CN109694904B - Detection method of BRCA1/2 gene mutation - Google Patents

Abstract

The invention provides a detection method of BRCA1/2 gene mutation. The invention adopts a multiple PCR mode and designs an optimized primer group, and only 2 PCR reactions can cover the following regions by using the primer group: the whole coding regions of BRCA1 and BRCA2 and the regions adjacent to +/-5-20 bp; sites of intronic regions closely related to the onset of breast cancer; the tagSNP site in the region of BRCA1/2 and the upstream and downstream 1M ranges. The method has the advantages of universality, high flux, high efficiency, wide coverage and simple operation.

Description

Detection method of BRCA1/2 gene mutation

Technical Field

The invention belongs to the field of gene detection, and particularly relates to detection of breast cancer susceptibility gene BRCA1/2 mutation.

Background

Currently, there are many factors that contribute to breast cancer, which may be genetic or environmental, or in most cases, a combination of both. About 60-70% of patients do not know exactly what caused their cancer. However, there are several established risk factors associated with breast cancer, such as: genetic factors (gene mutations), lifestyle habits (poor diet/alcohol consumption/overweight/obesity/chest CT/lack of physical exercise/HRT), and environmental changes, among others. Aiming at genetic factors, research of the center for preventing and treating breast cancer in Beijing university tumor hospital shows that: in 8085 patients with Chinese breast cancer who were serially enrolled, 46 cancer susceptibility genes were tested for germline mutations without restriction on age or family history. As a result, 9.2% of patients were found to carry pathogenic mutations; of these, 5.3% of patients carried BRCA mutations (1.8% for BRCA1 and 3.5% for BRCA 2), 2.9% carried other breast cancer susceptibility genes, and 1.0% carried other cancer susceptibility genes. It can be seen. BRCA1/2 gene mutation is the most common gene mutation that causes breast cancer.

BRCA1 and BRCA2 are human cancer suppressor genes, located on chromosomes 17 and 13, respectively. If the specific site of BRCA1/2 gene is mutated, the produced tumor suppressor protein will be changed, the function of normally repairing DNA will be limited, and the cell will accumulate harmful DNA damage more easily, and finally the tumor will occur. And on 20 d 6.2017, a large sample queue research report is formally published in journal of American medical society, and age-related risks of breast cancer, ovarian cancer and breast cancer on the opposite side of mutation-carrying women, family history of the cancer and the influence of mutation sites on the risks are evaluated. As a result, the risk of breast cancer accumulation is 72% and 69% and the risk of ovarian cancer is 44% and 17% respectively by the age of 80 in women carrying BRCA1 and BRCA2 mutations.

At present, Sanger sequencing and capture sequencing are used as mutation detection methods of BRCA 1/2. The Sanger sequencing experiment can only detect specific sites in one time, and the flux is low. Although capture sequencing can cover the entire coding region of BRCA1/2 in one experiment, it is costly, complex to perform, and requires relatively long experimental times.

In summary, the existing BRCA1/2 mutation detection method has the defects of complex operation, long time and high cost. Therefore, a new method for detecting BRCA1/2 mutation is needed to solve the above-mentioned deficiencies of the prior art.

Disclosure of Invention

Aiming at the technical difficulties, the invention adopts a multiple PCR mode and designs an optimized primer group, and only 2 PCR reactions can cover the following regions by using the primer combination: the whole coding regions of BRCA1 and BRCA2 and the regions adjacent to +/-5-20 bp; sites of intronic regions closely related to the onset of breast cancer; the tagSNP site in the region of BRCA1/2 and the upstream and downstream 1M ranges.

The first aspect of the invention provides a primer composition for detecting BRCA1 gene mutation, wherein the amplified fragment can cover the following genome regions:

the whole coding region of BRCA1 and the region adjacent to +/-5-20 bp;

sites of the intron region of BRCA1 that are closely associated with the onset of breast cancer; and

the region BRCA1 and the tagSNP site in the upstream and downstream 1M range.

The tagSNP in the invention refers to a representative SNP in a genome linkage disequilibrium region, and can represent other SNPs in the haplotype region, and the purpose of multiple PCR covering the tagSNP is to discover the haplotype closely related to breast cancer.

In one embodiment, the primer composition for detecting BRCA1 gene mutation comprises an upstream primer and a downstream primer respectively selected from SEQ ID NO (2n-1) and SEQ ID NO 2n, wherein n is a natural number from 1 to 86.

Preferably, the primer composition comprises all primers with the sequences of SEQ ID NO 1-172 respectively; most preferably, the primer composition consists of primers having the sequences of SEQ ID NO 1-172, respectively.

In a second aspect, the present invention provides a primer composition for detecting mutations in BRCA2 gene, wherein the amplified fragment covers the following genomic regions:

the whole coding region of BRCA2 and the region adjacent to +/-5-20 bp;

sites of the intron region of BRCA2 that are closely associated with the onset of breast cancer; and

the region BRCA2 and the tagSNP site in the upstream and downstream 1M range.

In one embodiment, the primer composition for detecting BRCA2 gene mutation comprises an upstream primer and a downstream primer respectively selected from SEQ ID NO (2n-1) and SEQ ID NO 2n, wherein n is a natural number ranging from 87 to 282.

Preferably, the primer composition comprises all primers with the sequences of SEQ ID NO 173-564 respectively; most preferably, the primer composition consists of primers having the sequences of SEQ ID NO 173-564, respectively.

The third part of the invention provides a primer composition for detecting BRCA1/2 gene mutation, which comprises the primer composition for detecting BRCA1 gene mutation and/or the primer composition for detecting BRCA2 gene mutation.

Furthermore, the invention also provides a detection reagent or a kit for detecting BRCA1/2 gene mutation, which comprises the primer composition for detecting BRCA1/2 gene mutation.

The detection reagent or kit may be used for diagnostic or non-diagnostic purposes. For example, due to the correlation between BRCA1/2 gene mutation and breast cancer, the gene can be used for screening BRCA1/2 mutation carriers in a human population and evaluating the breast cancer risk. In addition, the detection reagent or the kit can be used for studying the pathogenesis and the treatment method of the breast cancer in a laboratory, for example.

The fourth part of the invention provides a method for detecting the mutation of the BRCA1/2 gene, which comprises the following steps:

(1) extracting whole blood DNA;

(2) amplifying a target region by multiplex PCR using the primer composition for detecting BRCA1/2 gene mutation;

(3) constructing a library by using the amplification product;

(4) high-throughput sequencing;

(5) and processing and analyzing a sequencing result.

Preferably, the library construction in step (3) is performed according to the Illumina standard library construction procedure.

Preferably, the sequencing platform in the step (4) is an Illumina sequencing platform.

Preferably, in the step (5), the sequencing reading result is aligned with human genome Hg19 to obtain the position of the reading on a reference genome, and the variation sites and the situation of insertion deletion sequences are analyzed by using GATK software and annotated by VEP software.

In one embodiment, when the DNA molecules to be tested are from multiple test samples, the amplification products of each sample may be tagged with a different tag sequence (barcode) when constructing the library to allow for sample differentiation during sequencing, thereby allowing for simultaneous sequencing of multiple samples.

In a particular embodiment, the assay is a non-diagnostic assay for the in vitro detection of mutations in the BRCA1/2 gene.

The invention has the following advantages:

1. universality: the invention can be used for all samples needing BRCA1/2 mutation detection.

2. High flux: the invention relies on a second-generation sequencing technology sequencing and analyzing platform, and can complete a large amount of samples in a short time.

3. High efficiency: aiming at the technical difficulties of low sequencing flux, high cost and long time of a capture method in the first generation, the invention adopts a multiplex PCR method to combine with high-flux sequencing, thereby improving the efficiency and saving the cost.

4. The coverage is wide: the coverage range of the invention not only comprises a coding region of BRCA1/2 and a region adjacent to +/-5-20 bp, but also comprises sites of intron regions closely related to the onset of breast cancer, gene regions and tagSNP sites within the range of upstream and downstream 1M.

5. The operation is simple: using the primer composition of the present invention, only 2 PCR reactions are required to cover the above range.

Drawings

FIG. 1 is the distribution of the number of pathogenic mutations detected from susceptibility genes of breast cancer.

FIG. 2BRCA1/2 mutation carrier accumulates breast cancer risk with age.

FIG. 3 is a technical flowchart of the BRCA1/2 gene mutation detection method of the present invention.

Detailed Description

The embodiments of the present invention will be described in detail, but the present invention is not limited thereto, the described embodiments are only a part of the embodiments of the present invention, and all other embodiments obtained by those skilled in the art based on the embodiments of the present invention belong to the protection scope of the present invention; likewise, the drawings of the embodiments are only partial drawings of the embodiments of the invention, and other drawings obtained by those skilled in the art according to the drawings also belong to the protection scope of the invention.

The primer sets used in the following examples are primer compositions shown in Table 1 (wherein, the sequences of the upstream primer and the downstream primer are respectively SEQ ID NO: (2n-1) and SEQ ID NO:2n, wherein n is a natural number of 1-282), a large number of primer pairs are designed according to the range of a region to be detected (covering the whole coding region of BRCA1 and a region adjacent to +/-5-20 bp; sites of an intron region of BRCA1 closely related to the onset of breast cancer; and sites of tagSNP sites in the region of BRCA1 and the range of upstream and downstream 1M), and then the sequences of the primer compositions are determined through steps of homology screening, Tm value standardization and the like, and finally the primer compositions are obtained through chemical synthesis.

TABLE 1 primer set for detecting BRCA1/2 gene mutation

In the following examples, sample DNA was obtained by isolating a genome from a peripheral blood sample of a subject; building a library according to an Illumina standard library building process, sequencing by using an Illumina sequencing platform, and comparing a sequencing read result with human genome Hg19 to obtain the position of the read on a reference genome; the variation sites and insertion deletion sequence were analyzed using GATK software and annotated with VEP software.

The experimental procedures, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

Example 127 sample analysis consistency verification

Firstly, DNA extraction

Genomic DNA of 27 exception peripheral blood samples was extracted using a Tiangen blood genome extraction kit.

Second, capture the target sequence

In 27 cases, 20ng of each peripheral blood DNA sample was taken, and the target region was captured by multiplex PCR using the above multiplex primers.

Third, library construction

And constructing the library by adopting a kit of an Illumina standard library construction method.

Fourthly, sequencing

Sequencing the capture library using the Illumina platform.

Fifthly, processing and analyzing sequencing results

The sequencing reads (reads) result was aligned to the human genome Hg19, giving the position of the read on the reference genome. The variation sites and insertion deletion sequence were analyzed using GATK software and annotated with VEP software. The results of the experiment are shown in table 2.

TABLE 227 mutation results of BRCA1/2 gene in peripherical blood samples

Therefore, the detection result of the method of the invention on BRCA1/2 gene mutation in 27 cases of tumor DNA is completely consistent with the BRCA1/2 gene mutation situation obtained by the traditional method, which shows that the method of the invention has quite high detection precision.

Sequence listing

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<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 109

gatgggaaaa agtggtggta tacgatat 28

<210> 110

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 110

gtttcaaagc gccagtcatt tg 22

<210> 111

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 111

cttccctaga gtgctaactt ccag 24

<210> 112

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 112

atggaaggta aagaacctgc aact 24

<210> 113

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 113

agttccatat tgcttatact gctgctt 27

<210> 114

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 114

cccaacttaa gccatgtaac tgaaaatc 28

<210> 115

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 115

ccaatacatc agctactttg gcatttg 27

<210> 116

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 116

aaggctgaat tctgtaataa aagcaaacag 30

<210> 117

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 117

accatagggc tcataaaatt cacttcc 27

<210> 118

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 118

ttttccaggc atcatacatg ttagct 26

<210> 119

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 119

acgacatttg acagagaatg atactctaac 30

<210> 120

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 120

ctaaaggaga gagcagcttt cact 24

<210> 121

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 121

gaatgcgagg tgacaacgtg 20

<210> 122

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 122

ccggtgagaa ttcaagcgg 19

<210> 123

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 123

ccttttcatt tctaatacct gcctcagaa 29

<210> 124

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 124

ctcccttgga aatctgccat ga 22

<210> 125

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 125

cttgggatat tcaacactta cactcca 27

<210> 126

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 126

gcaatgcatt atatctgctg tggattt 27

<210> 127

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 127

gtttacctgt tatggatgaa actgacct 28

<210> 128

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 128

ctgtaactaa taaaacgtag gcacagaga 29

<210> 129

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 129

gcgcttgtac ttgtcaacag tt 22

<210> 130

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 130

agaagattgg ctcttaccac ttgtc 25

<210> 131

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 131

aacagcacaa catttacaaa acgtattttg 30

<210> 132

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 132

ccaaagacag tcttctaatt cctcattagt 30

<210> 133

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 133

tctgcagaaa tgggcaagtt cat 23

<210> 134

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 134

ctgaaaggca gtgggagcac at 22

<210> 135

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 135

gccaaaaaca caaccttcct gt 22

<210> 136

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 136

cgaagagaca gaaagtgtgt gtgt 24

<210> 137

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 137

cgcttctcct ccctggtttt g 21

<210> 138

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 138

cggaaccggg atctatttcg g 21

<210> 139

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 139

aagcagcata gcagcagaga tt 22

<210> 140

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 140

gctctaattg aggcctgtca caa 23

<210> 141

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 141

cattaatttg ctaaattgct ggctaagaca 30

<210> 142

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 142

ggctgatggg aaagagcaac at 22

<210> 143

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 143

ggagactagg accacatcag gt 22

<210> 144

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 144

ggactcccag gtggtgagaa ta 22

<210> 145

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 145

gatctgttct gggactcctg tt 22

<210> 146

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 146

agtgaggcag ggatctgagt ac 22

<210> 147

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 147

ggaagtaaag cttttaaaag ccacgta 27

<210> 148

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 148

cctgctgaag ggaatggaaa gaa 23

<210> 149

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 149

acaacaacac aaaaaccaag agaaatctt 29

<210> 150

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 150

ccactggtga cgacgtaaag at 22

<210> 151

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 151

caagcctcag gcttgtatgt ttg 23

<210> 152

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 152

gaggataatt aactctggaa catcaggt 28

<210> 153

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 153

aaacaataat tgcggaaagt gatgaaagt 29

<210> 154

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 154

tggagggaag tagcctgaag tt 22

<210> 155

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 155

cccgatcaat aattatcctg ggtttg 26

<210> 156

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 156

ctgtttattc tttcttctct agagagagca 30

<210> 157

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 157

ctatgagcat cgtgcagatg ga 22

<210> 158

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 158

tgtgtataaa tacttgccga aggtcaag 28

<210> 159

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 159

aaagaaatgg attacattct ggaaacttgc 30

<210> 160

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 160

ctgtcaaagc tatctgggtt cgat 24

<210> 161

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 161

tgaactgaat gaagacgcca ttaca 25

<210> 162

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 162

tgcttacaac cttgactccc ttt 23

<210> 163

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 163

aaataaaacc acccgttttc agatgg 26

<210> 164

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 164

gctggctaca gagagagatt gaaga 25

<210> 165

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 165

gggaatggag agaaggaaaa tctagt 26

<210> 166

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 166

gcctcttatt aaacatacag aaggaccttt 30

<210> 167

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 167

ccctccttgt tttattgact cttcagt 27

<210> 168

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 168

cacatttctg atccagatca catgtct 27

<210> 169

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 169

gcacgtttga caatcctgtg tct 23

<210> 170

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 170

atctcctgtg ctacatgcaa atatacaa 28

<210> 171

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 171

tgacctggga atctgcatct ttt 23

<210> 172

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 172

aacctattta ccagaaatgg ctttgttc 28

<210> 173

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 173

ccaaatttat ctccagaaat cttgcaca 28

<210> 174

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 174

ttaaggtcta tccaaaactt tattgccagt 30

<210> 175

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 175

tcaaaattcc aatgagcaga aaagcatc 28

<210> 176

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 176

caaatgctgc cttgtagaga agc 23

<210> 177

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 177

ccacacctgg ctacttttgt gtt 23

<210> 178

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 178

acagactgcc tggctgtg 18

<210> 179

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 179

catatttggc acagtgcatg ga 22

<210> 180

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 180

tcagtgccta acatctttct tgaagaaaat 30

<210> 181

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 181

aggtgagttt taattgtgta gaactgct 28

<210> 182

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 182

caatgtggtc tttgcagcta tttactt 27

<210> 183

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 183

tgggccaaac tcattatatg ctgg 24

<210> 184

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 184

ggtcactgaa gatgctgtgc tt 22

<210> 185

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 185

tgcaagtttt tcaggtcata tgactga 27

<210> 186

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 186

tgcaaatgta agtggtgctt caaaag 26

<210> 187

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 187

aaatgtcaga atctggaatt aaacccagat 30

<210> 188

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 188

actacacaga atagccctgg aaca 24

<210> 189

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 189

tcttttggaa agtaattcaa tagctgacga 30

<210> 190

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 190

cgtcaataat ttattgtcgc ctttgca 27

<210> 191

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 191

gaataggatt cagaggttcc tgagaga 27

<210> 192

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 192

gaactctgga tccattccac cat 23

<210> 193

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 193

agagaagcaa aatccactac taatgcc 27

<210> 194

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 194

atggagattc cataaactaa caagcactt 29

<210> 195

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 195

agcctacaca gtgagaaaca ctattataga 30

<210> 196

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 196

taggtttgta caatccagca tttaatagct 30

<210> 197

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 197

gggatgacac agctgcaaaa ac 22

<210> 198

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 198

aatgactgat ttttaccaag agtgcaaatt 30

<210> 199

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 199

cgcattgcat ttctgttgga cat 23

<210> 200

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 200

gggtagcaga agcataaggg aa 22

<210> 201

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 201

tagcttatct gtggtatctg gtagca 26

<210> 202

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 202

gctttttgaa gcctttaagg atttttcttg 30

<210> 203

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 203

gccttgccag ggattcaaat tc 22

<210> 204

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 204

ccttggaaag aaaaatcaga agggattcta 30

<210> 205

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 205

aatttgtcca gatttctgct aacagtact 29

<210> 206

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 206

gtggtggctc agctacttga g 21

<210> 207

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 207

gttgatgtta caaagctggc atca 24

<210> 208

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 208

tagaagtctg cacacacatc actc 24

<210> 209

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 209

cttatcttta aatctccctt ctttgggtgt 30

<210> 210

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 210

agcatttcaa catactcctt cctgt 25

<210> 211

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 211

gaaaagacca acaaaataga aagttaccca 30

<210> 212

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 212

agatgtaaat ctggaagaat atgttggca 29

<210> 213

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 213

agtttattca ctgtgttgat tgacctttc 29

<210> 214

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 214

cacatatagg accaggttta gagactttc 29

<210> 215

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 215

tatctgcaag gtgatgataa tgacactaac 30

<210> 216

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 216

acactggcta tttcccttta ttttctgt 28

<210> 217

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 217

ggtgcatagt cattatcaat ttgtgaatca 30

<210> 218

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 218

acgggaagtg ttaacttctt aacgtt 26

<210> 219

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 219

agaagggtaa atctgattgc ccaa 24

<210> 220

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 220

tccttggagg ctattaaaga atgaacc 27

<210> 221

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 221

cagatattag ccatctgtaa tgtagttggt 30

<210> 222

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 222

catttgcctg tgattattca aggct 25

<210> 223

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 223

tttgagagta gcttcaggca tcatc 25

<210> 224

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 224

acaaaagggt atcctctatg tattagctc 29

<210> 225

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 225

actgtgccca aacactacct tt 22

<210> 226

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 226

tgctttttgg atcattttca cactgtc 27

<210> 227

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 227

agcacaaaca aaccttatag cttaagtga 29

<210> 228

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 228

gtgatcattt gatacaggca tttaatgtgt 30

<210> 229

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 229

cttccaagga tgttctgtca aaccta 26

<210> 230

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 230

tcctttcatt agctacttgg aagacaaaat 30

<210> 231

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 231

ggtggatcac agtgtacgag aa 22

<210> 232

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 232

catagcttaa aatttaccat ttcagccgaa 30

<210> 233

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 233

ggtttatgtt cttgcagagg agaaca 26

<210> 234

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 234

ctgaggcttg ctcagtttct tttg 24

<210> 235

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 235

tgacttccaa gcatttttca agtgg 25

<210> 236

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 236

tgtgccgctc ccataaaaat attaga 26

<210> 237

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 237

gctacatatt gcagaagagt acatttgaag 30

<210> 238

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 238

tattggatgt acctctgcag aagtttc 27

<210> 239

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 239

aaatagtctg gccactgtgc at 22

<210> 240

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 240

tacacaggag tgttggagag aatca 25

<210> 241

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 241

tggcagtgat tcaagtaaaa atgatactgt 30

<210> 242

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 242

ttgagctttc gcaacttcca aaaa 24

<210> 243

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 243

gagttgagga gaggatgaga ctca 24

<210> 244

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 244

tggtaaaatg gttgtagttt gtctcttgt 29

<210> 245

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 245

atggacattc taagttatga ggaaacagac 30

<210> 246

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 246

tttgcccatt gatggctaaa actg 24

<210> 247

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 247

ctagtccaaa agagggccac ttt 23

<210> 248

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 248

tgactgctgc aaaccacaga ta 22

<210> 249

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 249

agcaaaaagt cctgcaactt gttac 25

<210> 250

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 250

gaatagctgt tagacatgct actgttact 29

<210> 251

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 251

caacagcaca cagtgtggaa att 23

<210> 252

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 252

gaagcatctg ttctttcctc tttttctg 28

<210> 253

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 253

cctgcaaaaa taaaaatgca gccattaaa 29

<210> 254

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 254

tccaatccag acatattttg gttatgttgt 30

<210> 255

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 255

ggaaatttac ccaagcttac ttcttcct 28

<210> 256

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 256

cagtagaacc caactaaaag tctcagaaa 29

<210> 257

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 257

tgcttcatta caaaacgcaa gacaag 26

<210> 258

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 258

ggtttctctt atcaacacga ggaagtattt 30

<210> 259

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 259

gaacttgaat tctgaaaaag agctcaaaga 30

<210> 260

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 260

agccacagtc tcagccatat atatagt 27

<210> 261

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 261

gaacaggctt cacctaaaaa cgtaaaaa 28

<210> 262

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 262

ggcaacacga aaggtaaaaa tgaaca 26

<210> 263

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 263

cccataagtg tactttcctc ttacatca 28

<210> 264

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 264

tgtatattcc caggataaag ccctgaa 27

<210> 265

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 265

aggacatcca ttttatcaag tttctgct 28

<210> 266

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 266

ggcaaaaatt catcacacaa attgtcatac 30

<210> 267

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 267

gagctgagta aaaattagga tgactggt 28

<210> 268

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 268

tcactcagga atacacacat gaaacc 26

<210> 269

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 269

gttgttgaat tcagtatcat cctatgtggt 30

<210> 270

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 270

cagactacac agaaacctta accatactg 29

<210> 271

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 271

tcttactcta ggttatggtc ataagcgt 28

<210> 272

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 272

ctggttgtga tctgtgtggt ct 22

<210> 273

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 273

cacattatta cagtggatgg agaagacat 29

<210> 274

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 274

tgttgctatt ctttgtctaa caccaaaaa 29

<210> 275

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 275

ctccataaat cctcacagcc tcat 24

<210> 276

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 276

gcatggattg gatgagcttg gt 22

<210> 277

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 277

gggatttgct ttgttttatt ttagtcctgt 30

<210> 278

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 278

tcacttaacg tttactgaat tgcctgt 27

<210> 279

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 279

cccagctttg aagactgtgt ga 22

<210> 280

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 280

tgacaaataa agtgagcttc attgtttga 29

<210> 281

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 281

aaacaaattt tccagcgctt ctga 24

<210> 282

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 282

gcaaacgatg attatgttgt taactggatt 30

<210> 283

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 283

tgcaaaggag cgaaatcctg at 22

<210> 284

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 284

ggtttcttca gcctgtctct actc 24

<210> 285

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 285

cctcacagta ccttcctatg gca 23

<210> 286

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 286

aaagactctg catttttgct gttaattttt 30

<210> 287

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 287

ttccagcttt gctcctagaa tcc 23

<210> 288

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 288

acttgagata gtcacctaat tcatagctca 30

<210> 289

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 289

acaactcccg gagagtcaga tt 22

<210> 290

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 290

gtgcctaaat ttgaacacta aggagaaaaa 30

<210> 291

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 291

cagacacaaa gctgggtgtc ta 22

<210> 292

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 292

acttgcatca tggttcaaga gca 23

<210> 293

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 293

gttagatgcc acacagctcc at 22

<210> 294

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 294

aaacaagttc caggaaatgt cctacaa 27

<210> 295

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 295

gcacatgtaa taatcaacag caaaagtcc 29

<210> 296

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 296

ttgcaaatgg tgtgcttcta attgg 25

<210> 297

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 297

ctaagtgtgg ataatttgca atcatatggg 30

<210> 298

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 298

agcagtgaga ctggagagaa gat 23

<210> 299

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 299

tgatttggtt tgcttacttt tcaccaatg 29

<210> 300

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 300

aaagatgtat gtaatcctgc tctcctcta 29

<210> 301

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 301

gggattctat ttctatttct tggatggctt 30

<210> 302

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 302

tgacatgtgg tctccaccta ct 22

<210> 303

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 303

gaaagctcag tgtctgtgca gta 23

<210> 304

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 304

ggatgtttct gaaccatgtg tccat 25

<210> 305

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 305

tgaatgattt tctaaacatg catcactca 29

<210> 306

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 306

tgtagcatga tacaagtcat tgatgca 27

<210> 307

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 307

agaggatctt gataaatgct gccaa 25

<210> 308

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 308

gtcagtcttt ccataccact tctttgt 27

<210> 309

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 309

tctgaatttg ttgtgaggat tccatga 27

<210> 310

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 310

tgcctagaaa aatacctttg attcagaact 30

<210> 311

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 311

aggaagtatc tgggccttat gct 23

<210> 312

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 312

catactttac acaaacagcc aattagacat 30

<210> 313

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 313

ggttggtctc atttgtttta ggacaa 26

<210> 314

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 314

tcttactcag ggctcagtgt gt 22

<210> 315

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 315

cctttagaga ataatgcagg ctttatctct 30

<210> 316

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 316

atttttgtga taacccagac tctgatgt 28

<210> 317

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 317

cgcggttttt gtcagcttac tc 22

<210> 318

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 318

caaggacctt tctctcaggc at 22

<210> 319

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 319

gtggaaccaa atgatactga tccattagat 30

<210> 320

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 320

ccactgtttc ctcatttaat ggcttct 27

<210> 321

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 321

tcttcatctt ccttcctttt catgtcattt 30

<210> 322

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 322

agttcttttg gtcatcaatc tctttctcc 29

<210> 323

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 323

cagatacaag cttcccaaaa attctgat 28

<210> 324

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 324

ggtatctaca accattttag ggaatccaa 29

<210> 325

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 325

ggagacgatg aaaataatgt ctcataggt 29

<210> 326

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 326

ttaacactct gaagaaaagg gaaagttca 29

<210> 327

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 327

gtttgttacc taccttggag ggttt 25

<210> 328

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 328

cccaacagtg ttgtagatat gtaagtgat 29

<210> 329

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 329

cttcgcacag tgaaaactaa aatgga 26

<210> 330

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 330

attgataatt caaggactga cttactgctt 30

<210> 331

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 331

aggtaaagaa agtggagaat atgttcaaca 30

<210> 332

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 332

gctagagaag acaaggttaa ggagaac 27

<210> 333

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 333

gattttgagc atttctaaag ttagactgct 30

<210> 334

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 334

ttccagaatg acagaattct aacagagaac 30

<210> 335

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 335

agaaaaagag tcatggcaac atatgaga 28

<210> 336

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 336

ccctatcgta gtgtccacag tca 23

<210> 337

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 337

taaaaataga ataagggcca cattcagct 29

<210> 338

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 338

gctaggtcag tccttccaca ga 22

<210> 339

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 339

tccagacttc caggctggta tt 22

<210> 340

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 340

ctgcccagag tatttcgaat atcctta 27

<210> 341

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 341

tccctctgct ctctgttatc atca 24

<210> 342

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 342

aaactcagct ttttcactgg atcct 25

<210> 343

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 343

tgaacaatgg aaggcatcca ga 22

<210> 344

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 344

ctcttgtctc tgtcttcggc at 22

<210> 345

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 345

ccatcgcaga gccatttgtt tg 22

<210> 346

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 346

aaggccttga caccactaca tc 22

<210> 347

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 347

aaaaatcaga actgaaagaa gcagtacac 29

<210> 348

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 348

ccatgattga tacacatatc tcaaggtttg 30

<210> 349

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 349

ctgtttcatc cagagatgtt tcaaacc 27

<210> 350

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 350

ccagaagaaa atcaaagttc aaagagacac 30

<210> 351

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 351

ccctggtaac tttttagctg cctt 24

<210> 352

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 352

tgactcttca ctgggtatca aaagg 25

<210> 353

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 353

ggattgaagg ccaggaggat tt 22

<210> 354

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 354

tcaggtgaaa tcaaagtgct tttgc 25

<210> 355

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 355

catgaaaact tcactctaga tctccgtt 28

<210> 356

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 356

gcagagtaaa ctctatatgg ttctcattcc 30

<210> 357

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 357

cttaacccac agatgcacct ttg 23

<210> 358

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 358

gtgtttattt aaccaaatca cacaaatgca 30

<210> 359

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 359

cccacgccca ttaaaaatac attaaaaga 29

<210> 360

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 360

ggtgcagagc ccagatttct ta 22

<210> 361

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 361

agatgacagc ttaccagaat atcagc 26

<210> 362

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 362

gatctctaat tgacacagcc ctttca 26

<210> 363

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 363

actgatagaa gaactctgct ttagtaaagc 30

<210> 364

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 364

ggcttctctt ccaagagtat gacaataat 29

<210> 365

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 365

cttgtactaa ctatgctgtt gagaaagct 29

<210> 366

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 366

cttacactga ataggctatg aaaccct 27

<210> 367

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 367

ccctttctca aactggcatt gaa 23

<210> 368

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 368

gcactttact tcacgactcc agat 24

<210> 369

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 369

aggagaaccc tcaatcaaaa gaaacttatt 30

<210> 370

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 370

tataaagagg tccttgatta ggcacagt 28

<210> 371

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 371

aaaagggaca ccttgcagat gt 22

<210> 372

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 372

ccatataaat ctgtgcatca caaatcatgt 30

<210> 373

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 373

cagtttccta gcaatcattt tggatca 27

<210> 374

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 374

agtgtttcta tttcatggac agtgcaa 27

<210> 375

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 375

ccctttcgtc tatttgtcag acgaa 25

<210> 376

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 376

aaatgtgtgg tgatgctgaa aagtaac 27

<210> 377

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 377

gctcaaaagc aagcaataca aaatcttg 28

<210> 378

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 378

agtggtgtat ctctcttttt atttgcatct 30

<210> 379

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 379

gtagctgtat acgtatggcg tttct 25

<210> 380

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 380

ggatgaggga atacataaaa gttaacacac 30

<210> 381

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 381

gttacccata ttgagcaaag atgtgttg 28

<210> 382

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 382

actgaattat tttgcttgtt gtgagatagc 30

<210> 383

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 383

gtgcctggcc tgatacaatt aac 23

<210> 384

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 384

ttcttcctct ctttcattgc gaaatatgta 30

<210> 385

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 385

ggatggcaat taaatgccat gtttttc 27

<210> 386

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 386

gggctttctt gggatggaag aa 22

<210> 387

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 387

gatatgcgaa ttaagaagaa acaaaggca 29

<210> 388

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 388

tgtcataaaa gccatcagta ttgtagacaa 30

<210> 389

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 389

gaagatgtaa tgtgagatgc catctgt 27

<210> 390

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 390

aaattgagaa tacaggtgtt aaagtgggat 30

<210> 391

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 391

agaactaatt aactgttcag cccagtt 27

<210> 392

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 392

cctaaacaat catgtataca gatgatgcct 30

<210> 393

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 393

gctgcccact gctaagtttt ct 22

<210> 394

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 394

gatgcccaag gtaaggagaa aga 23

<210> 395

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 395

gggattgcta ataaagcatt tttgcatt 28

<210> 396

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 396

gcgctcaatg aaattatgtc agaaacac 28

<210> 397

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 397

tctgtcatgc tgccctttga ttt 23

<210> 398

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 398

cagaaaaaca tgagagagga tgaacga 27

<210> 399

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 399

gagaatttga caatagcgtt atacctttgc 30

<210> 400

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 400

gagatgacaa ttatcaacct catctgct 28

<210> 401

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 401

aaagatggat tgcttttcct ctggt 25

<210> 402

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 402

cacctgaaca aaatgatttt tcttgtcatc 30

<210> 403

<211> 33

<212> DNA

<213> Artificial sequence ()

<400> 403

agaaacattt aaatagcatt aagaacttgt agc 33

<210> 404

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 404

aggaaagaat ccaagtttgg tataccag 28

<210> 405

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 405

tttttgagaa tacaaaacca gacaaaggt 29

<210> 406

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 406

agtcttattg ttagcatgac cgtagaaatc 30

<210> 407

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 407

aaaaactcag tatcaacaac taccggta 28

<210> 408

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 408

gatcactagt tagctagcaa atttgcc 27

<210> 409

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 409

gctataagag gcagagttgt aacttga 27

<210> 410

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 410

gtgattttag ctcagttata gaggtgtgaa 30

<210> 411

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 411

attttgcatc ggcatgtttg aca 23

<210> 412

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 412

tatacaacag aatatacgat ggcctccata 30

<210> 413

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 413

caaaatggtg atgggttctg cat 23

<210> 414

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 414

ccatcttaga tcactctctt aatctccaga 30

<210> 415

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 415

tcagcaaact gaaaaacctc ttcttaca 28

<210> 416

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 416

tcttctacca ggctcttagc caa 23

<210> 417

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 417

ctaaatattt tgctcagata gctccagtct 30

<210> 418

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 418

accaagaaca actgtcaaaa atacatcct 29

<210> 419

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 419

aatggtcaca gggttatttc agtgaa 26

<210> 420

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 420

gcattagtag tggattttgc ttctctgata 30

<210> 421

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 421

aaccaactag cctcatgagt ctaga 25

<210> 422

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 422

tctattaaac atcactagcc aggaaaatcc 30

<210> 423

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 423

cccagaagct gattctctgt catg 24

<210> 424

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 424

tctgacattt tgtatgattc tttgcctcta 30

<210> 425

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 425

ttagctctta ctcctgctct gtca 24

<210> 426

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 426

gtgtctgtaa gcacccatat atcaacat 28

<210> 427

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 427

tccagactct gaagaacttt tctcaga 27

<210> 428

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 428

ttgacctaga gtcattttta tatgctgctt 30

<210> 429

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 429

acagacaggc agttctctac ca 22

<210> 430

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 430

ggtggcctaa acttaaattt ttctgtcc 28

<210> 431

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 431

agcttgtgtt gaaattgtaa ataccttgg 29

<210> 432

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 432

ctcagaagtg gtctttaaga tagtcatctg 30

<210> 433

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 433

cccactttca tgtagcagcc aa 22

<210> 434

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 434

tgttagtgga tctattcaac tcaaaacaca 30

<210> 435

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 435

aagctgtgaa actgtttagt gatattgaga 30

<210> 436

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 436

atcagtaaat agcaagtccg tttcatctt 29

<210> 437

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 437

tttttcctcc cttccagaat atatatgct 29

<210> 438

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 438

ccatggtacc cggtgatttc ag 22

<210> 439

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 439

agggaaacac tcagattaaa gaagatttgt 30

<210> 440

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 440

caactgggac actttctttc agtattttg 29

<210> 441

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 441

caggtgtttg tcttccaaac tttgaa 26

<210> 442

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 442

gcatcaacat atctggttct gaattttcaa 30

<210> 443

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 443

cctttttgat gaaaaagagc aaggtact 28

<210> 444

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 444

ggctgaattt tcaatgactg aataaggg 28

<210> 445

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 445

gcctcagtga taagaaagat gcct 24

<210> 446

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 446

tcactgaaaa gatcattgtt tgttaggact 30

<210> 447

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 447

atgacaaaaa tcatctctcc gaaaaacaag 30

<210> 448

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 448

ggtggcccta cctcaaaatt attactatta 30

<210> 449

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 449

acagaaatat caggtctaag ccatttgt 28

<210> 450

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 450

gcataaggag caatatttct gcagataact 30

<210> 451

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 451

acataaggtt tttgctgaca ttcagagt 28

<210> 452

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 452

ggaaaagact tgcttggtac tatcttctat 30

<210> 453

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 453

atggccaaac ataagctaca cct 23

<210> 454

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 454

attctcaatt tctgtacttg tggatcagt 29

<210> 455

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 455

tcttcactat tcacctacgt ctagaca 27

<210> 456

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 456

cacaggaaca tcagaaaaag tttcagtttt 30

<210> 457

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 457

ggagatttga cgtgaataga aaaattcgt 29

<210> 458

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 458

aagttctttt agaaactgga ccctttct 28

<210> 459

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 459

aaagtcaaag gctagccttg aaaaatg 27

<210> 460

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 460

tgcctgtagt aatcaagtgt ctcattttt 29

<210> 461

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 461

cgggaagaaa tgatggagat gct 23

<210> 462

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 462

ttctgagaat aattgtgagg gcaaatct 28

<210> 463

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 463

atttccacat cctagtggtg caa 23

<210> 464

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 464

aaaaacctgt agttcaacta aacagagga 29

<210> 465

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 465

atcctgtaag ggaatggaca ttcttg 26

<210> 466

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 466

gcgaatgtaa gtttagggat aacataggaa 30

<210> 467

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 467

ctttttacag tcactgggaa gcaaa 25

<210> 468

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 468

ttctagtctc tcctctcttc atccatg 27

<210> 469

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 469

ccctagagtc ccataatgta gttttctac 29

<210> 470

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 470

ttgcacacgc agtagacaac aca 23

<210> 471

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 471

ttttagagag gcttcgatgt gatttca 27

<210> 472

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 472

cccttctgca acttaaggag tgt 23

<210> 473

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 473

tgttttgatt cataaggtaa cagtgctct 29

<210> 474

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 474

aatgtcatat ggaagttgag agatttgct 29

<210> 475

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 475

aagagatgtg agcacacgtg aa 22

<210> 476

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 476

tgatgcaaag tcatttatgt ccagaga 27

<210> 477

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 477

ccatggtaaa gttgtctcaa gtatttcttc 30

<210> 478

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 478

gcagctcaga gcactaagtc ac 22

<210> 479

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 479

gtggtaagcc agagaggaca ca 22

<210> 480

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 480

cagagcatgc agattcagag gta 23

<210> 481

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 481

tgcttgatgg gcttggaaat ct 22

<210> 482

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 482

atttttggaa caatgaacag cctgaaat 28

<210> 483

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 483

cctgtttggg ctctagcaac tt 22

<210> 484

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 484

agaatgaaat taaacacgtg actctttgc 29

<210> 485

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 485

ggttacagcc ctacctgatc ga 22

<210> 486

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 486

gcacagcagt gtcctgtgat ta 22

<210> 487

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 487

gaagtgcaga aaagctgctg at 22

<210> 488

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 488

tgaggcaaaa gaagcatgtg act 23

<210> 489

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 489

gggaaattta ctttgtttcg agaatgatgc 30

<210> 490

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 490

cagcaaattt gctgtgctca cta 23

<210> 491

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 491

atgtcctaac acaataatga aatcagcaaa 30

<210> 492

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 492

aaaattccag attagaaaag tagcccagat 30

<210> 493

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 493

ggtgacaaaa accaagagta tgttagattc 30

<210> 494

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 494

ccaagtgagt gaggattatg tctgt 25

<210> 495

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 495

ccttaacact ggcaaggagg aa 22

<210> 496

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 496

tcataaacat tcctgcgaga atgtaagt 28

<210> 497

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 497

tctgggtcac aaatttgtct gtca 24

<210> 498

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 498

aacacaggtt tgcctaaatt cctagt 26

<210> 499

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 499

agttaggagg aggaagatgc ttca 24

<210> 500

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 500

gagtaacagg acggagcctc ta 22

<210> 501

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 501

ctggccacct tgcttttaca at 22

<210> 502

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 502

cctcaaggca aattggtcca tg 22

<210> 503

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 503

tgacttaacc acagagagga ttcca 25

<210> 504

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 504

accatgttta aagtctacat acacacaca 29

<210> 505

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 505

cacaaaacaa caacagactc ttttatctct 30

<210> 506

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 506

cctgaaagaa tgtagtgaat ttttgagcaa 30

<210> 507

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 507

ggactgttct gacatcaagg ca 22

<210> 508

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 508

ctgggagtac cagtgcaaga tac 23

<210> 509

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 509

ggaatcctca gcaatcctgt gt 22

<210> 510

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 510

cctagcacct gcttggtaca tag 23

<210> 511

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 511

ctaagaagta gatgagcaca tgaaagaagt 30

<210> 512

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 512

cccttttgtt tgtttttcag aaatttttgg 30

<210> 513

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 513

ggttctaggg ccaacctcta ct 22

<210> 514

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 514

acaggacaaa tacaacaaaa ttagagctca 30

<210> 515

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 515

actttaacag gatttggaaa aacatcagg 29

<210> 516

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 516

ccactctcaa agggcttctg at 22

<210> 517

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 517

attctttgcc acgtatttct agcct 25

<210> 518

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 518

cactttcaga ggcttcagtt tcttttt 27

<210> 519

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 519

tctcagccca gatgacttca aag 23

<210> 520

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 520

ccagacaaaa gagcttgggt atttatca 28

<210> 521

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 521

tggtgatatc tgtccctata gtcacatg 28

<210> 522

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 522

atctttgcag tcttggatag attaccaa 28

<210> 523

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 523

cggtttccag cagctgaaat ttg 23

<210> 524

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 524

catacctgac ttatctcttt gtggtgt 27

<210> 525

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 525

acagacaaaa gcaaaacatt gatggac 27

<210> 526

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 526

cagcagtcct agattgtgtg tct 23

<210> 527

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 527

catgcctgag agaaaggtcc tt 22

<210> 528

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 528

gttaaagtga tcgtgtttcc aagaaatttc 30

<210> 529

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 529

tttttctctt gagacaccat gaatgttt 28

<210> 530

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 530

atgcatgaca ttaacaggca aatgt 25

<210> 531

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 531

agaaaggctt ctatttttgc tcatattcct 30

<210> 532

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 532

cgatagtact gcaggatggt gt 22

<210> 533

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 533

agacttggta tgctaacaat taagagtgtt 30

<210> 534

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 534

aagtcagagt ctgaacttgc tatgtatatg 30

<210> 535

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 535

gctcacaact caatcattta agtgtgtt 28

<210> 536

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 536

agcatttcag atagggatac tcaaccta 28

<210> 537

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 537

caaacatgtt ggtttaattc aacttcagga 30

<210> 538

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 538

tgcccaggat atactggagt ga 22

<210> 539

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 539

gcttggtagc tcaaaggtgg ta 22

<210> 540

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 540

aaatctcctt ttctgtggag catgt 25

<210> 541

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 541

aaaatcaggg caagagctga gt 22

<210> 542

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 542

gtgttgttcc aaggacagct gtata 25

<210> 543

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 543

tgatgttgaa tggatgctgt aaaaatgg 28

<210> 544

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 544

actccactta atagttcttc atcaaccag 29

<210> 545

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 545

ggattttcaa cttagcaaaa ttgcctttt 29

<210> 546

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 546

gcaagagtca cttctagcat attctatgc 29

<210> 547

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 547

agacaaaaca agcaaaggtt gacttt 26

<210> 548

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 548

agaaatggaa tatctccatt ctgggagta 29

<210> 549

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 549

agccctattg tcttgcagaa ctg 23

<210> 550

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 550

accaattgct ctaggacaag aatgttaat 29

<210> 551

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 551

ctctctgtac ttcctggtaa attttcttgt 30

<210> 552

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 552

tcatttctca gaatgtatcc ctgaggaa 28

<210> 553

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 553

gggagtgtaa tatgcagcaa attactg 27

<210> 554

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 554

actggtgcag ttccaaagtc at 22

<210> 555

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 555

gcatcaacat ataagacttt cctcttcgt 29

<210> 556

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 556

gggagatact cccatctcca ga 22

<210> 557

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 557

gatgggaaag gagaagtgag aaaca 25

<210> 558

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 558

tggactgaaa attgggagct tgt 23

<210> 559

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 559

aaatggtatt tcaaagcttg catgct 26

<210> 560

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 560

gaaattttga gcatgagttt ttaagtgcaa 30

<210> 561

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 561

aagctctatc atctctttcc caggta 26

<210> 562

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 562

gatctgcatg aagccaagca tt 22

<210> 563

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 563

gcattctgtt tgtgacccac ataaa 25

<210> 564

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 564

cacctccttt ccacttcaga gat 23

Claims (13)

1. A primer composition for detecting BRCA1 gene mutation is characterized in that: the fragments amplified by the primer composition can cover the following genome regions:

(1) the whole coding region of BRCA1 and the region adjacent to +/-5-20 bp;

(2) sites of the intron region of BRCA1 that are closely associated with the onset of breast cancer;

(3) the region BRCA1 and the tagSNP sites in the upstream and downstream 1M ranges;

the primer composition comprises all primers with sequences of SEQ ID NO 1-172.

2. The primer composition of claim 1, which consists of primers having the sequences of SEQ ID NOS: 1-172, respectively.

3. A primer composition for detecting BRCA2 gene mutation is characterized in that: the fragments amplified by the primer composition can cover the following genome regions:

(1) the whole coding region of BRCA2 and the region adjacent to +/-5-20 bp;

(2) sites of the intron region of BRCA2 that are closely associated with the onset of breast cancer;

(3) the region BRCA2 and the tagSNP sites in the upstream and downstream 1M ranges;

the primer composition comprises all primers with the sequences of SEQ ID NO 173-564 respectively.

4. The primer composition as claimed in claim 3, which comprises the primers having the sequences of SEQ ID NO 173-564, respectively.

5. A primer composition for detecting BRCA1/2 gene mutation, which is characterized in that: comprising the primer composition of any one of claims 1-2 and the primer composition of any one of claims 3-4.

6. A detection reagent for detecting a mutation in BRCA1/2 gene, comprising the primer composition of claim 5.

7. A kit for detecting a mutation in BRCA1/2 gene, comprising the primer composition of claim 5 or the detection reagent of claim 6.

8. A kit for predicting the risk of breast cancer, comprising the primer composition of claim 5 or the detection reagent of claim 6.

9. A non-diagnostic method for detecting mutations in the BRCA1/2 gene in vitro, comprising the steps of:

(1) extracting whole blood DNA;

(2) amplifying the target region by multiplex PCR using the primer composition of claim 5;

(3) constructing a library by using the amplification product;

(4) high-throughput sequencing;

(5) and processing and analyzing a sequencing result.

10. The method of claim 9, wherein the constructing a library in step (3) is performed according to the Illumina standard library construction protocol.

11. The method of claim 9, wherein, in step (3), when the DNA molecules to be tested are from a plurality of test samples, the amplification products of each sample are tagged with a different tag sequence in constructing the library.

12. The method of claim 9, wherein the sequencing platform in step (4) is an Illumina sequencing platform.

13. The method of claim 9 wherein in step (5) the sequencing reads are aligned to human genome Hg19 to obtain the position of the reads on the reference genome, and the variation sites and indel sequence context are analyzed using GATK software annotated by VEP software.

Images