阅读说明：本技术 一种同时检测基因突变与拷贝数变化的高通量方法 (High-throughput method for simultaneously detecting gene mutation and copy number change ) 是由 杨锋 余伟师 梁萌萌 于 2020-12-07 设计创作，主要内容包括：本发明涉及生物医药技术领域,具体涉及一种同时检测基因突变与拷贝数变化的高通量方法,包括：针对多个待检测位点或区域,分别设计一对正链探针和负链探针；使用所述正链探针和所述负链探针进行组合得到组合探针,并基于组合探针对待测DNA进行第一轮PCR扩增；对第一轮PCR扩增得到的产物,利用一对与二代测序平台测序引物相匹配的PCR引物对第一轮PCR扩增得到的产物进行第二轮PCR扩增；对第二轮PCR扩增得到的产物进行高通量双端测序；对高通量双端测序数据进行目标位点基因型判定及拷贝数分析。解决了现有技术中无法做到基因区域点突变及拷贝数变异在一种技术中同时检测的问题。(The invention relates to the technical field of biological medicines, in particular to a high-throughput method for simultaneously detecting gene mutation and copy number change, which comprises the following steps: aiming at a plurality of sites or regions to be detected, respectively designing a pair of positive strand probes and negative strand probes; combining the positive strand probe and the negative strand probe to obtain a combined probe, and performing a first round of PCR amplification on the DNA to be detected based on the combined probe; performing second-round PCR amplification on the product obtained by the first-round PCR amplification by utilizing a pair of PCR primers matched with the sequencing primer of the second-generation sequencing platform; performing high-throughput double-end sequencing on a product obtained by the second round of PCR amplification; and (4) carrying out target locus genotype judgment and copy number analysis on the high-throughput double-end sequencing data. Solves the problem that the simultaneous detection of point mutation and copy number variation in a gene region in one technology cannot be realized in the prior art.)

1. A high-throughput method for simultaneously detecting gene mutation and copy number variation, comprising:

aiming at a plurality of sites or regions to be detected, respectively designing a pair of positive strand probes and negative strand probes, wherein the positive strand probes in each pair of positive strand probes and negative strand probes are positioned on the positive strand of the genome sequence, and the negative strand probes in each pair of positive strand probes and negative strand probes are positioned on the negative strand of the genome sequence;

combining the positive strand probe and the negative strand probe to obtain a combined probe, and performing a first round of PCR amplification on the DNA to be detected by a low cycle amplification technology based on the combined probe; defining the amplification times of the low-cycle amplification technology to be between 12 and 18;

performing second-round PCR amplification on the product obtained by the first-round PCR amplification by utilizing a pair of PCR primers matched with the sequencing primer of the second-generation sequencing platform;

performing high-throughput double-end sequencing on a product obtained by the second round of PCR amplification;

carrying out target locus genotype judgment and copy number analysis on the high-throughput double-end sequencing data;

wherein the 5' -end part sequences of the positive strand probe and the negative strand probe have a universal sequence identical to the PCR amplification primers of the second round of PCR amplification;

the 3 'end parts of the positive strand probe and the negative strand probe are sequences which are specifically combined with the upstream region of the 5' end where the site to be detected is located.

2. The high-throughput method for simultaneously detecting gene mutation and copy number variation according to claim 1, wherein the length of the plus strand probe or the minus strand probe is 18-36 bp.

3. The high-throughput method for simultaneously detecting gene mutation and copy number variation according to claim 1, wherein the length of the plus strand probe or the minus strand probe is 20-27 bp.

4. The high-throughput method for simultaneously detecting gene mutation and copy number variation according to claim 1, wherein the sequencing read length of the product obtained by the second round of PCR amplification is PE150-300bp when performing high-throughput paired-end sequencing.

5. The high-throughput method for simultaneously detecting gene mutation and copy number variation according to claim 1, wherein the average sequencing depth of the products obtained by the second round of PCR amplification is greater than 5000X when the products are subjected to high-throughput paired-end sequencing.

6. The high-throughput method for simultaneously detecting gene mutation and copy number variation according to claim 1, wherein in the second round of PCR amplification, amplification products from different samples are amplified by PCR primers with different tag sequences.

7. The high-throughput method for simultaneously detecting gene mutation and copy number variation according to claim 1, wherein the target locus genotype determination and copy number analysis are performed on the high-throughput paired-end sequencing data, and the method comprises the following steps: firstly, classifying sequences obtained by sequencing into corresponding samples according to tag sequences with the length of several to tens of bases; then, according to the base composition of each sequence, the sequence is classified into the amplification product of the corresponding gene segment; counting the types of all the bases of the target sites, judging the genotypes of the target sites and analyzing the copy numbers.

Technical Field

The invention relates to the technical field of biological medicines, in particular to a high-throughput method for simultaneously detecting gene mutation and copy number change.

Background

A gene is the material basis for inheritance and is a specific nucleotide sequence with genetic information on a DNA or RNA molecule. Almost all genetic material of non-viral organisms is DNA, except that part of the viral genetic material is RNA. Different species have their specific gene sequences, so that the biological properties present in a sample can be determined by detecting the gene sequences in the sample. During the life process of a cell, a gene is transcribed into mRNA through DNA, and then the cell takes the mRNA as a template to translate a protein molecule with biological activity, thereby expressing genetic information stored in a DNA sequence. On one hand, the gene can faithfully replicate itself to maintain the basic characteristics of the organism; on the other hand, the gene is error-generated and "mutated" during the replication process. Such mutations include point mutations, large fragment deletions/duplications (referred to as copy number polymorphisms, CNVs), gene inversions or gene translocations, and the like. Some mutations seriously affect the function of key genes to cause diseases, and because of selection, the frequency of the mutations in a population is very low, while a considerable part of the mutations do not cause survival stress to individuals because the genes which do not seriously affect the gene function or influence the gene function, remain in the population and are changed in frequency because of random drift and founder effect, so that the mutations become genetic polymorphisms in the population, and the polymorphisms with single base or little base change are called Single Nucleotide Polymorphisms (SNPs), and the polymorphisms with deletion or repetition of a large segment are called Copy Number (CNPs).

Genetic polymorphism and genetic mutation analysis are the most common genetic analysis methods for studying gene function and pathogenesis of genetic diseases. Therefore, gene identification, gene expression analysis, DNA methylation analysis, mutation screening, SNP typing, CNP typing, and CNV detection are important molecular genetic research means, and have been widely used in clinical molecular diagnosis (Varela MA et al, 2010). Because of the importance of these genetic analyses, scientists and engineers have developed a variety of assays for each.

However, the applicant finds that the prior art means still have the following technical problems in practical application: early detection methods were mainly directed to limited target fragment analysis. Methods for SNP detection such as TaqMan probe allele detection technology, restriction enzyme Reaction (RFLP), high resolution melting curve reaction, single base extension technology and the like. The detection method of the CNV with medium and small flux mainly comprises real-time quantitative PCR, FISH, multiplex ligation probe amplification technology (MLPA) and the like. The method has high flexibility, but has the biggest defect that the flux is too low, different technologies are often combined to detect the gene region sites and copy numbers, and the method is useless for research projects or diagnosis requirements needing to detect a large number of gene sites.

Microarray chips (Microarray) are characterized by a high-density probe array, and are characterized in that various processed fluorescence labeling samples are hybridized with Microarray probes by utilizing a molecular hybridization principle, non-specific hybridization signals are removed by washing, and finally, fluorescence detection is carried out by a scanner, and the signal quantity related to a target gene is confirmed according to the intensity of the fluorescence signals and the array position where the fluorescence signals are located (Maskos U et al, 1992). The microarray chip has the greatest advantage of high throughput, and can analyze the change of genes on the whole genome level, but has the defects of low quantitative accuracy due to ubiquitous non-specific hybridization, expensive hybridization and scanning instruments, high cost, long time for customizing the chip and high cost, and can not detect unknown genes.

The advent of next generation sequencing technologies has revolutionized the field of gene detection. Custom synthesis of mixed oligonucleotides (probes) hybridization capture of sheared genomic DNA samples in liquid phase is a method that can capture large regions. The biggest advantage of high throughput sequencing technology is the large throughput. The enrichment technology is mainly used for mutation detection of candidate genes, but because the enrichment process eliminates the direct ratio relationship between products and original template quantity to a certain extent, quantitative analysis of enriched candidate gene fragments, such as expression quantity and copy number analysis, cannot be accurately realized.

Disclosure of Invention

The embodiment of the application provides a high-throughput method for simultaneously detecting gene mutation and copy number variation, and solves the problem that the gene region point mutation and the copy number variation cannot be simultaneously detected in one technology in the prior art.

The embodiment of the application provides a high-throughput method for simultaneously detecting gene mutation and copy number change, which comprises the following steps:

aiming at a plurality of sites or regions to be detected, respectively designing a pair of positive strand probes and negative strand probes, wherein the positive strand probes in each pair of positive strand probes and negative strand probes are positioned on the positive strand of the genome sequence, and the negative strand probes in each pair of positive strand probes and negative strand probes are positioned on the negative strand of the genome sequence; combining the positive strand probe and the negative strand probe to obtain a combined probe, and performing a first round of PCR amplification on the DNA to be detected based on the combined probe; performing second-round PCR amplification on the product obtained by the first-round PCR amplification by utilizing a pair of PCR primers matched with the sequencing primer of the second-generation sequencing platform; performing high-throughput double-end sequencing on a product obtained by the second round of PCR amplification; carrying out target locus genotype judgment and copy number analysis on the high-throughput double-end sequencing data; wherein the 5' -end part sequences of the positive strand probe and the negative strand probe have a universal sequence identical to the PCR amplification primers of the second round of PCR amplification; the 3 'end parts of the positive strand probe and the negative strand probe are sequences which are specifically combined with the upstream region of the 5' end where the site to be detected is located.

As an improved technical scheme of the application, the length of the positive strand probe or the negative strand probe is 18-36 bp.

As an improved technical scheme of the application, the length of the positive strand probe or the negative strand probe is 20-27 bp.

As an improved technical scheme of the application, the sequencing read length of the product obtained by the second round of PCR amplification is 150-300bp when the high-throughput double-end sequencing is carried out.

As an improved technical scheme of the application, the average sequencing depth of products obtained by second round PCR amplification is more than 5000X when the products are subjected to high-throughput paired-end sequencing.

As an improved technical scheme of the application, in the second round of PCR amplification process, amplification products from different samples are amplified by using PCR primers with different tag sequences.

As an improved technical scheme of the application, the method for carrying out target locus genotype judgment and copy number analysis on high-throughput double-end sequencing data comprises the following steps: firstly, classifying sequences obtained by sequencing into corresponding samples according to tag sequences with the length of several to tens of bases; then, according to the base composition of each sequence, the sequence is classified into the amplification product of the corresponding gene segment; counting the types of all the bases of the target sites, judging the genotypes of the target sites and analyzing the copy numbers.

One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages: 1. due to the adoption of a high-throughput sequencing technology, analysis of a plurality of target gene segments can be simultaneously realized, and the requirement of detecting a large number of gene loci is met. 2. By the low-cycle amplification technology, a direct ratio relationship exists between the amount of an amplification product and the amount of an original template, and meanwhile, as the amplification products of one round of different gene segments are amplified by adopting the universal primer, the amount of the amplification product well keeps the information of the amount of the original template, and the amount of the target gene of the original template can be obtained by utilizing the method. 3. The design of the probe does not need special modification or customization, thereby greatly reducing the detection cost.

In summary, the technical scheme of the application has the following advantages: 1. increase of detection flux: one reaction can detect thousands of sites simultaneously; 2. reduction of detection cost: the method is applied to a non-proprietary detection platform, no additional equipment is required to be invested, and meanwhile, analysis of dozens to hundreds of gene segments can be completed by one detection reaction, so that the detection cost of a single gene segment is greatly reduced; 3. the application is flexible: aiming at any target gene segment needing to be detected, a detection system can be quickly established; 4. the accuracy is improved: the digital counting is adopted for quantification, so that the accuracy is greatly improved. 5. Improvement of detection sensitivity: sequence identification using single molecule amplification product sequencing and digital counting quantification methods can provide great sensitivity.

Drawings

Fig. 1 is a technical flowchart of a medium-high throughput method according to an embodiment of the present application.

FIG. 2 results of copy number analysis of positive samples.

Detailed Description

The application aims to overcome the defects of the prior art and provide a method for realizing the application of the technology in the aspects of high-throughput single nucleotide polymorphism typing, copy number variation typing, mutation screening, gene expression and the like by sequencing after single molecule amplification or direct single molecule sequencing. In order to better understand the technical solution, the technical solution will be described in detail with reference to the drawings and the specific embodiments.

Example one high throughput method for simultaneously detecting gene mutations and copy number changes, as shown in FIG. 1, comprises: aiming at a plurality of sites or regions to be detected, respectively designing a pair of positive strand probes and negative strand probes, wherein the positive strand probes in each pair of positive strand probes and negative strand probes are positioned on the positive strand of the genome sequence, and the negative strand probes in each pair of positive strand probes and negative strand probes are positioned on the negative strand of the genome sequence; the length of the positive strand probe or the negative strand probe is 18-36bp, and preferably 20-27 bp.

Combining the positive strand probe and the negative strand probe to obtain a combined probe, and performing a first round of PCR amplification on the DNA to be detected based on the combined probe; amplifying products obtained by the first round of PCR amplification by using a pair of universal amplification primers matched with a sequencing primer of a second-generation sequencing platform, wherein the universal amplification primers have a label sequence with the length of several to tens of bases under the normal condition, and the amplification products from different samples can be amplified by using the universal amplification primers with different label sequences, so that the amplification products of different samples can be mixed together, and the sequences obtained by sequencing can be classified into different samples according to the label sequences in subsequent high-throughput sequencing data; performing high-throughput double-end sequencing on a product obtained by the second round of PCR amplification; and when the product obtained by the second round of PCR amplification is subjected to high-throughput double-end sequencing, the sequencing read length is 150-300bp PE, and the average sequencing depth is more than 5000X.

Carrying out target locus genotype judgment and copy number analysis on the high-throughput double-end sequencing data; wherein the 5' -end part sequences of the positive strand probe and the negative strand probe have a universal sequence identical to the PCR amplification primers of the second round of PCR amplification; performing target locus genotype judgment and copy number analysis on the high-throughput double-end sequencing data, wherein the steps comprise: firstly, classifying sequences obtained by sequencing into corresponding samples according to tag sequences with the length of several to tens of bases; then, according to the base composition of each sequence, the sequence is classified into the amplification product of the corresponding gene segment; counting the types of all the bases of the target sites, judging the genotypes of the target sites and analyzing the copy numbers. The 3 'end parts of the positive strand probe and the negative strand probe are sequences which are specifically combined with the upstream region of the 5' end where the site to be detected is located.

The technical scheme in the embodiment of the application at least has the following technical effects or advantages: 1. and the analysis of a plurality of target gene segments simultaneously has high detection flux. 2. The proportional relation between the amount of the amplification product and the amount of the original template is kept, and meanwhile, the gene copy number can be accurately detected due to the fact that the amplification products of one round of different gene segments are amplified by the universal primers. 3. The design of the probe does not need special modification or customization, thereby greatly reducing the detection cost.

Example two probes, probes and general primer information were designed for the coding regions of 4 genes (ATP7B, NF1, TSC1, TSC2) as shown in table 1:

TABLE 1

Carrying out PCR amplification on three known mutant positive samples by using a mixed solution of a positive strand probe and a negative strand probe of each site to obtain an amplification product; mixing the amplification products of the two panels, then amplifying by using PCR primers compatible with an illumina sequencing platform with different tag sequences, uniformly mixing the sample products, then sequencing by an illumina sequencing instrument in a PE150 mode, and carrying out subsequent analysis on sequencing data.

The experimental process comprises the following steps: (1) performing concentration quantification on the wild type sample and the mutant sample; (2) preparing a mixed solution of two panel plus strand probes and two panel minus strand probes, wherein the concentration of each primer is 2 uM; taking 10-100ng of genomic DNA as a template to carry out PCR reaction, wherein the reaction system comprises 20ul of 10u 12x HIFI multi PCR master mix, 2u1 Pmix1 for P1 or Pmix2 for P2, 2ul of connected purified products and 6ul of sterile water; the PCR program is that the temperature is 98 ℃ for 2 min; 18x (96 ℃ for 20s,60 ℃ for 4 min); ho1d at 10 ℃, P1 and P2 are mixed in equal proportion, and then a reaction product is purified by using a DNA purification magnetic bead kit according to the proportion of 1.8X; (4) using UNIPCRF/UDIRxxxx as well as the illiminia sequencing platform compatible PCR primers with different tag sequences, wherein the concentration of each primer is 2 uM; taking 2ul of the connection purification product as a template to carry out PCR reaction, wherein the reaction system is 20ul and comprises 10u 12x HIFI PCR master mix, 2u1 Pmix, 2ul of the connection purification product and 6ul of sterile water; the PCR program is that the temperature is 98 ℃ for 2 min; 12x (98 ℃ for 10s,60 ℃ for 30s, 72 ℃ for 30 s); ho1d at 10 ℃; (5) performing PE150 mode sequencing on the final product, namely an illumina sequencing platform, and performing subsequent analysis on sequencing data; (6) sequencing reads are divided into different samples according to the tag sequences, the sequencing depth of the target sites or regions is counted, mutation sites of the target regions are analyzed, and the gene copy number of the target regions is calculated at the same time.

The sequences of the universal primers used in this example were as follows:

joint universal primer F

TCAGACGTGTGCTCTTCCGATCTCAAGAACGGAATGTGTACTTGC

Joint universal primer R

TCAGACGTGTGCTCTTCCGATCTCTCTCGCTAACAAGCTCAGCTA

UNIPCRF

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC

UDIR0001

CAAGCAGAAGACGGCATACGAGAT AACCGCGG GTGACTGGAGTTCAGACGTG

UDIR0002

CAAGCAGAAGACGGCATACGAGAT GGTTATAA GTGACTGGAGTTCAGACGTG

UDIR0003

CAAGCAGAAGACGGCATACGAGAT CCAAGTCC GTGACTGGAGTTCAGACGTG

The results of the three positive results are shown in Table 2, Table 3 and FIG. 2.

TABLE 2 target sequencing depth statistics for each sample

TABLE 3 statistics of sequencing depth for target regions of each sample

Transcript	Region(s)	Location information (hg38)	Mutation information	Type of mutation
					NM_000053	exon8	chr13:51958356	c.2310C>G	Heterozygous mutations
NM_000053	exon8	chr13:51958333	c.2333G>T	Heterozygous mutations

The results of the three positive samples were matched with the known mutation types.

Sequence listing

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<210> 59

<211> 20

<212> DNA

<213> Positive Strand Probe (FP)

<400> 59

ggtcattctc tcctggctcc 20

<210> 60

<211> 28

<212> DNA

<213> negative strand Probe (RP)

<400> 60

atgatgtcat taagtgacct gttaccaa 28

<210> 61

<211> 30

<212> DNA

<213> Positive Strand Probe (FP)

<400> 61

tttatgtaca atatgtattc agagtatccc 30

<210> 62

<211> 22

<212> DNA

<213> negative strand Probe (RP)

<400> 62

cttggttgca gggatggatt at 22

<210> 63

<211> 30

<212> DNA

<213> Positive Strand Probe (FP)

<400> 63

caaaggtttt tataagttct gtggatcttt 30

<210> 64

<211> 28

<212> DNA

<213> negative strand Probe (RP)

<400> 64

gagaaccata aatatttggg agaagtga 28

<210> 65

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 65

cgagatgtgg ccctcgtt 18

<210> 66

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 66

attgctgccc acggagct 18

<210> 67

<211> 23

<212> DNA

<213> Positive Strand Probe (FP)

<400> 67

catgagcctg tgtgtaagtc ctg 23

<210> 68

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 68

agcaaatcca gggagggtgt 20

<210> 69

<211> 20

<212> DNA

<213> Positive Strand Probe (FP)

<400> 69

aggactgcgt tttcacctcc 20

<210> 70

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 70

gagacgggga tacctggctg 20

<210> 71

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 71

cacgagcttg gctctggc 18

<210> 72

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 72

tgacgccctg agcctcat 18

<210> 73

<211> 20

<212> DNA

<213> Positive Strand Probe (FP)

<400> 73

tgcggggact tggcctcagc 20

<210> 74

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 74

ggaggcccag caggcaggtg 20

<210> 75

<211> 20

<212> DNA

<213> Positive Strand Probe (FP)

<400> 75

tagggtccag aaggccctgt 20

<210> 76

<211> 24

<212> DNA

<213> negative strand Probe (RP)

<400> 76

gtcttttggg gaaaaaccct actg 24

<210> 77

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 77

ggctaccccg tgacctgg 18

<210> 78

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 78

atgtggggcc tacctggg 18

<210> 79

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 79

ggctgcctct gctgcaag 18

<210> 80

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 80

ggggactcac tggacaggaa 20

<210> 81

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 81

tctcagccac agccagca 18

<210> 82

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 82

ccctcaaagc caggaaggag 20

<210> 83

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 83

cttcagaggc gctgcacg 18

<210> 84

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 84

ttctgccgca aggcctag 18

<210> 85

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 85

tgtttccctg ctgccagg 18

<210> 86

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 86

acagccaagg gcaaagca 18

<210> 87

<211> 20

<212> DNA

<213> Positive Strand Probe (FP)

<400> 87

ttgccacccc tcactgtctg 20

<210> 88

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 88

accccacacc gactccag 18

<210> 89

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 89

gggagctggg ctctctgg 18

<210> 90

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 90

ccaagctcca gggtccgt 18

<210> 91

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 91

acctggcacc ctgaccct 18

<210> 92

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 92

tgtagtgccg cctggacc 18

<210> 93

<211> 22

<212> DNA

<213> Positive Strand Probe (FP)

<400> 93

tcttctccaa cttcacggct gt 22

<210> 94

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 94

aggaaggtgc agtcacctcg 20

<210> 95

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 95

ctggactcgg gggagctg 18

<210> 96

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 96

aggctcaccc gacatggaac 20

<210> 97

<211> 20

<212> DNA

<213> Positive Strand Probe (FP)

<400> 97

cgcctgccag cctcgacacc 20

<210> 98

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 98

gagggagccc cggtgcctgt 20

<210> 99

<211> 22

<212> DNA

<213> Positive Strand Probe (FP)

<400> 99

ggtaagtggt ggtcaccagt cc 22

<210> 100

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 100

cacataggcc gccaggtt 18

<210> 101

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 101

agaaggcctc agctggca 18

<210> 102

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 102

ccccaaatat cccaagaggg 20

<210> 103

<211> 22

<212> DNA

<213> Positive Strand Probe (FP)

<400> 103

gatgggtaag gggaggtact gg 22

<210> 104

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 104

tgccactcac ctgtgttgga 20

<210> 105

<211> 22

<212> DNA

<213> Positive Strand Probe (FP)

<400> 105

agccctgtac aagtcactgt cg 22

<210> 106

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 106

gcttcctgag cagggcag 18

<210> 107

<211> 20

<212> DNA

<213> Positive Strand Probe (FP)

<400> 107

gtagcccctc ctcctgctga 20

<210> 108

<211> 20

<212> DNA

<213> negative strand Probe (RP)

<400> 108

tggccaagcc aaagacattc 20

<210> 109

<211> 18

<212> DNA

<213> Positive Strand Probe (FP)

<400> 109

ccagccccac atccagca 18

<210> 110

<211> 18

<212> DNA

<213> negative strand Probe (RP)

<400> 110

agagccctgc ctccccta 18

<210> 111

<211> 45

<212> DNA

<213> Joint Universal primer (R)

<400> 111

tcagacgtgt gctcttccga tctctctcgc taacaagctc agcta 45

<210> 112

<211> 45

<212> DNA

<213> UNIPCRF

<400> 112

aatgatacgg cgaccaccga gatctacact ctttccctac acgac 45

<210> 113

<211> 52

<212> DNA

<213> UDIR0001

<400> 113

caagcagaag acggcatacg agataaccgc gggtgactgg agttcagacg tg 52

<210> 114

<211> 52

<212> DNA

<213> UDIR0002

<400> 114

caagcagaag acggcatacg agatggttat aagtgactgg agttcagacg tg 52

<210> 115

<211> 52

<212> DNA

<213> UDIR0003

<400> 115

caagcagaag acggcatacg agatccaagt ccgtgactgg agttcagacg tg 52

<210> 116

<211> 45

<212> DNA

<213> Joint Universal primer (F)

<400> 116

tcagacgtgt gctcttccga tctcaagaac ggaatgtgta cttgc 45