the computationally identified dpp mRNA molecules (light blue circles) and Engrailed expression detected by immunofluorescence (dark blue). FIG. 9E is an image containing enhanced dpp mRNA molecule signals (light blue) and Engrailed protein expression detected by immunofluorescence (dark blue). All images except the boxed portion of FIG. 9B are maximum merges of a z-stack of fluorescent images, and all scale bars are 5 µm long.

Figure 10 shows imaging single mRNA molecules in yeast and neurons. FIG. 10A and FIG. 10B illustrate STL1 mRNA particles in both unperturbed cells (FIG. 10A) and cells subjected to a 10 minute 0.4M NaCl salt shock, with nuclear DAPI counterstaining in purple (FIG. 10B). FIG. 10C illustrates expression of β-actin (green) and Map2 (red) mRNAs in rat hippocampus neurons in a dissociated neuron culture. FIG. 10D illustrates an enlarged and contrasted image of a segment of a dendrite enclosed by the red box in FIG. 10C. All scale bars are 5 µm long.

Figure 11 is an illustration of target sequences and probes used in the instant invention.


Detailed description of the invention



[0018] This invention relates in part to the development of an image analysis algorithm that utilizes a principled thresholding strategy and shows that we can accurately and unambiguously identify and count all the target mRNA molecules present in the cell. The simplicity and robustness of this approach permits reliable detection of three different mRNA species within the same cells. Using a rigorous set of criteria the inventors have demonstrated that the method allows extremely specific single mRNA imaging across a wide spectrum of cell types and model organisms.

[0019] The inventors have taken advantage of the availability of 96 well DNA synthesizers to synthesize many different terminally labeled smaller probes for the same target. The obtained results show that when a set of at least 30, preferably at least 40, more preferably, about 48 (half of a 96-well plate that is used for high throughput DNA synthesis) or more singly labeled probes bind to the same mRNA molecule, they render it sufficiently fluorescent that it becomes visible as a diffraction-limited spot in wide-field microscopy. The non-specific sites only associate with one or a few probes, yielding diffused signals, whereas the legitimate targets bind to all or most of the probes yielding a clearly detectable spot for each mRNA molecule.

[0020] The inventors have also developed an image analysis algorithm that utilizes a principled thresholding strategy and shows that it is possible to accurately and unambiguously identify and count the all target mRNA molecules present in the cell. The simplicity and robustness of this approach permits reliable detection of three different mRNA species within the same cells. Using a rigorous set of criteria the inventors demonstrate that the method allows extremely specific single mRNA imaging across a wide spectrum of cell types and model organisms.

[0021] Thus, 48 or more singly labeled oligonucleotide probes allow the detection of individual mRNA molecules. The mRNA molecules were visualized as diffraction limited spots that can be easily detected in a standard wide-field microscopic set up. The spots were bright enough to be accurately counted with the spot detection image processing algorithm of the instant invention. The inventors obtained quantitative counts of three different species of mRNA molecules within individual cells. Such analysis facilitates accurate multiplex gene expression profiling of even lowly expressed genes across a host of model organisms.

[0022] The basis of specificity of the instantly disclosed system is that most or all of the probes bind to the intended target mRNA and yield a particulate signal whereas the non-specific binding sites elsewhere in the cell associate with fewer probe molecules and give a diffused signal that the spot counting algorithm ignores. This highlights a key advantage of the instant method over other in situ hybridization methods that use heavily labeled probes such as dendrimers. If every probe molecule is detectable, each non-specific binding event will result in a false positive and any mRNA to which the probe does not bind will result in a false negative. The likelihood of false negatives and positives decreases, however, as the number of probes is increased, and in general, given a certain efficiency of hybridization, increasing the number of different probes will narrow the distribution of probes bound per molecule. The image analysis according to the instant invention showed that increasing the number of the probes resulted in robust spot detection that does not depend on arbitrarily chosen thresholds. This is crucial for accurately counting the number of mRNAs per cell, which is a key feature of the method of the invention.

[0023] In a related point, a potential factor in the design of the probe set is uniformity in hybridization affinities. Since oligonucleotide affinity is largely dominated by its relative GC content, the inventors have created a computer program to design a set of probes with optimally uniform overall GC content. This computer program is publicly available.

[0024] From a practical standpoint, the instantly claimed method also yields significant benefits over previous single molecule mRNA FISH method both in terms of time and cost. Due to advances in synthesis, researchers can easily and cheaply purchase large numbers of oligonucleotides with 3' amine modifiers. These can then be pooled, coupled, and purified en-masse, significantly reducing the labor associated with the multiple couplings and purifications required to generate multiply labeled probe. The resulting simplicity and cost-effectiveness of the instant method will facilitate genomics-scale studies involving the detection of many different mRNAs. Furthermore, the flexibility of the hybridization procedure allows for it to be combined with other standard techniques, such as immunofluorescence.

[0025] In another embodiment, the fluorophores can be incorporated into the probes during automated DNA synthesis.

[0026] Other methods for quantifying the number of mRNAs in individual cells include single-cell RT-PCR and digital RT-PCR. One problem with these methods is the practical difficulties associated with assembling large numbers of individual reactions that require the use of microfluidic or robotic devices. Moreover, those methods suffer from concerns about stochastic variations in exponential amplification when the target inputs are just a few molecules. Such stochastic behavior complicates the analysis of single cell gene expression, which itself is subjected to stochastic forces. Moreover, these methods do not provide any information about the spatial location of the mRNAs.

[0027] Given the simplicity and broad applicability of our single-molecule mRNA detection method, such method is suitable for a variety of studies. By obtaining exact mRNA counts in individual cells, one can make accurate determinations of both expression differences in different conditions and the cell-to-cell variability in gene expression. By yielding quantitative, spatial measurements of individual mRNAs in single cells, this method is valuable in many studies in systems biology, cell biology, neurobiology and developmental biology.

[0028] Accordingly, this method may be utilized for multiple assays, including, without limitation a screening assay. In one embodiment, the screening assay determines whether a test compound affects an amount of a distribution of a target sequence of messenger ribonucleic acid molecules (mRNA's) said target sequence including at least 30 non-overlapping probe binding regions of 15-100 nucleotides in a cell. The assay generally comprises the following steps: incubating a cell with a test compound for a period of time sufficient to elicit a response; permeabilizing the cell; immersing said cell in an excess of at least 30 nucleic acid hybridization probes, each singly labeled with the same fluorescent label and each containing a nucleic acid sequence that is complementary to a different probe binding region of said target sequence; washing said fixed cell to remove unbound probes detecting an amount of a distribution of fluorescence from said probes, comparing said amount or said distribution with an amount of a distribution, respectively, obtained from a control cell, treated as described above, but with the exception of being incubated with the test compound.

[0029] Suitable test compound candidates include, without limitation, peptide-based compounds (e.g., antibodies or nanobodies), RNA interference agents (i.e., siRNA, shRNA, miRNA etc), and small molecules. All these compounds may be made according to the methods known in the art. For example Naito (US 20080113351) and Khvorova (US 20070031844) provide methods of selecting active RNA interference compounds. Antibodies may also be prepared by known techniques including the use of hybridomas, selection of monoclonal antibodies, use of phage display libraries, antibody humanization and the like.

[0030] Small molecule compounds may be selected from screening of the appropriate libraries. In one aspect, small molecule libraries are synthesized according to methods well known and routinely practiced in the art. See, for example, Thompson and Ellman, Chem. Rev. 1996, 96, 555-600, Shipps, et al., Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 11833-11838, October 1997, and Combinatorial Library Design and EvaluationPrinciples, Software Tools and Applications in Drug Discovery, Ghose and Viswanadhan (eds), Marcel Dekker 2001. Alternatively, small libraries are obtained from any of a number of sources including, for example, the NIH Molecular Libraries Small Molecule Repository. Alternative sources include AnalytiCon Discovery GmbH (Potsdam, Germany) which makes available MEGAbolite®, pure natural product small molecule libraries and NatDiverse™, semi-synthetic natural product analogue small molecule libraries; Quantum Pharmaceuticals Ltd. (Moscow, Russian Federation); and Praecis Pharmaceuticals Incorporated (Waltham, Mass.).

[0031] In yet another aspect, the invention provides software implementing the thresholding algorithm as described above. Thus, in one embodiment, provided is a computer readable medium, comprising instructions for: obtaining a 3-D stack of 2-D fluorescent images; filtering said 3-D stack using a 3-D filter; counting a total number of 3-D spots in said filtered 3-D stack for each of a plurality of intensity thresholds; obtaining an optimum intensity threshold representative of a plateau region in a plot of said total number of 3-D spots verses the intensity threshold at which said total number was counted; and using the total number of 3-D spots obtained at said optimum threshold as representative of a number of fluorescing particles detected in said 3-D stack.

[0032] In one embodiment, the thresholding is accomplished using three dimensional linear Laplacian of Gaussian filter.

[0033] In another aspect, a kit is provided. The kit comprises a computer-readable media implementing the thresholding algorithm, as described above, and a set of probes against a pre-selected target sequence. The probes described in connection with the claimed method are also suitable for the instant kit.

[0034] Specific embodiments according to the methods of the present disclosure will now be described in the following examples. The examples are illustrative only, and are not intended to limit the remainder of the disclosure in any way.

EXAMPLES


Example 1: Materials and Methods



[0035] The procedures described in this section are applicable to all examples unless indicated otherwise.

Probe Design



[0036] The sets of probes were designed to consist of at least 48 oligonucleotides each with lengths varying from 17 to 22 nucleotides long with a 3'-amine modification (FKBP5, FLJ11127, and Map2 mRNAs were probed using 63, 53 and 72 oligonucleotides respectively). Additionally, the GC content of the oligonucleotides was kept close to 45% when possible. The oligonucleotides were pooled and coupled to a fluorophore in a single reaction, after which the uncoupled oligonucleotides and remaining free fluorophores were removed by HPLC purification.

Fluorescence in situ hybridization



[0037] In preparation for FISH, all samples were fixed with 3.7% formaldehyde and permeabilized with ethanol. The hybridization was performed using buffers and conditions similar to those outlined by Femino et al., with the key difference being the stringency of the hybridization, which was lowered by reducing the amount of formamide used to 10%. The concentration of the probe that gave optimal signal was determined empirically.

Imaging and data analysis



[0038] All images were acquired using a standard wide-field fluorescence microscope. Computer-aided detection and counting of particles was performed with linear filters designed for enhancing particulate signals.

Example 2: Probing repeated and unique sequences present in the same mRNA molecule



[0039] Utilizing small oligonucleotide probes labeled with a single fluorophore moiety, the inventors have shown that individual mRNA molecules that were engineered to contain 32-96 tandem copies of a probe-binding sequence can be detected by in situ hybridization. The inventors also demonstrated that the individual spots in the image represent single mRNA molecules, utilizing a number of different approaches, including correlating the average mRNA copy number obtained by directly counting the diffraction-limited spots to a measurement of the number of target molecules obtained by real-time RT-PCR. Thus, if many different probes are utilized, each targeted to a distinct region of a natural mRNA, it would be possible to obtain single-molecule sensitivity without resorting to the use of engineered genes.

[0040] For the initial test of this hypothesis, the inventors constructed a doxycycline-controlled gene that produced an mRNA encoding green fluorescent protein and possessed 32 tandemly repeated 80 nucleotide-long sequences in its 3 '-UTR; and then this engineered gene was stably integrated into the genome of a Chinese hamster ovary cell line. The mRNA expressed from this gene was probed simultaneously with 48 different oligonucleotides, each complementary to a unique region in the coding sequence, and a set of four oligonucleotides, each having a complementary sequence in the repeated motif (a total of 128 probes bound) (FIG. 1A). Each oligonucleotide in the probe set that was specific for the coding sequence was labeled with a single Alexa-594 fluorophore, and each oligonucleotide in the set specific for the repeat sequence was labeled with a single tetramethylrhodamine (TMR) fluorophore. The use of appropriate filter sets ensured that the fluorescence emitted from TMR fluorophores was not detected in the Alexa-594 channel and vice versa, as described below.

[0041] After performing FISH with these probes, the inventors have found that many "particles" with a diameter of about 0.25 micrometers were visible in both the TMR and Alexa-594 channels (FIG. 1B). The particles were identified computationally using an image processing program (described in the next section) that categorizes the particles as being labeled with either the GFP-coding-sequence probes (TMR), the UTR-specific probes (Alexa-594), or both (FIG. 1C). Upon identifying and localizing particles in four fields of view similar to the ones shown in Figure 1c, a total of 599 particles corresponding to GFP-coding sequence-specific probes and 565 particles corresponding to the UTR-specific probes were counted. Of these particles, 85% of the "UTR particles" co-localized with the "GFP particles," whereas 81% of the GFP particles co-localized with the UTR particles. The high degree of co-localization between particles detected by the previously established tandem repeat detection method and the particles detected via simultaneous probing with 48 different singly-labeled oligonucleotides demonstrates the validity of using multiple single-labeled probes for the detection of endogenous transcripts. The fraction of particles that did not display co-localization likely correspond to mRNA molecules that lost either their coding sequence or their 3'-UTR in the natural processes of mRNA degradation.

[0042] The inventors also analyzed the fluorescent intensity of the co-localized spots in both the TMR and Alexa-594 channel and found that the spot intensities displayed a unimodal distribution (FIG. 2), arguing that the particles detected are not clumps of many mRNAs but rather individual molecules. The spot intensities displayed a strong correlation between the two channels (FIG. 3). Since there is no cross talk between the two channels, this indicates that the variability in spot intensity was not primarily due to random variability in probe hybridization (which would be uncorrelated between different probe sets) but rather other factors, such as mRNA integrity or accessibility, that affect both probes equally.

[0043] The inventors also explored how the signal intensity would vary with the number of probes by performing in situ hybridization using either first 12, 24, 36 probes or all 48 probes in the set. For this particular target mRNA, it was found that particles could be detected with fewer numbers of probes, albeit with decreased intensity (FIG. 3A). However, the automatic spot detection algorithm (described in details below) performed particularly well with 48 probes, detecting the same number of spots over a broad range of thresholds (FIG. 3B, see further discussion below). The number of probes required for robust signal is likely to depend on the target sequence, though, as the inventors have obtained clear mRNA signals using as few as 30 probes. When the instant method was compared to the method of Femino et al. by using a 45 bp long oligonucleotide labeled with 5 fluorophores and complementary to a sequence repeated 32 times in the 3'UTR of a gene, potentially yielding 160 fluorophores per mRNA (FIG. 4A), it was found that the signal to background were about the same in both methods (FIG. 4B), indicating that the instantly claimed method is at least as sensitive despite using fewer fluorophores.

[0044] Moreover, CHO cells lacking the reporter gene yielded no signals while CHO cells having the reporter gene that was turned off by addition of doxycycline, yielded mRNA particles in only a few cells, indicating that the signals observed were specific.

Example 3: Computational algorithm for spot detection



[0045] In order to reliably identify large numbers of mRNA molecules, the inventors developed a semiautomated computational algorithm for finding spots in a three-dimensional stack of fluorescent images. One of the difficulties associated with spot detection is the nonuniform background arising from cellular autofluoresence and low levels of non-specific probe hybridization. To circumvent these issues, the inventors filtered image stacks using a three dimensional linear Laplacian of Gaussian filter designed to enhance spot-like signals of the correct size and shape (FIG. 5A and FIG. 5B) while removing the slowly varying background. In the next step in the algorithm, the inventors applied a threshold to the filtered image in order to define the spots. In order to make a rational choice of threshold, the number of spots in three dimensions for all thresholds ranging from zero to the maximum pixel intensity in the filtered image was counted. When the inventors plotted the number of particles as a function of the threshold, a wide plateau was found, indicating that there is a region over which the number of particles detected is fairly insensitive to the particular threshold chosen (FIG. 5C). When a threshold in this region is chosen, the spots detected correspond very well with those identified by eye, demonstrating the efficacy of the spot detection algorithm (FIG. 5D).

Example 4: Gene expression profiling of three different mRNA species



[0046] A potential use of the instantly claimed method is the simultaneous detection of single molecules of multiple mRNAs in individual cells. To demonstrate this capability, the inventors designed probes specific to three mRNAs encoding FK506 binding protein 5 (FKBP5), Cox-2 and FLJ11127 in the human carcinoma cell line A549. These probes were coupled to the spectrally distinct fluorophores Cy5, Alexa 594 and TMR, respectively. Upon performing FISH with all three probes simultaneously, individual spots were visible in the three different fluorescence channels (FIG. 6A-FIG. 6F); an intensity analysis showed that fluorescent spots did not bleed through into other channels (FIG. 7).

[0047] To demonstrate that the claimed method of mRNA detection was specific and quantitative, the cells were incubated with the cell-permeable glucocorticoid dexamethasone, thus upregulating the expression of FKBP5 and FLJ1127 while mildly downregulating the expression of Cox-2 in this particular cell-line. The inventors found that the mean number of FKBP5 and FLJ1127 mRNAs measured by combining FISH with the instantly disclosed spot detection algorithm increased while the mean number of Cox-2 mRNAs decreased (compare FIG. 6A-FIG. 6C to FIG. 6D- FIG. 6F). These numbers corresponded well to RT-PCR measurements of the fold induction and repression of these genes performed on the same samples, demonstrating that the fluorescent spots are the appropriate mRNAs and that a majority of the mRNA molecules (FIG. 6G) was detected using the instantly claimed methods. Moreover, this further demonstrates the effectiveness of the spot detection method for accurate gene expression quantification.

[0048] One technical challenge that arose in imaging multiple mRNAs simultaneously was fluorophore photolability, particularly in the case of Cy5. In order to image all of the mRNA molecules within a single cell, 10 to 30 "z-section" images for each visual field were acquired, utilizing a one-to-three second exposure for each image and a high numerical aperture objective. Only TMR and (to a lesser extent) Alexa-594 could withstand this intense and relatively prolonged exposure to light; Cy5, for instance, proved extremely photolabile under these conditions (FIG. 8). To overcome this problem, the inventors employed a special mounting medium in which fluorophores are much more photostable. This method was adapted from Yildiz et al. with minor modifications. In this medium, a mixture of catalase, glucose oxidase, and glucose enzymatically removes molecular oxygen from the medium, thereby inhibiting oxygen-dependent, light-initiated pathways that destroy fluorophores. The use of these enzymes lead to a dramatic 10-fold enhancement of Cy5 photostability while not adversely affecting the imaging of TMR and Alexa-594, thus facilitating the acquisition of multiple z-sections when performing three color imaging.

Example 5: mRNA detection in model organisms and cell types



[0049] One of the canonical uses for in situ hybridization has been for the detection of mRNA localization during development. The inventors tested the instantly claimed method for efficacy in two commonly studied developmental systems: the nematode, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster. In the nematode, the inventors constructed probes to detect mRNA molecules from the gene elt-2, a transcription factor that is expressed only in the nematode gut, and only after the nematode embryo has developed to the 45-cell stage. After hybridization of the probe set to both embryos and larvae, it was found that elt-2 mRNA molecules were present only within the gut region (FIG. 9A) of both the embryos and the larvae (FIG. 9B). However, consistent with the known timing of the onset of expression, elt-2 mRNAs were only detected in the gut of embryos older than the 45-cell stage, again highlighting the specificity of the instantly claimed method. Furthermore, at those early stages, only a few transcripts were detected, showing that this method is sensitive enough to detect even small numbers of transcripts in complex tissues.

[0050] In the fruit fly, one of the most well-studied examples of the localization of gene expression occurs in wing imaginal disc development. The wing discs of fruit fly larvae display a remarkable set of gene expression patterns, one of which is the formation of a stripe of expression of the gene dpp in response to gradients of the proteins Hedgehog and Engrailed. In particular, Engrailed, which negatively regulates dpp mRNA synthesis, is high in the posterior compartment of the wing disc and low in the anterior compartment of the wing disc. Similarly, Hedgehog, which positively regulates dpp mRNA synthesis, is high in the posterior compartment of the wing disc and low in the anterior compartment of the wing disc. However, there is a region between the posterior and the anterior where the levels of Hedgehog is high enough to activate dpp but not high enough to activate engrailed, resulting in the synthesis of dpp mRNA in a narrow stripe (FIG. 9C).

[0051] To check whether this narrow stripe of dpp mRNA synthesis can be imaged, the inventors constructed a set of singly labeled probes against dpp mRNA and performed in situ hybridization on imaginal wing discs isolated from third-instar larvae. Moreover, this in situ procedure was combined with immunofluorescence against Engrailed protein (shown in blue). FIG. 9D shows a full image, in which the locations of the algorithmically identified mRNA molecules are presented as blue circles; and FIG. 9E shows an enlarged portion of the image with enhanced mRNA signals. The images show that mRNA molecules were found only at the anterior edge of the area of Engrailed expression, again confirming the specificity of detection.

[0052] The inventors also tested the instantly claimed method in Saccharomyces cerevisae by designing a set of probe to target transcripts from the gene STL1. STL1 is one among a number of yeast genes whose expression is significantly up-regulated by the addition of salt to the growth medium. It was found that non-shocked cells contain virtually no STL1 mRNA molecules (FIG. 10A), while cells subjected to a ten minute 0.4 M salt shock possessed a large numbers of STL1 mRNA molecules (FIG. 10B).

[0053] Another cell type in which mRNA localization is commonly studied is neurons. To show efficacy of the instantly claimed method in that system the inventors imaged β-actin mRNA and Map2 mRNA in cultured hippocampal neurons. FIG. 10C shows that a β-actin probe set (labeled with TMR) and a differently colored Map2 probe set (labeled with Alexa-594) can be used to image and distinguish their targets with a single molecule resolution. A fraction of these mRNAs migrate to distant reaches of dendrites (FIG. 10D). Particle counts indicated that 14% of the 791 β-actin mRNA molecules were located in dendrites, whereas 37% of the 140 Map2 mRNA molecules were located in the dendrites, which is similar to the previously reported distributions.

References



[0054] 
  1. 1. Kaufmann, B.B. & van Oudenaarden, A. Stochastic gene expression: from single molecules to the proteome. Curr Opin Genet Dev 17, 107-112 (2007).
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  6. 6. Raap, A.K. et al. Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum Mol Genet 4, 529-534 (1995).
  7. 7. Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA transcripts in situ. Science 280, 585-590 (1998).
  8. 8. Tsokas, P. et al. Local protein synthesis mediates a rapid increase in dendritic elongation factor 1A after induction of late long-term potentiation. J Neurosci 25, 5833-5843 (2005).
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  11. 11. Randolph, J.B. & Waggoner, A.S. Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes. Nucleic Acids Res 25, 2923-2929 (1997).
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  17. 17. Wang, J.C. et al. Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes. Proc Natl Acad Sci U S A 101, 15603-15608 (2004).
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  22. 22. Sanicola, M., Sekelsky, J., Elson, S. & Gelbart, W.M. Drawing a stripe in Drosophila imaginal disks: negative regulation of decapentaplegic and patched expression by engrailed. Genetics 139, 745-756 (1995).
  23. 23. Rep, M., Krantz, M., Thevelein, J.M. & Hohmann, S. The transcriptional response of Saccharomyces cerevisiae to osmotic shock. Hot1p and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathwaydependent genes. J Biol Chem 275, 8290-8300 (2000).
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SEQUENCE LISTING



[0055] 

<110> University of Medicine and Dentistry of New Jersey

<120> Imaging Individual mRNA Molecules Using Multiple Singly Labeled Probes

<130> 96738.00072

<150> 61/191,724
<151> 2008-09-10

<160> 482

<170> PatentIn version 3.5

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gcctcggtga gcagcacagg   20

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cagcctggat ggctacgtac   20

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acagcactgt gttggcatag   20

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tcctgcttgc tgatccacat   20

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cgcaggatgg catgagggac gcaggatggc atgagggagg   40

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atcgcactta tactggtcaa   20

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gtgttgagca gttttctcca   20

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<220>
<223> Probe of COX-2

<400> 66
agtgctgggc aaagaatgca   20

<210> 67
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of COX-2

<400> 67
cgcttatgat ctgtcttgaa   20

<210> 68
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of COX-2

<400> 68
atggcccagc ccgttggtga   20

<210> 69
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of COX-2

<400> 69
gccagagttt caccgtaaat   20

<210> 70
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of COX-2

<400> 70
atccttgaaa aggcgcagtt   20

<210> 71
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 71
gaggatacat ctctccatca   20

<210> 72
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for COX-2

<400> 72
tcatctctgc ctgagtatct   20

<210> 73
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 73
tcagaccagg caccagacca   20

<210> 74
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 74
gttgtgttcc cgcagccaga   20

<210> 75
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 75
tcctgtttaa gcacatcgca   20

<210> 76
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 76
aacaactgct catcacccca   20

<210> 77
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 77
atagtctctc ctatcagtat   20

<210> 78
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for COX-2

<400> 78
ccactcaagt gttgcacata   20

<210> 79
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 79
gttctgggtc aaatttcagt   20

<210> 80
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 80
gtactggaat tgtttgttga   20

<210> 81
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 81
gggtgttaaa ttcagcagca   20

<210> 82
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 82
gttgtatttc tggtcatgaa   20

<210> 83
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 83
ggtaattcca tgttccagca   20

<210> 84
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 84
gcctggtgaa tgattcaaca   20

<210> 85
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for COX-2

<400> 85
caatggaagc ctgtgatact   20

<210> 86
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for COX-2

<400> 86
gcgtttgcgg tactcattaa   20

<210> 87
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for COX-2

<400> 87
cctgtaagtt cttcaaatga   20

<210> 88
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 88
caactctgca gacatttcct   20

<210> 89
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 89
ccacagcatc gatgtcacca   20

<210> 90
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 90
tttctaccag aagggcagga   20

<210> 91
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 91
cttctaccat ggtttcacca   20

<210> 92
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 92
cataagtcct ttcaaggaga   20

<210> 93
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for COX-2

<400> 93
cagtaggcag gagaacatat   20

<210> 94
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for COX-2

<400> 94
aaaacccact tctccaccaa   20

<210> 95
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 95
attgcagatg agagactgaa   20

<210> 96
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 96
ctggaacact gaatgaagta   20

<210> 97
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for COX-2

<400> 97
cttgcattga tggtgactgt   20

<210> 98
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for COX-2

<400> 98
cagttcagtc gaacgttctt   20

<210> 99
<211> 958
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for d2EGFP

<400> 99

<210> 100
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 100
cgcccttgct caccatc   17

<210> 101
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 101
caccccggtg aacagct   17

<210> 102
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 102
agctcgacca ggatggg   17

<210> 103
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 103
ggccgtttac gtcgccg   17

<210> 104
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 104
gccggacacg ctgaact   17

<210> 105
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 105
gtggcatcgc cctcgcc   17

<210> 106
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 106
tcagggtcag cttgccg   17

<210> 107
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 107
gccggtggtg cagatga   17

<210> 108
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 108
ggccagggca cgggcag   17

<210> 109
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 109
tcagggtggt cacgagg   17

<210> 110
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 110
gaagcactgc acgccgt   17

<210> 111
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 111
atgtggtcgg ggtagcg   17

<210> 112
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 112
tgaagaagtc gtgctgc   17

<210> 113
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 113
gccttcgggc atggcgg   17

<210> 114
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 114
atggtgcgct cctggac   17

<210> 115
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 115
tgccgtcgtc cttgaag   17

<210> 116
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 116
ctcggcgcgg gtcttgt   17

<210> 117
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 117
gtgtcgccct cgaactt   17

<210> 118
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 118
gctcgatgcg gttcacc   17

<210> 119
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 119
cttgaagtcg atgccct   17

<210> 120
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 120
cccaggatgt tgccgtc   17

<210> 121
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 121
agttgtactc cagcttg   17

<210> 122
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 122
atagacgttg tggctgt   17

<210> 123
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 123
ttctgcttgt cggccat   17

<210> 124
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 124
agttcacctt gatgccg   17

<210> 125
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 125
gatgttgtgg cggatct   17

<210> 126
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 126
agctgcacgc tgccgtc   17

<210> 127
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 127
tctgctggta gtggtcg   17

<210> 128
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 128
gccgtcgccg atggggg   17

<210> 129
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 129
ttgtcgggca gcagcac   17

<210> 130
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 130
actgggtgct caggtag   17

<210> 131
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 131
ggggtctttg ctcaggg   17

<210> 132
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 132
atgtgatcgc gcttctc   17

<210> 133
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 133
tcacgaactc cagcagg   17

<210> 134
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 134
gagagtgatc ccggcgg   17

<210> 135
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 135
ttgtacagct cgtccat   17

<210> 136
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 136
ggaagccatg gctaagc   17

<210> 137
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 137
ctgctcctcc acctccg   17

<210> 138
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 138
atgggcagcg tgccatc   17

<210> 139
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 139
cgctctcctg ggcacaa   17

<210> 140
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 140
tgcagggtga cggtcca   17

<210> 141
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 141
atcctagcag aagcaca   17

<210> 142
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 142
acgaattcct acacatt   17

<210> 143
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 143
tcaatgtatc ttatcat   17

<210> 144
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 144
ttgtggtttg tccaaac   17

<210> 145
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 145
tttttttcac tgcattc   17

<210> 146
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe of d2EGFP

<400> 146
caaatttcac aaataaa   17

<210> 147
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe of d2EGFP

<400> 147
caaataaagc aatagca   17

<210> 148
<211> 1767
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 148



<210> 149
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 149
ggagtagaag ccatgcgcgc   20

<210> 150
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 150
gtttgaaaag tcgccagcac   20

<210> 151
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 151
cctcggtgct agcaactcga   20

<210> 152
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 152
cgggcgctat ggcggcgatg   20

<210> 153
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 153
ctgatgctga tgccagcgg   19

<210> 154
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 154
gctggtcgag gctcctaccg   20

<210> 155
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 155
tgaacgaggc gggctcgctg   20

<210> 156
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 156
ctccgatggc ttttatcact   20

<210> 157
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 157
cgttgaactg tcggttcgcg   20

<210> 158
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 158
ggtggctcct ctgctccttg   20

<210> 159
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 159
ggctgcgatg gtggtggctc   20

<210> 160
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 160
ctgtggatgc actggcctgc   20

<210> 161
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 161
ccagcgtcgg ctcctccacg   20

<210> 162
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 162
ccttggcgtt ggcgggcacg   20

<210> 163
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 163
ggtggacggg ccctgctcgg   20

<210> 164
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 164
ggaggggtct ggcttcagct   20

<210> 165
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 165
agcgagagca ggctcttctc   20

<210> 166
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 166
gagcggtcga tcttgggcgg   20

<210> 167
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 167
cttcatcggc tcggggatga   20

<210> 168
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 168
tcgtggccca tgatctcggc   20

<210> 169
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 169
gcagacccgg cttggggatg   20

<210> 170
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 170
cgcactgtgt tggccgactt   20

<210> 171
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 171
gcagccgaaa ccggtggtgg   20

<210> 172
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 172
ccgccgcctt cagcttctcg   20

<210> 173
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 173
agtgcgtccc gggtcagctg   20

<210> 174
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 174
ccgacgatct gctggccacc   20

<210> 175
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 175
agcacctggt agcgcgtccg   20

<210> 176
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 176
cacgcacccc gacgcgcgtg   20

<210> 177
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 177
gccggaccgt cttggtgtcc   20

<210> 178
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 178
gaggctcacc gtgtccgtgc   20

<210> 179
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 179
gccagccacc ggtccacggc   20

<210> 180
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 180
cagcagtccg tagttgcgct   20

<210> 181
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 181
gggagcggac cgtccgcacc   20

<210> 182
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 182
caggcgtaca tggtggtgtg   20

<210> 183
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 183
cgtgcgcctc gtccgcgctg   20

<210> 184
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 184
gagcggctgc ttgtgctgcc   20

<210> 185
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 185
ccgcccgtcg tccgtgtagg   20

<210> 186
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 186
gccaccgccc tctccgccag   20

<210> 187
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 187
cgtaggccgt ctcggctgcc   20

<210> 188
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 188
gcgagtgccg ccggcaggtg   20

<210> 189
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 189
cgtcccagcc cacgtccgag   20

<210> 190
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 190
cgtagcccag aggcgccaca   20

<210> 191
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 191
cggccagcgg gaaggggcac   20

<210> 192
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 192
gggtctgcac cacggcgtga   20

<210> 193
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 193
gccttcggca ccttgccggg   20

<210> 194
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 194
gtccagttgc gtgggcacgc   20

<210> 195
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for dpp

<400> 195
gcaccaccgt actttggtcg   20

<210> 196
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for dpp

<400> 196
gccacagccc accacggtca   20

<210> 197
<211> 1302
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 197



<210> 198
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 198
ccatttcggc ccagccgttg   20

<210> 199
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 199
gacctcccat tggttgagat   20

<210> 200
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 200
ccatgttctg agttggtagg   20

<210> 201
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 201
actcattatt ttgctctggt   20

<210> 202
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 202
ttctcggtag ttcactcaat   20

<210> 203
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 203
taacactttg ccgttcaatt   20

<210> 204
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 204
catagttatt tgtgccactt   20

<210> 205
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 205
atgtaacagt ttccactttc   20

<210> 206
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 206
agtctatgcc agtatggaaa   20

<210> 207
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 207
ggtccaacat tccaaagttt   20

<210> 208
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 208
gataaaacgg ttgcatggta   20

<210> 209
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 209
ttacgggaat tccactgtaa   20

<210> 210
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 210
atcccgaaaa agttccaaga   20

<210> 211
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 211
gtttgtcgta tatggagttt   20

<210> 212
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 212
gaatactggg gtcgtacaga   20

<210> 213
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 213
aagtagaagg gatgttaatg   20

<210> 214
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 214
cgtaagttgg agccacagtt   20

<210> 215
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 215
aactttgtga gcatttgacg   20

<210> 216
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 216
ttactgcctt catcccggcc   20

<210> 217
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 217
agttgacgca catcattcct   20

<210> 218
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 218
aatacgtggt ttttggtgtt   20

<210> 219
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 219
aagtgctata cgcgactgga   20

<210> 220
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 220
ccagaatcgg gggttgtccc   20

<210> 221
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 221
cagttggctg ctctgaaggt   20

<210> 222
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 222
tagaggattg cttggcaatt   20

<210> 223
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 223
ccctatttga gctacttgac   20

<210> 224
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 224
gacgggacgc agatccgttt   20

<210> 225
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 225
aattggagca cacaagtccc   20

<210> 226
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 226
agagagttgt gttggtacca   20

<210> 227
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 227
gatctccttc agcatttctt   20

<210> 228
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 228
aaagcccgca agcattgcag   20

<210> 229
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 229
gagggatgtg atggagtttg   20

<210> 230
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 230
cttctttctt cattgaggtc   20

<210> 231
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 231
tggaagagtc tccgcttttt   20

<210> 232
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 232
ctcgggcccg tgacgttgat   20

<210> 233
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 233
aagaggctct ctcaaacttc   20

<210> 234
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 234
acctttgagc cttttcggtc   20

<210> 235
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 235
cacttcccgc acgccggtta   20

<210> 236
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 236
tgctcagttc tcggtctgct   20

<210> 237
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 237
agtcggcatg tgacacatat   20

<210> 238
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 238
cggcagctga ggaaacggga   20

<210> 239
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 239
tgtacgtttg atctggcaag   20

<210> 240
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 240
ccatcatcaa cccagctgta   20

<210> 241
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 241
cagtctggta gtttgttgct   20

<210> 242
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 242
ggatgttatc ggcaggtctt   20

<210> 243
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for elt-2

<400> 243
cctgaactgg catcacatga   20

<210> 244
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 244
gagccgcagc tttggtttca   20

<210> 245
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for elt-2

<400> 245
ctccgtcgac cgcttccaaa   20

<210> 246
<211> 1374
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 246



<210> 247
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 247
gttcttggca ccttcat   17

<210> 248
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 248
aatcatcggc gtttcct   17

<210> 249
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 249
tggacataaa ctttgtc   17

<210> 250
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 250
ttgacaattt tcctttg   17

<210> 251
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 251
ggaatcaaac ttctttc   17

<210> 252
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 252
ggttcatttc tatcatg   17

<210> 253
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 253
tgccaagact aaagaca   17

<210> 254
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 254
tgccttgatg acttggc   17

<210> 255
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 255
gtagccaccc caatgtc   17

<210> 256
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 256
atatctctcc tttcttc   17

<210> 257
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 257
tggtttgcac agtaaat   17

<210> 258
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 258
gccgagccat atgcata   17

<210> 259
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 259
gaattttagg gagactg   17

<210> 260
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 260
aaagagagtt gcattcg   17

<210> 261
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 261
tcaaggagct caatctc   17

<210> 262
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 262
ataaatcctc tcctttg   17

<210> 263
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 263
gataatgcct ccatctt   17

<210> 264
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 264
cctttccgtt tggttct   17

<210> 265
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 265
ttggatttga atatccc   17

<210> 266
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 266
ttctactgtt gctcctt   17

<210> 267
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 267
cagcggcctt ccaggtg   17

<210> 268
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 268
agtcaaacat ccttcca   17

<210> 269
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 269
agtgaatgcc acatctc   17

<210> 270
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 270
tggtcttctc cttcgcc   17

<210> 271
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 271
caattccaat tggaatg   17

<210> 272
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 272
cattttctcc agagctt   17

<210> 273
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 273
atacattgtt cttcccg   17

<210> 274
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 274
atcttggtcc aagatat   17

<210> 275
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 275
ccctgcctct ccaaaac   17

<210> 276
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 276
tcaatgccaa atttagg   17

<210> 277
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 277
atataagctc agcatta   17

<210> 278
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 278
gctcttaagt gtaactt   17

<210> 279
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 279
gattctttgg ccttttc   17

<210> 280
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 280
ctttggtatc catctcc   17

<210> 281
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 281
ggcagcctgc tccaatt   17

<210> 282
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 282
gttcccttct ctttgac   17

<210> 283
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 283
tgcctccctt gaagtat   17

<210> 284
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 284
aatcaccgcc tgcatgt   17

<210> 285
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 285
gacactatct tcccata   17

<210> 286
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 286
catattccat ctctaac   17

<210> 287
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 287
cgattccttt tctgata   17

<210> 288
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 288
agaaatgatt cagaagc   17

<210> 289
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 289
ggttcagaaa ggcagca   17

<210> 290
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 290
cttcaggtag cacatgg   17

<210> 291
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 291
gctttggtgt attctct   17

<210> 292
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 292
ccttgtcaca gcattca   17

<210> 293
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 293
ggcactgtcc agtccaa   17

<210> 294
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 294
ctatacaagc ctttctc   17

<210> 295
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 295
gcagctgggc ttcaccc   17

<210> 296
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 296
tgactcaaac tcgttca   17

<210> 297
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 297
ttctggcaca tggagat   17

<210> 298
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 298
cgttgtgctc cttggcc   17

<210> 299
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 299
gtatatcctg cggtccc   17

<210> 300
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 300
aacttcttga acatgtt   17

<210> 301
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 301
ccttggcatc ctgctct   17

<210> 302
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FKBP5

<400> 302
cattgcttta ttggcct   17

<210> 303
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 303
ccttctgaag tcttctt   17

<210> 304
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 304
ttcctttttc attagtg   17

<210> 305
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 305
ttccattgct tgactgt   17

<210> 306
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FKBP5

<400> 306
stggccctca ggtttctc   18

<210> 307
<211> 1071
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<4> 307

<210> 308
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 308
cccgcgtggg gctcctt   17

<210> 309
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 309
agaccgctcc cgctccc   17

<210> 310
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 310
cttcctgcgg cgggagc   17

<210> 311
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 311
tccaggagtg aacttgg   17

<210> 312
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 312
ggcttggctt gtagcta   17

<210> 313
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 313
attctccaga cagtgtc   17

<210> 314
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 314
acatcacaaa gcctttt   17

<210> 315
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 315
caccagaaat gaaactg   17

<210> 316
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 316
cggaagtagc agatggc   17

<210> 317
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 317
ctgaatataa atgtagc   17

<210> 318
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 318
ccaccatttc agcttgt   17

<210> 319
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 319
tttctctgca gatatcc   17

<210> 320
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 320
cactgaggtt ccttttg   17

<210> 321
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 321
taaatcaacc tctgcct   17

<210> 322
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 322
tctcttgcac aataact   17

<210> 323
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 323
ggggtgtctc tcctttc   17

<210> 324
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 324
cttcctcatc agcttgt   17

<210> 325
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 325
caaaatagct cctcata   17

<210> 326
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 326
cacatttaat gtgatgc   17

<210> 327
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 327
atctctcctt acttgtc   17

<210> 328
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FUJ1127

<400> 328
gatctgagag catcata   17

<210> 329
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FUJ1127

<400> 329
tgaatatctg aaataac   17

<210> 330
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 330
tggaaaagag atgccct   17

<210> 331
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 331
tccttttctt tcatcca   17

<210> 332
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 332
tttcaggaag cttaaca   17

<210> 333
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 333
accttgtgaa aacagca   17

<210> 334
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 334
tactgctgaa tccaatt   17

<210> 335
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 335
acttctcagg accaaaa   17

<210> 336
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 336
aaacacattc gagcctg   17

<210> 337
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 337
acatatttcc gtagttt   17

<210> 338
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 338
actgtgtttt caataat   17

<210> 339
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 339
aatgccatta aattcag   17

<210> 340
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 340
cctctcttgt gataatc   17

<210> 341
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 341
aaagggtgtt gcacata   17

<210> 342
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 342
ttccagaatg gcatctg   17

<210> 343
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 343
aaagcttcat aaagttt   17

<210> 344
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 344
gatacagcat gatgaac   17

<210> 345
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 345
ttcataaact tcagtga   17

<210> 346
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 346
acctttttag tcttcat   17

<210> 347
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 347
gtctaaaaag actggga   17

<210> 348
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 348
tgtctccctg gaaaaca   17

<210> 349
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 349
aagctcaaag gatcaga   17

<210> 350
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 350
aattcaggtg attcatc   17

<210> 351
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 351
tccacatgtg tcgccta   17

<210> 352
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 352
aacatatcaa tctgctc   17

<210> 353
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 353
caagggagta tccaagt   17

<210> 354
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 354
gaacactttt atcttta   17

<210> 355
<211> 17
<212> DNA
<213> Artificial sequence

<220> Probe for FLJ11127

<400> 355
gagttaaact tgaacag   17

<210> 356
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 356
agcagacttc aaagtct   17

<210> 357
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FLJ11127

<400> 357
cctgagaggc tcctctg   17

<210> 358
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 358
agggagatct ccggcca   17

<210> 359
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for FLJ11127

<400> 359
ggcggtcgtt ctcggtc   17

<210> 360
<211> 17
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for FUJ1127

<400> 360
aaagactgga atgtggt   17

<210> 361
<211> 5478
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 361







<210> 362
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 362
tgtggtgcct ttccttcgtc   20

<210> 363
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 363
gctgcctctg tgagtgaggc   20

<210> 364
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 364
tccttcatct ctggcgagtg   20

<210> 365
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 365
cggctcagcc cttcccctga   20

<210> 366
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 366
tcctctctgt atggaaat   18

<210> 367
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 367
gacccgtgct ccccaaaggc   20

<210> 368
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 368
ttctctttgg tatctgaata   20

<210> 369
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 369
tcagctgagg tcagctctcc   20

<210> 370
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 370
cttgcagaca cttcctctgc   20

<210> 371
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 371
acagcttcag ctgtgactac   20

<210> 372
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 372
tccttctctt gttcaccttt   20

<210> 373
<211> 20
<212> DNA
<213> Artificial sequence

<220> Probe for Map2

<400> 373
agagctgcag gctgatcctt   20

<210> 374
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 374
agattaactg tttcttcagc   20

<210> 375
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 375
gatgctggcg atggtggtgg   20

<210> 376
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 376
gaggcttctt ccagtgcagc   20

<210> 377
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 377
aatttctgct gctcagggaa   20

<210> 378
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 378
ttgtctaaag gctcagcgaa   20

<210> 379
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 379
ttactttgca tcttaaactc   20

<210> 380
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 380
ctgaaacttg tacgacggaa   20

<210> 381
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 381
ggagttttac ttgtgtccgg   20

<210> 382
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 382
tccatgtctt ggggatcctt   20

<210> 383
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 383
gcaaatggac tggcaggcga   20

<210> 384
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 384
atgtcttcca ggttggtacc   20

<210> 385
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 385
actgttatgc ttggttctgt   20

<210> 386
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 386
aggggctctg cggagaggcc   20

<210> 387
<211> 20
<212> DNA
<213> Artificial Sequence

<220> Probe for Map2

<400> 387
atgaaccagt ctttctgatc   20

<210> 388
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 388
tcatccttct ttgattccac   20

<210> 389
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 389
ccaggagata ttggggcagc   20

<210> 390
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 390
acatcttttt ccctcatggg   20

<210> 391
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 391
tttccttccc atcttgggat   20

<210> 392
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 392
aaggggctag gcatgggaga   20

<210> 393
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 393
tctaagggaa gagtgaaact   20

<210> 394
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 394
ccttctgtga ctctctcatc   20

<210> 395
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 395
tggaagaaga caggggcaaa   20

<210> 396
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 396
gtgtcctgca gagacatttt   20

<210> 397
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 397
gaactctctt tggaagtagc   20

<210> 398
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 398
gctttatctt tctgtggctc   20

<210> 399
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 399
tctgagacag gaacatctgc   20

<210> 400
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 400
ctgtgaacat ctcccagtac   20

<210> 401
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 401
ttctccccga caaagccttc   20

<210> 402
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 402
tctgtggtac ccttttcttc   20

<210> 403
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 403
ctgggtgtcg aagtctcttt   20

<210> 404
<211> 20
<212> DNA
<213> Artificial Sequence

<220> Probe for Map2

<400> 404
tcagtttcag tgagtgtagg   20

<210> 405
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 405
tttgatgtct cttcaagctt   20

<210> 406
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 406
tcttttgcca cagtttcttc   20

<210> 407
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 407
ttatcatctt ttaatttcaa   20

<210> 408
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 408
tgctcggtgg aagtctgaat   20

<210> 409
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 409
tcgcctttct ggtcttcttt   20

<210> 410
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 410
cagacccagc gccttcgagc   20

<210> 411
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 411
gcccaaccca gtgcttctgg   20

<210> 412
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 412
gctggaggac taggctggga   20

<210> 413
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 413
ccgcccagcc ccaagcttcg   20

<210> 414
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 414
gatcatggcc ccggccaaag   20

<210> 415
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 415
caggtgtggt ggggggcagg   20

<210> 416
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 416
ggctgggaag ggtgactcgg   20

<210> 417
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 417
ggcaggtcag gggccatgac   20

<210> 418
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 418
ctggacctgg tccctgctag   20

<210> 419
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 419
cacagcctcc gatgggactg   20

<210> 420
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 420
gaactggcga tgggagaggg   20

<210> 421
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 421
gccctcatca ggcttcaggg   20

<210> 422
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 422
ccatctggac ctccgccttc   20

<210> 423
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 423
tcaggctgtg gctgggtgac   20

<210> 424
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 424
tctggagggt gtctgagcgg   20

<210> 425
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 425
gctcccctgg gcaggtctcc   20

<210> 426
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 426
agcgcacact gtgggatccc   20

<210> 427
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 427
tccttgggct ctgcctgggc   20

<210> 428
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 428
gctggagcat ctggagagcc   20

<210> 429
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 429
tccacccaca ggctgtcggc   20

<210> 430
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for Map2

<400> 430
gagacggtct ccgagcttcc   20

<210> 431
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 431
tccctggaga ccgtgctagg   20

<210> 432
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 432
cgcactggga gcctgagctg   20

<210> 433
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for Map2

<400> 433
ggaggaaggt cttgggaggg   20

<210> 434
<211> 1710
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 434



<210> 435
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 435
gcctttgaaa ttcgataatt   20

<210> 436
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 436
cagtgactgg ttctgcttat   20

<210> 437
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 437
taccgcaact tcttacccgt   20

<210> 438
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 438
gaagcccgtc atagatgcga   20

<210> 439
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 439
tcaacccttg gtcgtatcca   20

<210> 440
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 440
gttgaactgt ttaccagtaa   20

<210> 441
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 441
tgtctgtcat gatcgccatt   20

<210> 442
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 442
gaacctgcga aacaacctaa   20

<210> 443
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 443
ttcaccgcag aacataacga   20

<210> 444
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 444
tacggaaccc atcaggatta   20

<210> 445
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 445
cacgaaatgc gcatgtagaa   20

<210> 446
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 446
tccgatgata aactggccta   20

<210> 447
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 447
ttctgattgc caaacgggaa   20

<210> 448
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 448
gcaaccctct attttcagct   20

<210> 449
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 449
gcaattgtgg aaccttctaa   20

<210> 450
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 450
caacccaaaa tcaatccaat   20

<210> 451
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 451
cactgaacag aactgttggt   20

<210> 452
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 452
caggaagaga gcaaaaacga   20

<210> 453
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 453
gtggcgattc aggtagttta   20

<210> 454
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 454
cttctgttcg actttgagaa   20

<210> 455
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 455
ctgttataac ttcctcatca   20

<210> 456
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 456
gttaacagca tcgtgaagca   20

<210> 457
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 457
acagtgaatg tttctcgtgt   20

<210> 458
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 458
ggacctgcct ctggagaaca   20

<210> 459
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 459
caatcaaagc cctctgaaga   20

<210> 460
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 460
gcagcgttac aaccagtaaa   20

<210> 461
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 461
gaataataca gtagagtagt   20

<210> 462
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 462
ccccacctat gatcattgat   20

<210> 463
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 463
aatagtagat aaggcgtaga   20

<210> 464
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 464
agcttacgtc tacctagctt   20

<210> 465
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 465
ttgacctgtg gcacctaata   20

<210> 466
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 466
gaccaagcat gcaaatgtaa   20

<210> 467
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 467
gcacctcttg cgttttcttt   20

<210> 468
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 468
ccaaagaatg taatgaacaa   20

<210> 469
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 469
tggtgggtat atccatggta   20

<210> 470
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 470
cgtttgttga tgcacgaact   20

<210> 471
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 471
ccgcaaagtt acacaaccaa   20

<210> 472
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 472
caaccggact gtccaataaa   20

<210> 473
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 473
aattcataac agcaaaaaat   20

<210> 474
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 474
tcagggtaga aaaagaagat   20

<210> 475
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 475
gatgatgtcg atttcctcca   20

<210> 476
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 476
agtgccatcc tcgtatgctt   20

<210> 477
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 477
ggataacttg ggcaaatggt   20

<210> 478
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 478
cgtcataaga gcccaatgca   20

<210> 479
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 479
ccaaagtcct ctttttccat   20

<210> 480
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 480
ttataggtgt cttctactct   20

<210> 481
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Probe for STL1

<400> 481
ctagacgaat tatcgccgtt   20

<210> 482
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Probe for STL1

<400> 482
cgttcactgt atcttcattt   20




Claims

1. A probe set for in situ hybridization that enables detection of individual mRNA molecules in fixed, permeabilized cells, comprising at least 30 non-overlapping nucleic acid hybridization probes singly labelled with the same first fluorophore label of a first color, wherein the probes have sequences complementary to different probe binding regions of a target sequence of an individual mRNA molecule, wherein said probes are 7-30 nucleotides in length.
 
2. The probe set of Claim 1 wherein the probe set comprises 40 to 60 non-overlapping nucleic acid hybridization probes singly labelled with the same first fluorophore label of a first color.
 
3. The probe set of Claim 1 or Claim 2 wherein the probes have target-complementary sequences 15-30 nucleotides in length.
 
4. The probe set of any preceding claim wherein all probes in the set are labelled with a fluorophore at their 3' end.
 
5. A hybridization solution that enables detection of individual mRNA molecules in fixed, permeabilized cells comprising an excess of a first set of at least one probe set wherein the probe set comprises at least 30 non-overlapping nucleic acid hybridization probes singly labelled with the same first fluorophore label of a first color, wherein the probes have sequences complementary to different probe binding regions of a first target sequence of an individual mRNA molecule, wherein said probes are 7-30 nucleotides in length.
 
6. The hybridization solution of Claim 5 wherein the probe set comprises 40-60 non-overlapping nucleic acid hybridization probes singly labelled with the same first fluorophore label of a first color.
 
7. The hybridization solution of Claim 5 or 6 wherein the hybridization solution may comprise a mixture of more than one set of probes labelled where each set is labelled with a distinct fluorophore.
 
8. The hybridization solution of any of Claims 5 to 7, wherein each probe is included in the range of 0.2-1.0 nanograms per microliter.
 
9. A kit that enables detection of individual mRNA molecules in fixed, permeabilized cells comprising:

a) computer readable medium, comprising instructions, which when executed on a computer cause the computer to carry out steps of obtaining a 3-D stack of 2-D fluorescent images; filtering said 3-D stack using a 3-D filter; counting a total number of 3-D spots in said filtered 3-D stack for each of a plurality of intensity thresholds; obtaining an optimum intensity threshold representative of a plateau region in a plot of said total number of 3-D spots verses the intensity threshold at which said total number was counted; using the total number of 3-D spots obtained at said optimum threshold as representative of a number of fluorescing particles detected in said 3-D stack; and

b) the probe set of any one of claims 1-4.


 
10. The kit of Claim 9, wherein said probe set includes at least 30 probes singly labelled with a first fluorescent label and at least 30 probes labelled with a second fluorescent label.
 
11. The kit of Claim 9 or 10, wherein said mRNA molecule includes at least two target regions, and the fluorescent labels for each target region are distinguishable.
 
12. The probe set of any one of claims 1-4, the hybridization solution of any one of claims 5-8, or the kit of any one of claims 9-11, wherein one or more of the probes are DNA probes.
 


Ansprüche

1. Sondensatz für in-situ-Hybridisierung, der den Nachweis von einzelnen mRNA-Moleküle in fixierten permeabilisierten Zellen ermöglicht, der mindestens 30 nicht überlappende Nukleinsäure-Hybridisierungssonden umfasst, die einzeln mit der gleichen ersten Fluorophormarkierung einer ersten Farbe markiert sind, wobei die Sonden Sequenzen aufweisen, die zu anderen Sonden-bindenden Regionen einer Zielsequenz eines einzelnen mRNA-Moleküls komplementär sind, wobei die Sonden eine Länge von 7-30 Nukleotiden aufweisen.
 
2. Sondensatz nach Anspruch 1, wobei der Sondensatz 40 bis 60 nicht überlappende Nukleinsäure-Hybridisierungssonden umfasst, die einzeln mit der gleichen ersten Fluorophormarkierung einer ersten Farbe markiert sind.
 
3. Sondensatz nach Anspruch 1 oder Anspruch 2, wobei die Sonden zum Ziel komplementäre Sequenzen von einer Länge von 15-30 Nukleotiden aufweisen.
 
4. Sondensatz nach einem vorstehenden Anspruch, wobei alle Sonden in dem Satz mit einem Fluorophor an ihrem 3'-Ende markiert sind.
 
5. Hybridisierungslösung, die den Nachweis von einzelnen mRNA-Molekülen in fixierten permeabiliserten Zellen ermöglicht, die einen Überschuss eines ersten Satzes von mindestens einem Sondensatz umfasst, wobei der Sondensatz mindestens 30 nicht überlappende Nukleinsäure-Hybridisierungssonden umfasst, die einzeln mit der gleichen ersten Fluorophormarkierung einer ersten Farbe markiert sind, wobei die Sonden Sequenzen aufweisen, die zu anderen Sonden-bindenden Regionen einer ersten Zielsequenz eines einzelnen mRNA-Moleküls komplementär sind, wobei die Sonden eine Länge von 7-30 Nukleotiden aufweisen.
 
6. Hybridisierungslösung nach Anspruch 5, wobei der Sondensatz 40-60 nicht überlappende Nukleinsäure-Hybridisierungssonden umfasst, die einzeln mit der gleichen ersten Fluorophormarkierung einer ersten Farbe markiert sind.
 
7. Hybridisierungslösung nach Anspruch 5 oder 6, wobei die Hybridisierungslösung eine Mischung von mehr als einem Satz von markierten Sonden umfassen kann, wobei jeder Satz mit einem unterschiedlichen Fluorophor markiert ist.
 
8. Hybridisierungslösung nach einem der Ansprüche 5 bis 7, wobei jede Sonde in dem Bereich von 0,2-1,0 Nanogramm pro Mikroliter eingeschlossen ist.
 
9. Kit, der den Nachweis von einzelnen mRNA-Molekülen in fixierten permeabilisierten Zellen ermöglicht, der Folgendes umfasst:

a) computerlesbares Medium, das Anweisungen umfasst, welche bei Ausführung an einem Computer bewirken, dass der Computer die folgenden Schritte durchführt: Erhalten eines 3-D-Stapels von 2-D-Fluoreszenzbildern; Filtern des 3-D-Stapels unter Verwendung eines 3-D-Filters; Zählen einer Gesamtanzahl von 3-D-Flecken in dem gefilterten 3-D-Stapel für jeden einer Vielzahl von Intensitätsschwellenwerten; Erhalten eines optimalen Intensitätsschwellenwerts, der einen Plateaubereich in einem Diagramm der Gesamtanzahl von 3-D-Flecken gegenüber dem Intensitätsschwellenwert, bei dem die Gesamtanzahl gezählt wurde, repräsentiert; Verwenden der Gesamtanzahl von 3-D-Flecken, die bei dem optimalen Schwellenwert erhalten wurden, als repräsentativ für eine Anzahl von fluoreszierenden Partikeln, die in dem 3-D-Stapel nachgewiesen werden; und

b) den Sondensatz nach einem der Ansprüche 1-4.


 
10. Kit nach Anspruch 9, wobei der Sondensatz mindestens 30 Sonden, die einzeln mit einer ersten Fluoreszenzmarkierung markiert sind, und mindestens 30 Sonden, die mit einer zweiten Fluoreszenzmarkierung markiert sind, einschließt.
 
11. Kit nach Anspruch 9 oder 10, wobei das mRNA-Molekül mindestens zwei Zielregionen einschließt und die Fluoreszenzmarkierungen für jede Zielregion unterscheidbar sind.
 
12. Sondensatz nach einem der Ansprüche 1-4, die Hybridisierungslösung nach einem der Ansprüche 5-8 oder der Kit nach einem der Ansprüche 9-11, wobei eine oder mehrere der Sonden DNA-Sonden sind.
 


Revendications

1. Ensemble de sondes destiné à l'hybridation in situ qui permet la détection de molécules d'ARNm individuelles dans des cellules fixes, perméabilisées, comprenant au moins 30 sondes d'hybridation d'acide nucléique non chevauchantes à marqueur unique avec le même premier marqueur à fluorophore d'une première couleur, dans lequel les sondes comportent des séquences complémentaires à différentes régions de liaison de sonde d'une séquence cible d'une molécule d'ARNm individuelle, dans lequel lesdites sondes ont une longueur comprise dans la plage allant de 7 à 30 nucléotides.
 
2. Ensemble de sondes selon la revendication 1, dans lequel l'ensemble de sondes comprend 40 à 60 sondes d'hybridation d'acide nucléique non chevauchantes à marqueur unique avec le même premier marqueur à fluorophore d'une première couleur.
 
3. Ensemble de sondes selon la revendication 1 ou la revendication 2, dans lequel les sondes possèdent des séquences complémentaires cibles de longueur comprise dans la plage allant de 15 à 30 nucléotides.
 
4. Ensemble de sondes selon l'une quelconque des revendications précédentes, dans lequel toutes les sondes dans l'ensemble sont marquées par un fluorophore au niveau de leur extrémité 3'.
 
5. Solution d'hybridation qui permet la détection de molécules d'ARNm individuelles dans des cellules fixes, perméabilisées comprenant un excès d'un premier ensemble d'au moins un ensemble de sondes, dans laquelle l'ensemble de sondes comprend au moins 30 sondes d'hybridation d'acide nucléique non chevauchantes à marqueur unique avec le même premier marqueur à fluorophore d'une première couleur, dans laquelle les sondes possèdent des séquences complémentaires à différentes régions de liaison de sonde d'une première séquence cible d'une molécule d'ARNm individuelle, dans laquelle lesdites sondes ont une longueur comprise dans la plage de 7 à 30 nucléotides.
 
6. Solution d'hybridation selon la revendication 5, dans laquelle l'ensemble de sondes comprend 40 à 60 sondes d'hybridation d'acide nucléique non chevauchantes à marqueur unique avec le même premier marqueur à fluorophore d'une première couleur.
 
7. Solution d'hybridation selon la revendication 5 ou la revendication 6, dans laquelle la solution d'hybridation peut comprendre un mélange de plus d'un ensemble de sondes marquées où chaque ensemble est marqué par un fluorophore distinct.
 
8. Solution d'hybridation selon l'une quelconque des revendications 5 à 7, dans laquelle chaque sonde est comprise dans la plage allant de 0,2 à 1,0 nanogramme par microlitre.
 
9. Kit qui permet la détection de molécules d'ARNm individuelles dans des cellules fixes, perméabilisées comprenant :

a) un support lisible par ordinateur, comprenant des instructions, qui lorsqu'elles sont exécutées sur un ordinateur amènent l'ordinateur à effectuer les étapes d'obtention d'un empilement tridimensionnel d'images fluorescentes bidimensionnelles ; de filtration dudit empilement tridimensionnel en utilisant un filtre tridimensionnel ; de comptage d'un nombre total de points tridimensionnels dans ledit empilement tridimensionnel filtré pour chacun d'une pluralité de seuils d'intensité ; d'obtention d'un seuil d'intensité optimum représentatif d'une région de plateau dans un graphique dudit nombre total de points tridimensionnels comparativement au seuil d'intensité auquel ledit nombre total a été compté ; de l'utilisation du nombre total de points tridimensionnels obtenus au niveau dudit seuil optimal comme représentatif d'un nombre de particules fluorescentes détectées dans ledit empilement tridimensionnel ; et

b) l'ensemble de sondes selon l'une quelconque des revendications 1 à 4.


 
10. Kit selon la revendication 9, dans lequel ledit ensemble de sondes comprend au moins 30 sondes à marqueur unique avec un premier marqueur fluorescent et au moins 30 sondes marquées par un second marqueur fluorescent.
 
11. Kit selon la revendication 9 ou la revendication 10, dans lequel ladite molécule d'ARNm comprend au moins deux régions cibles, et les marqueurs fluorescents pour chaque région cible sont distinguables.
 
12. Ensemble de sondes selon l'une quelconque des revendications 1 à 4, solution d'hybridation selon l'une quelconque des revendications 5 à 8, ou kit selon l'une quelconque des revendications 9 à 11, une ou plusieurs des sondes étant des sondes ADN.
 




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.

Patent documents cited in the description




Non-patent literature cited in the description