High-throughput detection kit for human BRCA1/2 gene mutation

文档序号：1553691 发布日期：2020-01-21 浏览：4次中文

阅读说明：本技术 人类brca1/2基因突变的高通量检测试剂盒 (High-throughput detection kit for human BRCA1/2 gene mutation ) 是由何曼文魏汉敏黎美燕陈芳蒋慧于 2018-07-12 设计创作，主要内容包括：本发明公开了人类BRCA1/2基因突变的高通量检测试剂盒。所述试剂盒包括195个特异引物对和74个通用引物对；195个特异引物对的上游特异引物的核苷酸序列分别见表1中第2行至第196行,与之对应的下游特异引物的核苷酸序列依次见表1中第197行至第391行。74个通用引物对的上游通用引物的核苷酸序列具体见表2中第2行,下游通用引物的核苷酸序列分别见表2中第3行至第76行。本发明提供的试剂盒基于BGISEQ-500测序平台多重PCR检测BRCA1/2基因突变,其不仅检测性能优良,能提供全面的变异位点解析,而且检测时间大大缩短,检测成本大大降低。本发明具有重大的应用价值。(The invention discloses a high-throughput detection kit for human BRCA1/2 gene mutation. The kit comprises 195 specific primer pairs and 74 universal primer pairs; the nucleotide sequences of the upstream specific primers of the 195 specific primer pairs are shown in Table 1, respectively, at lines 2 to 196, and the nucleotide sequences of the corresponding downstream specific primers are shown in Table 1, sequentially, at lines 197 to 391. The nucleotide sequences of the upstream universal primers of the 74 universal primer pairs are shown in the 2 nd row in Table 2, and the nucleotide sequences of the downstream universal primers are shown in the 3 rd to 76 th rows in Table 2. The kit provided by the invention is used for detecting BRCA1/2 gene mutation based on BGISEQ-500 sequencing platform multiplex PCR, has excellent detection performance, can provide comprehensive analysis of mutation sites, greatly shortens the detection time and greatly reduces the detection cost. The invention has great application value.)

1. A kit for detecting human BRCA1/2 gene mutation comprises a plurality of specific primer pairs and at least one universal primer pair;

each specific primer pair consists of an upstream specific primer and a downstream specific primer;

the upstream specific primer sequentially comprises a DNA fragment A, an Ad153 adaptor sequence 1 and a DNA fragment B from a5 'end to a 3' end; the DNA fragment A and the DNA fragment B are respectively identical with two segments on BRCA1 gene or BRCA2 gene, and the distance between the two segments on BRCA1 gene or BRCA2 gene is 12-15 nucleotides;

the downstream specific primer sequentially comprises a DNA fragment C, an Ad153 joint sequence 2 and a DNA fragment D from the 5 'end to the 3' end; the DNA fragment C and the DNA fragment D are respectively identical with two segments on BRCA1 gene or BRCA2 gene, and the distance between the two segments on BRCA1 gene or BRCA2 gene is 12-15 nucleotides;

neither Ad153 linker sequence 1 nor Ad153 linker sequence 2 can bind to BRCA1 gene or BRCA2 gene;

the upstream specific primer and the downstream specific primer of each specific primer pair have different nucleotide sequences and can be combined to different positions of BRCA1 gene or BRCA2 gene; the length of the target region detected by each specific primer pair is 81-146 bp; the target areas of all the specific primer pairs can cover the coding regions of the BRCA1 gene and the BRCA2 gene after being spliced;

each universal primer pair consists of an upstream universal primer and a downstream universal primer;

the upstream universal primer comprises a d153 adaptor sequence 3;

the downstream universal primer comprises a sequencing adaptor sequence and a DNA fragment 3 from the 5 'end to the 3' end;

the d153 linker sequence 3 is partially identical to the nucleotide sequence of Ad153 linker sequence 1; the DNA fragment 3 is partially identical to the nucleotide sequence of Ad153 linker sequence 2.

2. The kit of claim 1, wherein:

the DNA fragment A is a single-stranded DNA molecule consisting of 25-30 nucleotides;

the Ad153 linker sequence 1 is a single-stranded DNA molecule consisting of 28-32 nucleotides;

the DNA fragment B is a single-stranded DNA molecule consisting of 12-15 nucleotides;

the DNA fragment C is a single-stranded DNA molecule consisting of 25-30 nucleotides;

the Ad153 linker sequence 2 is a single-stranded DNA molecule consisting of 28-32 nucleotides;

the DNA fragment D is a single-stranded DNA molecule consisting of 12-15 nucleotides.

3. The kit of claim 1 or 2, wherein: the nucleotide sequences of the Ad153 linker sequence 1 and the Ad153 linker sequence 2 may be different or the same.

4. The kit of claim 1, wherein: and carrying out phosphorylation modification on the 5' end of the upstream universal primer.

5. The kit of claim 1, wherein: the downstream universal primer also comprises a specific segment; the specific segment is used for sliding recognition of a template when downstream universal primers are used for PCR amplification; the specific segment is located downstream of the sequencing adaptor sequence and upstream of the DNA fragment 3.

6. The kit of claim 1 or 5, wherein: the downstream universal primer also comprises a Barcode marking sequence; the Barcode tag sequence is located downstream of the sequencing adapter sequence, upstream of the DNA fragment 3 or the specific segment.

7. The kit of any one of claims 1 to 6, wherein:

the nucleotide sequence of the Ad153 linker sequence 1 is GCTCACAGAACGACATGGCTACGATCCGACTT;

the nucleotide sequence of the Ad153 linker sequence 2 is GTCTTCCTAAGACCGCTTGGCCTCCGACTT;

the nucleotide sequence of the sequencing linker sequence is AGCCAAGGAGTTG;

the nucleotide sequence of the specific segment is TT;

the nucleotide sequence of the Barcode marker sequence is any one of a1) -a 74): a1) CGGATTGCCG, respectively; a2) CATCACTCAC, respectively; a3) CAGCTGACTC, respectively; a4) TTCGCAGACA, respectively; a5) TTGTACCAAT, respectively; a6) ACCACAATCG, respectively; a7) GGAAGTCTGT, respectively; a8) AGAGTGTGGA, respectively; a9) GCTTGTGGTG, respectively; a10) TTGTCCTCTA, respectively; a11) ATTCGCTAGG, respectively; a12) CGATGACTAC, respectively; a13) ACAGCTCAGC, respectively; a14) TATCTAGGTT, respectively; a15) GAGATGGCAA, respectively; a16) CGCAAGATCT, respectively; a17) GCCGATAGCG, respectively; a18) CCATCGTTGC, respectively; a19) TGAACGATTA, respectively; a20) TAGAGCGAAC, respectively; a21) ATGTGTGAGA, respectively; a22) ATCCTAACAG, respectively; a23) CGCGTCTGCG, respectively; a24) GATGATCCTT, respectively; a25) GCTCAACGCT, respectively; a26) ATGCATCTAA, respectively; a27) AGCTCTGGAC, respectively; a28) CTATCACGTG, respectively; a29) GGACTAGTGG, respectively; a30) GCCAAGTCCA, respectively; a31) CCTGTCAAGC, respectively; a32) TAGAGGTCTT, respectively; a33) TATGGCAACT, respectively; a34) CTGCGTACAT, respectively; a35) ATCTCATTAA, respectively; a36) AAGTGGCGCA, respectively; a37) GGCCTTAATG, respectively; a38) TCTGAGGCGG, respectively; a39) CGAGCCGATT, respectively; a40) GATAACCGGC, respectively; a41) TCAATATTCC, respectively; a42) TCCGTTGAAT, respectively; a43) CAGTACAGTT, respectively; a44) ATTGAGGTAC, respectively; a45) ATTAGAAGTC, respectively; a46) CAACGCTTCA, respectively; a47) GGATCGCACG, respectively; a48) TGCCTTCCGA, respectively; a49) GCGACATCGG, respectively; a50) CATTCTAAGT, respectively; a51) CAGGCTTGGA, respectively; a52) ATCATCGTCT, respectively; a53) GTCTTGTGAG, respectively; a54) AGTAGGAACG, respectively; a55) TCACAACCAC, respectively; a56) GCAGGCCTTC, respectively; a57) TGGCAAGCTA, respectively; a58) GAGCATTGTC, respectively; a59) TGTGATTAGC, respectively; a60) CCTATGGACT, respectively; a61) TAGGCGATAG, respectively; a62) AGACCACGAT, respectively; a63) GTATTAGCCA, respectively; a64) CTCTGCACTG, respectively; a65) ACCAGCCTGA, respectively; a66) GCGTGAGTAT, respectively; a67) CGCGGAGCAT, respectively; a68) CAAGTTCACA, respectively; a69) AGCACCTCTC, respectively; a70) TTACAGTGCA, respectively; a71) TTGCCTAGGC, respectively; a72) GCTATGATGG, respectively; a73) AATTACCATG, respectively; a74) AGACATGGTG are provided.

8. The kit of any one of claims 1 to 7, wherein: the kit comprises 195 specific primer pairs and 74 universal primer pairs;

the nucleotide sequences of the upstream specific primers of the 195 specific primer pairs are b1) -b195 in sequence):

b1)ACATGTCTTTTCTTCCCTAGTATGTAAGGTGCTCACAGAACGACATGGCTACGATCCGACTTGCATAGGAGATAATC；

b2)AAACAAAAGCTAATAATGGAGCCACATAACGCTCACAGAACGACATGGCTACGATCCGACTTTGCAAAATATGTGGT；

b3)TTTCGTTCTCACTTAATTGAAGAAAGTAAAGCTCACAGAACGACATGGCTACGATCCGACTTGGTGTTTCCTGGGTT；

b4)AATTATATACCTTTTGGTTATATCATTCTTGCTCACAGAACGACATGGCTACGATCCGACTTTGAAGGCCCTTTCTT；

b5)ATGGTTTTATAGGAACGCTATGTTATTAAAGCTCACAGAACGACATGGCTACGATCCGACTTCCTACTGTGGTTGCT；

b6)CATAGAAAGTAATTGTGCAAACTTCCTGAGGCTCACAGAACGACATGGCTACGATCCGACTTCTTGAGTGTCATTCT；

b7)AGGAAGGATTTTCGGGTTCACTCTGTAGAAGCTCACAGAACGACATGGCTACGATCCGACTTTTCTGTAGCCCATAC；

b8)AAGGGGGCTAAGGCAGGAGGACTGCTTCTAGCTCACAGAACGACATGGCTACGATCCGACTTCAAGACTCCATCTCA；

b9)TCCAGCAATTATTATTAAATACTTAAAAAAGCTCACAGAACGACATGGCTACGATCCGACTTCAATTCAATGTAGAC；

b10)TTTTTTATAACTCACCATAGGGCTCATAAAGCTCACAGAACGACATGGCTACGATCCGACTTCTGCCTACCACAAAT；

b11)ACATCAATCCTTAATATTAACTAAATAGGAGCTCACAGAACGACATGGCTACGATCCGACTTAGACAAAGGTTCTCT；

b12)AGAATAATCTAATTACAGTACTGTATCTACGCTCACAGAACGACATGGCTACGATCCGACTTTGCCTGTTAAGTTGG；

b13)AACTACCCTGATACTTTTCTGGATGCCTCTGCTCACAGAACGACATGGCTACGATCCGACTTCAGTGGTGTTCAAAT；

b14)TGTCTTTAGTGAGTAATAAACTGCTGTTCTGCTCACAGAACGACATGGCTACGATCCGACTTTGGCATGAGTATTTG；

b15)TACATGTTTCCTTACTTCCAGCCCATCTGTGCTCACAGAACGACATGGCTACGATCCGACTTCTAAGCCAGGCTGTT；

b16)CACAGGGGATCAGCATTCAGATCTACCTTTGCTCACAGAACGACATGGCTACGATCCGACTTGTCCGCCTATCATTA；

b17)AATGCTGCTATTTAGTGTTATCCAAGGAACGCTCACAGAACGACATGGCTACGATCCGACTTAGGATTCTCTGAGCA；

b18)AATACATCAGCTACTTTGGCATTTGATTCAGCTCACAGAACGACATGGCTACGATCCGACTTGAGTCATCAGAACCT；

b19)ATTTGGAGTGAACTCTTTCACTTTTACATAGCTCACAGAACGACATGGCTACGATCCGACTTGATCACTGGCCAGTA；

b20)CTATAATTAGATTTTCAGTTACATGGCTTAGCTCACAGAACGACATGGCTACGATCCGACTTCCTTCTTCCGATAGG；

b21)ACGCTTTAATTTATTTGTGAGGGGACGCTCGCTCACAGAACGACATGGCTACGATCCGACTTCTCAGTAACAAATGC；

b22)CTGCTCCGTTTGGTTAGTTCCCTGATTTATGCTCACAGAACGACATGGCTACGATCCGACTTTTGAACTGCCAAATC；

b23)TGGGTTAGGATTTTTCTCATTCTGAATAGAGCTCACAGAACGACATGGCTACGATCCGACTTATTCTCATGACCACT；

b24)TTTTTAGGTGCTTTTGAATTGTGGATATTTGCTCACAGAACGACATGGCTACGATCCGACTTTTGCTTATACTGCTG；

b25)CAACTATCAATTTGCAATTCAGTACAATTAGCTCACAGAACGACATGGCTACGATCCGACTTCTACTGACTACTAGT；

b26)TCTTTACCTTCCATGAGTTGTAGGTTTCTGGCTCACAGAACGACATGGCTACGATCCGACTTATTTGGTTGTACTTT；

b27)TGGGAAAGTATCGCTGTCATGTCTTTTACTGCTCACAGAACGACATGGCTACGATCCGACTTCTTGTTACTCTTCTT；

b28)CTTTAAGTTCACTGGTATTTGAACACTTAGGCTCACAGAACGACATGGCTACGATCCGACTTCATTTGTTAACTTCA；

b29)AACCCTTTCTCCACTTAACATGAGATCTTTGCTCACAGAACGACATGGCTACGATCCGACTTATTAGACACTTTAAC；

b30)TTCCCTAGAGTGCTAACTTCCAGTAACGAGGCTCACAGAACGACATGGCTACGATCCGACTTCCATAATCAGTACCA；

b31)TTCTATTATCTTTGGAACAACCATGAATTAGCTCACAGAACGACATGGCTACGATCCGACTTCAAATGCTGCACACT；

b32)AAATACTGAGCATCAAGTTCACTTTCTTCCGCTCACAGAACGACATGGCTACGATCCGACTTTCCCGACTGTGGTTA；

b33)CTTTAAGGACCCAGAGTGGGCAGAGAATGTGCTCACAGAACGACATGGCTACGATCCGACTTTGCATTTCCTGGATT；

b34)AGGAAAGCCTGCAGTGATATTAACTGTCTGGCTCACAGAACGACATGGCTACGATCCGACTTAGACTCATTCTTTCC；

b35)ATGAGTCCAGTTTCGTTGCCTCTGAACTGAGCTCACAGAACGACATGGCTACGATCCGACTTCTAGAGCCTCCTTTG；

b36)CCTCAAAGTTTTCCTCTAGCAGATTTTTCTGCTCACAGAACGACATGGCTACGATCCGACTTCAAATGACTTGATGG；

b37)TTTAAAAACATTTTCTCTAATGTTATTACGGCTCACAGAACGACATGGCTACGATCCGACTTTGTACTTGGAATGTT；

b38)TATTTCATTAATACTGGAGCCCACTTCATTGCTCACAGAACGACATGGCTACGATCCGACTTTTCATTAATATTGCT；

b39)GACCTCAGGTTGCAAAACCCCTAATCTAAGGCTCACAGAACGACATGGCTACGATCCGACTTTGGCCCTCTGTTTCT；

b40)AAATCAGATATGGAGAGAAATCTGTATTAAGCTCACAGAACGACATGGCTACGATCCGACTTCTTCATATTCTTGCT；

b41)CAAAACTAGTATCTTCCTTTATTTCACCATGCTCACAGAACGACATGGCTACGATCCGACTTCAGGTGTCTCAGAAC；

b42)GAAAGGGCTAGGACTCCTGCTAAGCTCTCCGCTCACAGAACGACATGGCTACGATCCGACTTGCTAAAAACAGCAGA；

b43)AGCAGGGAAGCTCTTCATCCTCACTAGATAGCTCACAGAACGACATGGCTACGATCCGACTTACTCTAATTTCTTGG；

b44)CTCCTCTGTGTTCTTAGACAGACACTCGGTGCTCACAGAACGACATGGCTACGATCCGACTTCCTAGTAGACTGAGA；

b45)TGATGTTCCTGAGATGCCTTTGCCAATATTGCTCACAGAACGACATGGCTACGATCCGACTTTCATTTAAGCTATTC；

b46)AGAACCAATCAAGAAAGGATCCTGGGTGTTGCTCACAGAACGACATGGCTACGATCCGACTTGTCTTCCAATTCACT；

b47)TTTTCTTCCAAGCCCGTTCCTCTTTCTTCAGCTCACAGAACGACATGGCTACGATCCGACTTTCCTTGTCACTCAGA；

b48)AAAGCATAAACATTTAGCTCACTTCTATAAGCTCACAGAACGACATGGCTACGATCCGACTTCACAAAAACCTGGTT；

b49)CTGAATGCAAAGGACACCACACACACGCATGCTCACAGAACGACATGGCTACGATCCGACTTCGCTTTTTACCTGAG；

b50)TTTAAGGAGACAATGAACCACAAACAATTGGCTCACAGAACGACATGGCTACGATCCGACTTGAGATGATGTCAGCA；

b51)TGGATTTCGCAGGTCCTCAAGGGCAGAAGAGCTCACAGAACGACATGGCTACGATCCGACTTAGGGTAGCTGTTAGA；

b52)TAGGTCCTTACTCTTCAGAAGGAGATAAAGGCTCACAGAACGACATGGCTACGATCCGACTTTTGCTTAAGATATCA；

b53)AGAGGGAAGGCTCAGATACAAACACAGCTAGCTCACAGAACGACATGGCTACGATCCGACTTTCCTTTTGGCCAGAA；

b54)CTTACCTTTCCACTCCTGGTTCTTTATTTTGCTCACAGAACGACATGGCTACGATCCGACTTCTGCAGACACCTCAA；

b55)TGAAAAAAATTAACAATCAGAGTTCAATATGCTCACAGAACGACATGGCTACGATCCGACTTATACCACAGCATCTT；

b56)ATGTTTCCGTCAAATCGTGTGGCCCAGACTGCTCACAGAACGACATGGCTACGATCCGACTTCCTCCACATCAACAA；

b57)AACCAGAATATCTTTATGTAGGATTCAGAGGCTCACAGAACGACATGGCTACGATCCGACTTTGTTCCAATACAGCA；

b58)GGTTGAAGATGGTATGTTGCCAACACGAGCGCTCACAGAACGACATGGCTACGATCCGACTTGTCTTCAGAAGGATC；

b59)TACCCAGCAGTATCAGTAGTATGAGCAGCAGCTCACAGAACGACATGGCTACGATCCGACTTGATTCTGCAACTTTC；

b60)ATTCTTCTGGGGTCAGGCCAGACACCACCAGCTCACAGAACGACATGGCTACGATCCGACTTTGACCCTTTCTGTTG；

b61)CAGAACTGTGATTGTTTTCTAGATTTCTTCGCTCACAGAACGACATGGCTACGATCCGACTTTGACAATACCTACAT；

b62)AGGCATGCGCCACCGTGCCTCGCCTCATGTGCTCACAGAACGACATGGCTACGATCCGACTTATGCAAGGTATTCTG；

b63)AAGGGAGGAGGGGAGAAATAGTATTATACTGCTCACAGAACGACATGGCTACGATCCGACTTCTACCCATTTTCCTC；

b64)ATAAAAGTAGTTTAGTATTACAATTAAAGAGCTCACAGAACGACATGGCTACGATCCGACTTGACTCAGCATCAGCA；

b65)TCATGGAAAATTTGTGCATTGTTAAGGAAAGCTCACAGAACGACATGGCTACGATCCGACTTAAGGAAGCAAATACA；

b66)GGGAGTGGAATACAGAGTGGTGGGGTGAGAGCTCACAGAACGACATGGCTACGATCCGACTTGGGAGGGAGCTTTAC；

b67)CTTATTTATGTGGTTGGGATGGAAGAGTGAGCTCACAGAACGACATGGCTACGATCCGACTTGAAAGTATCTAGCAC；

b68)TCTCCCAGGCTCTTACCTGTGGGCATGTTGGCTCACAGAACGACATGGCTACGATCCGACTTCAACAGATTTCTAGC；

b69)AGAATAGCCTCTAGAACATTTCAGCAATCTGCTCACAGAACGACATGGCTACGATCCGACTTTGGGATCTTGCTTAT；

b70)TAGGGACTGACAGGTGCCAGTCTTGCTCACGCTCACAGAACGACATGGCTACGATCCGACTTGTCCTCCCTCTCTGA；

b71)AAGAACTGTGCTACTCAAGCACCAGGTAATGCTCACAGAACGACATGGCTACGATCCGACTTCCCATGCAAAAGGAC；

b72)TCTGGGGTATCAGGTAGGTGTCCAGCTCCTGCTCACAGAACGACATGGCTACGATCCGACTTCTACACTGTCCAACA；

b73)CAGGGCCTGGAAAGGCCACTTTGTAAGCTCGCTCACAGAACGACATGGCTACGATCCGACTTTGGCTCTGTACCTGT；

b74)TGGGACTGAATTAGAATTCAAACAAATTTTGCTCACAGAACGACATGGCTACGATCCGACTTTTTACCTCAGTCACA；

b75)GACTTATTTACCAAGCATTGGAGGAATATCGCTCACAGAACGACATGGCTACGATCCGACTTTATTGGATCCAAAGA；

b76)TCCATAGTCAAGATCTTAAGCATTTTTTTCGCTCACAGAACGACATGGCTACGATCCGACTTTGTTCTGGGTCACAA；

b77)TTTTAAATAGATTTAGGACCAATAAGTCTTGCTCACAGAACGACATGGCTACGATCCGACTTCTTTCTTCAGAAGCT；

b78)TACGAACCAAACCTATTTAAAACTCCACAAGCTCACAGAACGACATGGCTACGATCCGACTTAATCAGCTGGCTTCA；

b79)GGGGGTAATCAGCAAACTGAAAAACCTCTTGCTCACAGAACGACATGGCTACGATCCGACTTACATTCTCATTCCCA；

b80)CAAATTTATAATCCAGAGTATATACATTCTGCTCACAGAACGACATGGCTACGATCCGACTTCTGTTTCAGGAAGGA；

b81)AGACATAAAAGTCTTCGCACAGTGAAAACTGCTCACAGAACGACATGGCTACGATCCGACTTGATGATGTTTCCTGT；

b82)GATAAACTAGTTTTTGCCAGTTTTTTAAAAGCTCACAGAACGACATGGCTACGATCCGACTTGCTTTGTTTTATTTT；

b83)GATTTGCTTTGTTTTATTTTAGTCCTGTTGGCTCACAGAACGACATGGCTACGATCCGACTTATGTAACACCACAAA；

b84)GTTATACCTTTGCCCTGAGATTTACAAATCGCTCACAGAACGACATGGCTACGATCCGACTTGCCTCATACAGGCAA；

b85)GCATTTCTATAAAAAATAAACTATTTTCTTGCTCACAGAACGACATGGCTACGATCCGACTTCAGACACCAAAACAT；

b86)AAGTACTTGAATCAATTCATTTTGTTTCAAGCTCACAGAACGACATGGCTACGATCCGACTTAAATAGTAGATGTGC；

b87)ACAATACACATAAATTTTTATCTTACAGTCGCTCACAGAACGACATGGCTACGATCCGACTTTCTGAAACTGTATTT；

b88)TTTTTGGACCTAGGTTGATTGCAGATAACTGCTCACAGAACGACATGGCTACGATCCGACTTGAAACCATGGATAAG；

b89)CTATAATTTTTGCAGAATGTGAAAAGCTATGCTCACAGAACGACATGGCTACGATCCGACTTGAAAGTCTGAAGAAA；

b90)AAAGGTTGTGAGAATAATATAAATTATATGGCTCACAGAACGACATGGCTACGATCCGACTTATGTGCTTCTGTTTT；

b91)ACATCAGGGAATTCATTTAAAGTAAATAGCGCTCACAGAACGACATGGCTACGATCCGACTTGGAAAGTCAATGCCA；

b92)TTCATTATGTTTTTCTAAATGTAGAACAAAGCTCACAGAACGACATGGCTACGATCCGACTTAAGAACTAGCAAGAC；

b93)AAGTAAGAACTAGCAAGACTAGGAAAAAAAGCTCACAGAACGACATGGCTACGATCCGACTTACGCTGATGAATGTG；

b94)TCCATTAGATTCAAATGTAGCAAATCAGAAGCTCACAGAACGACATGGCTACGATCCGACTTAAGTGACAAAATCTC；

b95)AGGTCTAAATGGAGCCCAGATGGAGAAAATGCTCACAGAACGACATGGCTACGATCCGACTTTTCTTCATGTGACCA；

b96)GAACAAAAGAAAGAAAGATTTTCTTACTTCGCTCACAGAACGACATGGCTACGATCCGACTTACGTATTTCTAGCCT；

b97)AAATCAGAGAAGCCATTAAATGAGGAAACAGCTCACAGAACGACATGGCTACGATCCGACTTGATGAAGAGCAGCAT；

b98)TGCATTCTTGCAGTAAAGCAGGCAATATCTGCTCACAGAACGACATGGCTACGATCCGACTTGCTTCTTCATTTCAG；

b99)GTTTTTCAGGTCATATGACTGATCCAAACTGCTCACAGAACGACATGGCTACGATCCGACTTAAGCCTCTGAAAGTG；

b100)CTTATGTCCAAATTTAATTGATAATGGAAGGCTCACAGAACGACATGGCTACGATCCGACTTCACACAGAATTCTGT；

b101)CTATACATGATGAAACATCTTATAAAGGAAGCTCACAGAACGACATGGCTACGATCCGACTTACCAAAAATCAGAAC；

b102)TCAGCCTCCCAAAAGTGCTGAGATTACAGGGCTCACAGAACGACATGGCTACGATCCGACTTCCAAACACTACCTTT；

b103)CTTTAATTTTGTCACTTTGTGTTTTTATGTGCTCACAGAACGACATGGCTACGATCCGACTTTCTTCTGTGAAAAGA；

b104)AACCAACTTTGTCCTTAACTAGCTCTTTTGGCTCACAGAACGACATGGCTACGATCCGACTTAATGTTCTAGAAATG；

b105)GCAAAATGTAATAAGGAAAAACTACAGTTAGCTCACAGAACGACATGGCTACGATCCGACTTGCTGATTCTCTGTCA；

b106)CAAAAAAGTTTCAGATATAAAAGAAGAGGTGCTCACAGAACGACATGGCTACGATCCGACTTTCACCCAGTACAACA；

b107)GATACTGACTTTCAATCCCAGAAAAGTCTTGCTCACAGAACGACATGGCTACGATCCGACTTAATGCCAGCACTCTT；

b108)ATTTCTAGAGGCAAAGAATCATACAAAATGGCTCACAGAACGACATGGCTACGATCCGACTTGGTAACAATTATGAA；

b109)AAAGAATCAAGATGTATGTGCTTTAAATGAGCTCACAGAACGACATGGCTACGATCCGACTTTGAGCTGTTGCCACC；

b110)AAAAAATCAAGAAGAAACTACTTCAATTTCGCTCACAGAACGACATGGCTACGATCCGACTTTCCAGACTCTGAAGA；

b111)AGTAGCTAATGAAAGGAATAATCTTGCTTTGCTCACAGAACGACATGGCTACGATCCGACTTACTTCATGAAACAGA；

b112)ATTTTCAAGAACTCTACCATGGTTTTATATGCTCACAGAACGACATGGCTACGATCCGACTTAAACAAGCAACCCAA；

b113)AATTAAAAAAGATTTGGTTTATGTTCTTGCGCTCACAGAACGACATGGCTACGATCCGACTTTAGTGTAAAGCAGCA；

b114)ATAGATAAAATACCAGAAAAAAATAATGATGCTCACAGAACGACATGGCTACGATCCGACTTGCAGGACTCTTAGGT；

b115)CTTCAGAACAGCTTCAAATAAGGAAATCAAGCTCACAGAACGACATGGCTACGATCCGACTTCATTAAGAAGAGCAA；

b116)ACAATATCCTACTAGTTTAGCTTGTGTTGAGCTCACAGAACGACATGGCTACGATCCGACTTGGCATTAGATAATCA；

b117)GTTGTTTCTGATTGTAAAAATAGTCATATAGCTCACAGAACGACATGGCTACGATCCGACTTTTTTCCAAGCAGGAT；

b118)AGAAGAATCAGGAAGTCAGTTTGAATTTACGCTCACAGAACGACATGGCTACGATCCGACTTAAGCTACATATTGCA；

b119)AATGCAGAGATGCTGATCTTCATGTCATAAGCTCACAGAACGACATGGCTACGATCCGACTTTTGGTCAGGTAGACA；

b120)GTTGAAAAATGACTGTAACAAAAGTGCTTCGCTCACAGAACGACATGGCTACGATCCGACTTTGAAAATGAAGTGGG；

b121)AAAAGCTGTGAAACTGTTTAGTGATATTGAGCTCACAGAACGACATGGCTACGATCCGACTTAACTTCTGCAGAGGT；

b122)ACTGTAAGTGAAAAAAATAATAAATGCCAAGCTCACAGAACGACATGGCTACGATCCGACTTAATATTGAAATGACT；

b123)TACAAGAGAAATACTGAAAATGAAGATAACGCTCACAGAACGACATGGCTACGATCCGACTTAGTAGAAATTCTCAT；

b124)TGGCAGTGATTCAAGTAAAAATGATACTGTGCTCACAGAACGACATGGCTACGATCCGACTTTGAAACGGACTTGCT；

b125)TGTCTTAAATTATCTGGCCAGTTTATGAAGGCTCACAGAACGACATGGCTACGATCCGACTTATTAAAGAAGATTTG；

b126)TGTCAGATTTAACTTTTTTGGAAGTTGCGAGCTCACAGAACGACATGGCTACGATCCGACTTGTCATGGTAATACTT；

b127)AAAAGATTTTGAGACTTCTGATACATTTTTGCTCACAGAACGACATGGCTACGATCCGACTTGAAAAATATTAGTGT；

b128)ATTTAATAAAATTGTAAATTTCTTTGATCAGCTCACAGAACGACATGGCTACGATCCGACTTGCATAACTTTTCCTT；

b129)ACATAGTTAAACACAAAATACTGAAAGAAAGCTCACAGAACGACATGGCTACGATCCGACTTCTGGAAATCAACTAG；

b130)GGGTTTTCATACAGCTAGCGGGAAAAAAGTGCTCACAGAACGACATGGCTACGATCCGACTTATCTTTGGACAAAGT；

b131)AGGTACTAGTGAAATCACCAGTTTTAGCCAGCTCACAGAACGACATGGCTACGATCCGACTTCCTAAAGTACAGAGA；

b132)GTGTAAAGAAATGCAGAATTCTCTCAATAAGCTCACAGAACGACATGGCTACGATCCGACTTTTCTATTGAGACTGT；

b133)TATCTTTTTGAAAGTTAAAGTACATGAAAAGCTCACAGAACGACATGGCTACGATCCGACTTAGCAAAAAGTCCTGC；

b134)AGCCTTAGCTTTTTACACAAGTTGTAGTAGGCTCACAGAACGACATGGCTACGATCCGACTTTCAGACTTCATTACT；

b135)AAAAATGGCTTAGAGAAGGAATATTTGATGGCTCACAGAACGACATGGCTACGATCCGACTTTAAATACTGCAGATT；

b136)TCCGAAAAACAAGATACTTATTTAAGTAACGCTCACAGAACGACATGGCTACGATCCGACTTAGCTATTCCTACCAT；

b137)TGATTCAGGATATCTCTCAAAAAATAAACTGCTCACAGAACGACATGGCTACGATCCGACTTGCCAGTATTGAAGAA；

b138)ATATCCAATGTAAAAGATGCAAATGCATACGCTCACAGAACGACATGGCTACGATCCGACTTGAAGATATTTGCGTT；

b139)TAAATTGTCCATATCTAATAGTAATAATTTGCTCACAGAACGACATGGCTACGATCCGACTTTGCATTTAGGATAGC；

b140)ACAGTTTCAGTAAAGTAATTAAGGAAAACAGCTCACAGAACGACATGGCTACGATCCGACTTAAATTTGCCAAACGA；

b141)CAAACGAAAATTATGGCAGGTTGTTACGAGGCTCACAGAACGACATGGCTACGATCCGACTTGAGGATATTCTTCAT；

b142)ACATAACCAAAATATGTCTGGATTGGAGAAGCTCACAGAACGACATGGCTACGATCCGACTTACCTTGTGATGTTAG；

b143)ATGTAAATGTAGTATAGGGAAGCTTCATAAGCTCACAGAACGACATGGCTACGATCCGACTTAAATACTTGTGGGAT；

b144)AGATGCTTCATTACAAAACGCAAGACAAGTGCTCACAGAACGACATGGCTACGATCCGACTTAGATAGTACCAAGCA；

b145)ATACTGCTATACGTACTCCAGAACATTTAAGCTCACAGAACGACATGGCTACGATCCGACTTTTTCATATAATGTGG；

b146)AGTGGAAAGCAAGTTTCCATTTTAGAAAGTGCTCACAGAACGACATGGCTACGATCCGACTTAAGGGAGTGTTAGAG；

b147)ATTCACCTACGTCTAGACAAAATGTATCAAGCTCACAGAACGACATGGCTACGATCCGACTTTTGATAAGAGAAACC；

b148)AAAAACCTGCAGTAAAGAATTTAAATTATCGCTCACAGAACGACATGGCTACGATCCGACTTTGAAGGTGGTTCTTC；

b149)AACCAAAGTGTCACTTGTTGAGAACATTCAGCTCACAGAACGACATGGCTACGATCCGACTTACAGGCTTCACCTAA；

b150)TATAGAAGTTTGTTCTACTTACTCCAAAGAGCTCACAGAACGACATGGCTACGATCCGACTTTGAAACAGAAGCAGT；

b151)GACTTTTGAGAAATAAAACTGATATTATTTGCTCACAGAACGACATGGCTACGATCCGACTTATGAAATATTTCTTT；

b152)ACAGTAACATGGATATTCTCTTAGATTTTAGCTCACAGAACGACATGGCTACGATCCGACTTAAAATAATTGTTTCC；

b153)TTTGGAATGGCAACCATGGTGAATACAAAAGCTCACAGAACGACATGGCTACGATCCGACTTTATCACCATGTAGCA；

b154)TTATATGTGTACTAGTCAATAAACTTATATGCTCACAGAACGACATGGCTACGATCCGACTTCAGCACAACTAAGGA；

b155)CAAGAGATACAGAATCCAAATTTTACCGCAGCTCACAGAACGACATGGCTACGATCCGACTTCTGTCTAAATCTCAT；

b156)TTGGAAAAATCTTCAAGCAATTTAGCAGTTGCTCACAGAACGACATGGCTACGATCCGACTTTATCAAGTTTCTGCT；

b157)TTTGTTCCACCTTTTAAAACTAAATCACATGCTCACAGAACGACATGGCTACGATCCGACTTCAGTGTGTTAGGAAT；

b158)TAAAAATAAGATTAATGACAATGAGATTCAGCTCACAGAACGACATGGCTACGATCCGACTTCAACTCCAATCAAGC；

b159)GCCAGGGGTTGTGCTTTTTAAATTTCAATTGCTCACAGAACGACATGGCTACGATCCGACTTATTTATTCTTTGATA；

b160)AATTAAGAAGAAACAAAGGCAACGCGTCTTGCTCACAGAACGACATGGCTACGATCCGACTTTCTGTATCTTGCAAA；

b161)TGTGTGATACATGTTTACTTTAAATTGTTTGCTCACAGAACGACATGGCTACGATCCGACTTGTTTATTTTGTGTAG；

b162)TCTTTTCAGTTTCACACTGAAGATTATTTTGCTCACAGAACGACATGGCTACGATCCGACTTTGGACTGGAAAAGGA；

b163)AATGTAGTTTTTGTACAGAGAATAGTTGTAGCTCACAGAACGACATGGCTACGATCCGACTTATCATCCTATGTGGT；

b164)TTATTTGTTCAGGGCTCTGTGTGACACTCCGCTCACAGAACGACATGGCTACGATCCGACTTGCTTATTTCTAGAAT；

b165)GGGTTTATAATCACTATAGATGGATCATATGCTCACAGAACGACATGGCTACGATCCGACTTTGGAATGTGCCTTTC；

b166)TTTTATTCTCAGTTATTCAGTGACTTGTTTGCTCACAGAACGACATGGCTACGATCCGACTTAGAGTCACACTTCCT；

b167)AAATTGATAGAAGCAGAAGATCGGCTATAAGCTCACAGAACGACATGGCTACGATCCGACTTGGGATGACACAGCTG；

b168)TTCATTGAGCGCAAATATATCTGAAACTTCGCTCACAGAACGACATGGCTACGATCCGACTTTAGTGCAGATACCCA；

b169)GGGTGGTATGCTGTTAAGGCCCAGTTAGATGCTCACAGAACGACATGGCTACGATCCGACTTGTCTTAAAGAATGGC；

b170)TTTACTGTCTTACTAATCTTCCTAAGACTTGCTCACAGAACGACATGGCTACGATCCGACTTTTTAAGGCAGTTCTA；

b171)ATTTATTAATTTGTCCAGATTTCTGCTAACGCTCACAGAACGACATGGCTACGATCCGACTTCGCTGGTATACCAAA；

b172)GCCTCCCAAAGTTCTGGGATTACAGATGTGGCTCACAGAACGACATGGCTACGATCCGACTTCCTGATACAATTAAC；

b173)TTTTGGTGTGTGTAACACATTATTACAGTGGCTCACAGAACGACATGGCTACGATCCGACTTATCTGGATTATACAT；

b174)AGCAGTTATATAGTTTCTTATCTTTAAATCGCTCACAGAACGACATGGCTACGATCCGACTTTTTTATGCTTGGTTC；

b175)TTTTCTTAGAAAACACAACAAAACCATATTGCTCACAGAACGACATGGCTACGATCCGACTTTAACAAGACAGCAAG；

b176)GCACCTGAGAATATTATGTGAGAAACTGATGCTCACAGAACGACATGGCTACGATCCGACTTCTTAAGATGAGCTCT；

b177)TTTTGTTCTGATTGCTTTTTATTCCAATATGCTCACAGAACGACATGGCTACGATCCGACTTGGTTATTTCAGTGAA；

b178)GAGCAGTTAAGAGCCTTGAATAATCACAGGGCTCACAGAACGACATGGCTACGATCCGACTTAAGAAACAAGCTCAG；

b179)ACAAAAGGAACAAGGTTTATCAAGGGATGTGCTCACAGAACGACATGGCTACGATCCGACTTGTTGCGTATTGTAAG；

b180)TGTATTTATTTTGAAACAAACATTTAAATGGCTCACAGAACGACATGGCTACGATCCGACTTATTGCATCTTTCTCA；

b181)AGTATTTGGCGTCCATCATCAGATTTATATGCTCACAGAACGACATGGCTACGATCCGACTTGGAAAGAGATACAGA；

b182)CAAACCTTTCATTGTAATTTTTCAGTTTTGGCTCACAGAACGACATGGCTACGATCCGACTTTTTATGGAATCTCCA；

b183)TTTACCAGCCACGGGAGCCCCTTCACTTCAGCTCACAGAACGACATGGCTACGATCCGACTTCAGACTTTCAGCCAT；

b184)GCATATACCAAAATAAATAGGCATATTAGAGCTCACAGAACGACATGGCTACGATCCGACTTTCTTAAAATTCATCT；

b185)TCCATTCTAGGACTTGCCCCTTTCGTCTATGCTCACAGAACGACATGGCTACGATCCGACTTTACAATTTACTGGCA；

b186)TCATATGTTAATTGCTGCAAGCAACCTCCAGCTCACAGAACGACATGGCTACGATCCGACTTCAAATCAGGCCTTCT；

b187)AACTTTTCATTTCTGCTTTTAAAGGAAATAGCTCACAGAACGACATGGCTACGATCCGACTTATATGTGGGTTTGCA；

b188)CATACTTTGCAATGAAGCAGAAAACAAGCTGCTCACAGAACGACATGGCTACGATCCGACTTTGCAAATGATCCCAA；

b189)GAACTGAAATCACCTAACCTATTAGGAGTTGCTCACAGAACGACATGGCTACGATCCGACTTTGTGTAATATTTGCG；

b190)ACATAATTATGATAGGCTACGTTTTCATTTGCTCACAGAACGACATGGCTACGATCCGACTTTCTCCTAATTGTGAG；

b191)TGAGATATATTATCAAAGTCCTTTATCACTGCTCACAGAACGACATGGCTACGATCCGACTTGAAGTCTGTTTCCAC；

b192)GGGAGAAAGAGATTGATGACCAAAAGAACTGCTCACAGAACGACATGGCTACGATCCGACTTCCTTGGATTTCTTGA；

b193)TACATTTGTTTCTCCGGCTGCACAGAAGGCGCTCACAGAACGACATGGCTACGATCCGACTTGAGTTGTGGCACCAA；

b194)AAAAATTCAATGAAATTTCTCTTTTGGAAAGCTCACAGAACGACATGGCTACGATCCGACTTACGAAGAACTTGCAT；

b195)CAATTTATATCTGTCAGTGAATCCACTAGGGCTCACAGAACGACATGGCTACGATCCGACTTTCAGAAGATTATCTC；

the nucleotide sequence of the downstream specific primer corresponding to the downstream specific primer is b196) to b 390):

b196)AAATGAAGTTGTCATTTTATAAACCTTTTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTATGTTTTTCTAATGT；

b197)CAGACATTTAATAAATATTGAACGAACTTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTTCAGTCATAACAGCT；

b198)GAGCCTCATTTATTTTCTTTTTCTCCCCCCGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGAGTTGATCAAGGA；

b199)TTTTTAAATGGCTCTTAAGGGCAGTTGTGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCTATTTGCCTTTTG；

b200)TACAAAAGGAAGTAAATTAAATTGTTCTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTATAGATTTTGCATGC；

b201)TATTTGTTTACATGTCTTTTCTTATTTTAGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGATAATCACTTGCTG；

b202)TAGGGTTTCTCTTGGTTTCTTTGATTATAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTAACTGCAAACATA；

b203)GTATTTTACAGATGCAAACAGCTATAATTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTAACTCTCCTGAACA；

b204)GACTGATGATGGTCAATTTATTTTGTCCATGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTCAGGAGGAAAAGC；

b205)TAGCAGGAAACCAGTCTCAGTGTCCAACTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTGAGAACTCTGAGG；

b206)AACTAGCATTGTACCTGCCACAGTAGATGCGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGTTGAATATCTGTT；

b207)TTCTGTAATCGAAAGAGCTAAAATGTTTGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGTTCTGCATACATG；

b208)AGTTTATGAGGTTAGTTTCTCTAATATAGCGTCTTCCTAAGACCGCTTGGCCTCCGACTTCACCTCCAAGGTGTA；

b209)TTGTTATTTTTGTATATTTTCAGCTGCTTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGATGTAACAAATAC；

b210)CTGAGAAGCGTGCAGCTGAGAGGCATCCAGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTCTGTTTCAAACT；

b211)ACTCATGCCAGCTCATTACAGCATGAGAACGTCTTCCTAAGACCGCTTGGCCTCCGACTTACTAAAGACAGAATG；

b212)GCTGAATTCTGTAATAAAAGCAAACAGCCTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAACATAACAGATGG；

b213)CGGACTCCCAGCACAGAAAAAAAGGTAGATGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTGTGTGAGAGAAAA；

b214)GAGATACTGAAGATGTTCCTTGGATAACACGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGAAAGTTAATGAGT；

b215)CAAAGTAGCTGATGTATTGGACGTTCTAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTCTGGTTCTTCAGA；

b216)TTACTGGCCAGTGATCCTCATGAGGCTTTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGAGTTCACTCCAAA；

b217)GAAGACAAAATATTTGGGAAAACCTATCGGGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCCAACTTAAGCCAT；

b218)AGCGTCCCCTCACAAATAAATTAAAGCGTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAGGCCTTCATCCTG；

b219)AGTTCAAAAGACTCCTGAAATGATAAATCAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGAGCAGAATGGTCA；

b220)GAGAAAAATCCTAACCCAATAGAATCACTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTCAAAACGAAAGCT；

b221)AATTAAATATCCACAATTCAAAAGCACCTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGAGGAAGTCTTCTA；

b222)CTAGTAGTCAGTAGAAATCTAAGCCCACCTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAAATTGATAGTTGT；

b223)GCAAATTGATAGTTGTTCTAGCAGTGAAGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTACAACCAAATGCC；

b224)AAGAGTAACAAGCCAAATGAACAGACAAGTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGATACTTTCCCAGAG；

b225)TTACTAAGTGTTCAAATACCAGTGAACTTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTAGCCTTCCAAGAG；

b226)GAAGACCCCAAAGATCTCATGTTAAGTGGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTACTGAAAGATCTGTA；

b227)GGAAAGTATCTCGTTACTGGAAGTTAGCACGTCTTCCTAAGACCGCTTGGCCTCCGACTTAACAGAACCAAATAA；

b228)AAACCCCAAGGGACTAATTCATGGTTGTTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGACACAGAAGGCTT；

b229)GTGAACTTGATGCTCAGTATTTGCAGAATAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGCGCCAGTCATTTG；

b230)CCCACTCTGGGTCCTTAAAGAAACAAAGTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTAATGTGAACAAAAGG；

b231)CCTGTACAGACAGTTAATATCACTGCAGGCGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAGAAAGATAAGCCA；

b232)TGTCTATCATCTCAGTTCAGAGGCAACGAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCAAATAAACATGGA；

b233)GTTAAAACTAAATGTAAGAAAAATCTGCTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGAACATTCAATGTCA；

b234)TCACCTGAAAGAGAAATGGGAAATGAGAACGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGCACAATTAGCCGT；

b235)GTAGGTTCCAGTACTAATGAAGTGGGCTCCGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGTTCCAGTGATGAA；

b236)GCTATGCTTAGATTAGGGGTTTTGCAACCTGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGTCTTCCTGGAAGT；

b237)AGTAGTTCAGACTGTTAATACAGATTTCTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGATAACTTAGAACA；

b238)TTTGTTCTGAGACACCTGATGACCTGTTAGGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGGAAGATACTAGTT；

b239)AAGCGTCCAGAAAGGAGAGCTTAGCAGGAGGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCATACACATTTGGC；

b240)AGTCCTCAGAAGAGAACTTATCTAGTGAGGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCTTCCAACACTTGT；

b241)AATATACCTTCTCAGTCTACTAGGCATAGCGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGTCTGTCTAAGAAC；

b242)CCAGGTAATATTGGCAAAGGCATCTCAGGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGAAACAAAATGTTC；

b243)GCAAATACAAACACCCAGGATCCTTTCTTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAAATGAGGCATCAG；

b244)CCAATGAGAAGAAAAAGACACAGCAAGTTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCTTTTACATCTGAA；

b245)CAGCATCTGGGTGTGAGAGTGAAACAAGCGGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAGGGCTATCCTCTC；

b246)ATTTGTATGATATATTTTCATTTAATGGAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTCATTTTCTTGGTG；

b247)TACCATGCAACATAACCTGATAAAGCTCCAGTCTTCCTAAGACCGCTTGGCCTCCGACTTACTAGAAGCTGTGTT；

b248)CTTGTGGGGCATTCCTTTTTGAACAGTACCGTCTTCCTAAGACCGCTTGGCCTCCGACTTTACAATTTCACCTTT；

b249)TTGTGTATCATAGATTGATGCTTTTGAAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCTGAATTATCACTA；

b250)TTAAAGCAGTATTAACTTCACAGAAAAGTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCCAGAATCCAGAAG；

b251)GCTTTGGCTGCCCAGCAAGTATGATTTGTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCGATGGTTTTCTCC；

b252)CATTAGATGATAGGTGGTACATGCACAGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGAATAGAAACTACC；

b253)CTTAACCTAACTTTATTGGTCTTTTTAATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTAACTTTGTAATTCAA；

b254)TTAAACTTCTCCCATTCCTTTCAGAGGGAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTGGAATCAGCCTCT；

b255)ATCCTTCTGAAGACAGAGCCCCAGAGTCAGGTCTTCCTAAGACCGCTTGGCCTCCGACTTTACCATCTTCAACCT；

b256)CTGCTCATACTACTGATACTGCTGGGTATAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTGTGAGCAGGGAGA；

b257)ATAGGTAAACATATGCCATGGTGGAATAACGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGTGCTAGAGGTAAC；

b258)ATCTTGGGAGTGTAAAAAACTGAGGCTCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCACTTCCTGATTTT；

b259)CTTCTAATCCTTTGAGTGTTTTTCATTCTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGTGTGAACGGACAC；

b260)TCCTGTGCTCTTTTGTGAATCGCTGACCTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGAGCACGTTCTTCT；

b261)GTGTCTGCTCCACTTCCATTGAAGGAAGCTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTGATGGGTTGTGTT；

b262)ATTTTGAAGTCAGAGGAGATGTGGTCAATGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTCCAAAGCGAGCAA；

b263)GGAAGTAGCAGCAGAAATCATCAGGTGGTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTAAAGCTCTTCCTTTT；

b264)ATATTCTTTTATAACTAGATTTTCCTTCTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTCTCTCTTCCTCTC；

b265)CTTGCTATAAGCCTTCATCCGGAGAGTGTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTAAGTATGCAGAT；

b266)GCAAGACCCTGTCTCAAAAACAAACAAAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGTTCCAGTAGTCCT；

b267)ATCCTAAGAACTCATACAACCAGGACCCTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGCCTAGTCCAGGAG；

b268)GGAGAATGAATTGACACTAATCTCTGCTTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCAATTGGGCAGATG；

b269)CGGTGTAATTTATAAAGTTATATAAAATTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTGCAGCGTGTCTT；

b270)TATACACACATAAGGAACAGTTTATGGTTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTCGTACTGGGTTTTTA；

b271)TTTGTGGAGTTTTAAATAGGTTTGGTTCGTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTCAGATTCTTCTG；

b272)TTTGAATTTATCTAATTCTTTTACAGGAGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGTCAGCCCTTGCTC；

b273)GCCCAGCATGACACAATTAATGAATGAGCTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTTGCCTAAATTCC；

b274)TAGAAGTGGACAGGAAACATCATCTGCTTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTCACTGTGCGAAGACT；

b275)AAATATATGTAGGAAAATGTTTCATTTAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCTTCATCATACCTT；

b276)AAGGACTCAACCTAAAAGATTATTTAAAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAGGCTCTTAGCCAA；

b277)TAAACCAGGTAGAATATTTACCTTCACAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAAACTCCCACATAC；

b278)TAACGCTATTGTCAAATTCTCAATTACTAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAGGTAGAATATTTA；

b279)TAAGGGTGGGTGGTGTAGCTAAAGAACTTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGATCCACCTCAGCTC；

b280)TTTCCTTTTAACAGAAGTATTAGAGATGACGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTGCTCTTTCTTGTA；

b281)ACAAGGCATTCCAAAATTGTTAGCAATTTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTCATATTTACTTAC；

b282)GCAGAGTTTCACAGGAAGTTAAAATCACATGTCTTCCTAAGACCGCTTGGCCTCCGACTTGAGACTTTCTCAAAG；

b283)CAGAGGACTTACCATGACTTGCAGCTTCTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTCACTGTCTGTCACAG；

b284)GAGATCACGGGTGACAGAGCAAGACTCCACGTCTTCCTAAGACCGCTTGGCCTCCGACTTTCAAAACAACAACAA；

b285)TTGTTCTACATTTAGAAAAACATAATGAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGGTATCTACAACTG；

b286)AAATGAGTATTTTTCTTTCACTTGGTTTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTATCAGCGTTTGCTTC；

b287)AGGGCTTCTGATTTGCTACATTTGAATCTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTGGTTCCACTTCAG；

b288)TTCTCCATCTGGGCTCCATTTAGACCTGAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCATTCACAGGCCAAA；

b289)ACGTGGCAAAGAATTCTCTGAAGTAAGAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTCTCTGTGTCTAA；

b290)AGTCTGTATGAGATTCAAGATGCTGCTCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCACTGTTTCCTCAT；

b291)TACCCTGAAATGAAGAAGCCACTGGAGAAGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCTTTACTGCAAGAA；

b292)GTTTCTTTTTTAAAGTTTGGATCAGTCATAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCATTGAAAGTCTCT；

b293)TGGCTGGCCAGCTTCCATTATCAATTAAATGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCTCCTTCTGTGAGC；

b294)ATGTTTCATCATGTATAGCATAAATAAACTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTCAAAGTGGATATTA；

b295)CCTGAATCAGCATTTGCAAATGTAAGTGGTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCTTCAAACTGGGCT；

b296)AAATATAAGATATGAAGATTTTAAAAAGCAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGAATCGTCATCTAT；

b297)ATTTCTAGAACATTTCCTCAGAATTGTCCCGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGACAAAGTTGGTTC；

b298)TAAATAACTGTAGTTTTTCCTTATTACATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAAGATCCTGAGAGA；

b299)GCAGCCAAGACCTCTTCTTTTATATCTGAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGATCATTTTCACAC；

b300)TAAAATAAGAGTGCTGGCATTTTCATGATCGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTGGGATTGAAAGTC；

b301)GCTTGTCTGACATTTTGTATGATTCTTTGCGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTAGGTTTGACAGAA；

b302)TTCAGGTGGCAACAGCTCAACGTTTTTATAGTCTTCCTAAGACCGCTTGGCCTCCGACTTACATACATCTTGATT；

b303)AGTTTCTTCTTGATTTTTTTGGATTACTCTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTGAATTGTACCTT；

b304)AGCAAGATTATTCCTTTCATTAGCTACTTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTCATTGTCTGAGAA；

b305)CACTTGGGTTGCTTGTTTATCACCTGTGTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGTAGAGTTCTTGAA；

b306)GACCTAGAGTCATTTTTATATGCTGCTTTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCTCTGCAAGAACAT；

b307)TCATGTAATCATTATTTTTTTCTGGTATTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGATGTCCGATTTTA；

b308)TTAATGTTATGTTCAGAGAGCTTGATTTCCGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTGAAGCTACCTCCA；

b309)TGAGGCTTGCTCAGTTTCTTTTGATTATCTGTCTTCCTAAGACCGCTTGGCCTCCGACTTACAATTTCAACACAA；

b310)ACATCTGAGGGGTTATATGACTATTTTTACGTCTTCCTAAGACCGCTTGGCCTCCGACTTCACTACTCTGTAAAT；

b311)TGACTTCCTGATTCTTCTAATATAGTAGAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCCTTTTGGCTAGGT；

b312)TTCCTCAGAAGTGGTCTTTAAGATAGTCATGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTCAAATGTACTCTT；

b313)ACCAGAAGCACTTTTGTTACAGTCATTTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTTCCGTTTAATTTC；

b314)ACTAAACAGTTTCACAGCTTTTTGCAGAGCGTCTTCCTAAGACCGCTTGGCCTCCGACTTCAGTTTTGTGCCATG；

b315)ATTTTCTATCTTAAACATTGAAACAACAGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGAAGATAAACTTAT；

b316)TCTTCATTTTCAGTATTTCTCTTGTAATTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTACAAAAGTGCCAGTA；

b317)GCAAGTCCGTTTCATCTTTATGAATACAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTGAATCACTGCCAT；

b318)TGACAAATCTTCTTTAATCTGAGTGTTTCCGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCCAGATAATTTAAG；

b319)AGCAGTTAACTGTTCTTTATTTGAAGTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTGAGCTTTCGCAAC；

b320)GCAGTCTGAAAAAATGTATCAGAAGTCTCAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGCTCCGTTTTAGTA；

b321)GTTATGCAATTCTTCTGGTTTCTGATCAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTATTAAATGACTCTTT；

b322)AGTACCAACTGGGACACTTTCTTTCAGTATGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTCTGTTTCCTCATA；

b323)AAAACCCAATAGAGTAGGTTCTTTGATCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTGTCCCTGGAAGGT；

b324)TCTGTACTTTAGGGTCTTTGCCCATTGATGGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTCACTAGTACCTTG；

b325)GAGAGAATTCTGCATTTCTTTACACTTTGGGTCTTCCTAAGACCGCTTGGCCTCCGACTTAATGGTCTCACATGC；

b326)TTTCAAAAAGATACTTTTTGATGTTTTGAGGTCTTCCTAAGACCGCTTGGCCTCCGACTTACATAAATTATCACT；

b327)GTAAAAAGCTAAGGCTGAATTTTCAATGACGTCTTCCTAAGACCGCTTGGCCTCCGACTTATTTGTGTAACAAGT；

b328)ACAAATAATTTCCTACATAATCTGCAGTATGTCTTCCTAAGACCGCTTGGCCTCCGACTTGACCATCAAATATTC；

b329)GCTGTTAGACATGCTACTGTTACTTAAATAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGAGAGATGATTTTT；

b330)CAATACCAGAATCAAGTTTATTTTTTGAGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTATATACCTCATCAG；

b331)TAGTCACAAGTTCCTCAACGCAAATATCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGTATGCATTTGCAT；

b332)ATTATTACTATTAGATATGGACAATTTAATGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTTGCAGGGTGAAGA；

b333)ACTTTACTGAAACTGTCTGTAAATATGTCTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTTCATGTGAAACA；

b334)CATTCATCATTATCTAGAGAGTTATGAAGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTCCAATGCCTCGTAA；

b335)ATCCAGACATATTTTGGTTATGTTGTAAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGTCAGCAAAAACCT；

b336)GATGAGACTGACTTATGAAGCTTCCCTATAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTCTGAAGTTTCCAAA；

b337)TTCTATTTCAGAAAACACTTGTCTTGCGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGATACCTGGACAGA；

b338)TATTAAATGTTCTGGAGTACGTATAGCAGTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGAGCTGGTCTGAATG；

b339)CTTAACTTTGTGTAAGGAACTTTCTAAAATGTCTTCCTAAGACCGCTTGGCCTCCGACTTACTTGCTGTACTAAA；

b340)TCAACACGAGGAAGTATTTTTGATACATTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGAATAGTGAAGACTA；

b341)AACATTTAAGTTATTTGATAATTTAAATTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTCCATTTCTGAGTT；

b342)GTTCTCAACAAGTGACACTTTGGTTCCTAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTCTTGTTGAAATTG；

b343)ATTTGTTTTCACAGGAACATCAGAAAAAGTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTCCATTTTTACGTT；

b344)ACATGTAAAAAGAGAATGTGTGGCATGACTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGTCAGTTCATCATC；

b345)TACTGAATAAACACTTTAAAAATAGTGATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTAAAAATGAACACTTA；

b346)GCACTTTGAGAGGCAGGTGGATCACCTGAGGTCTTCCTAAGACCGCTTGGCCTCCGACTTACCATACCTATAGAG；

b347)TCATTATTTTTAGAAATGTTCATTTATAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTATACTGTATTAGAAT；

b348)AGTCAGATGTTCATACAAATGAGATTTAGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGGTGCGGTAAAATT；

b349)TTTCTTGTAGCAGAAACTTGATAAAATGGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAAATTGCTTGAAGAT；

b350)AATGTGATTTAGTTTTAAAAGGTGGAACAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGCCTGTAGTAATCA；

b351)GTCATTAATCTTATTTTTACTATCATCAGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTTTGCTTTTGTCT；

b352)GCAAAAATTCATCACACAAATTGTCATACAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTCTTCACACTTTGTG；

b353)AATCTAAAACATTAAAAAGGGCTTTAAAATGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGGAAAACCATCAGG；

b354)ACCTGTTTATGAGAACACGCAGAGGGAACTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCTTTCAGAGAGATT；

b355)ACAAAGCCATTTGTAGATACTAGTTAATGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGTCATAAAAGCCAT；

b356)GAGTACCTATAAAATTCTTCTTTTCCAGCCGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGTATGAGCCATCCA；

b357)TCTAGCCAACTTTTTAGTTCGAGAGACAGTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGATGAGGGAATACA；

b358)GGCATCTATTAGCAAATTCCTTAGGAAAGGGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCAGTTTCCATATGA；

b359)ATGTAATGCTTTAAACTTGCCTGTATTTTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTTCTGGGCTTAGGC；

b360)TGCAACATTTTGACATGGAAGTCACAGACTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCATACTGCCGTATA；

b361)GTATCTGCACTACTAGTTTTATTGCTAGAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCGCTCAATGAAATT；

b362)TTCTTTAAGACAGCTAAGAGGGGAGGATCTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCATACCACCCATCT；

b363)GGGGCTTCAAGAGGTGTACAGGCATCAGGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCTCCATGAAGAATA；

b364)AGCATTTAAAATTTAAATGTAAAACTTCTAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAATGACTGATTTTTA；

b365)CACAACCAACATTTCCTCCATCACTGAAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTGAGGAAAAGGTCTAG；

b366)AAACAAATTAATTGTATCAAAAGAAAGAAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAAGAATACATCATAC；

b367)TCCTCCTGAATTTTAGTGAATAAGGCTTCTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCCTCCACATATTTT；

b368)ACAATTTACTCATTAAAAATGTTAAATTCAGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGTTCTCATATTAGA；

b369)ACTCTCTCACCTCAAGGTAAGCTGGGTCTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTTCATAAAGCTCTG；

b370)TGTGATGGCCAGAGAGTCTAAAACAGCTTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTATCAAGCCTCATTAT；

b371)CTTCCTAATTTCCAACTGGATCTGAGCTTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTCATTTGCCTGTGATT；

b372)CAATACGCAACTTCCACACGGTTGTGACATGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTCCTTTTGTTCAG；

b373)TTGTTAGTAAGGTCATTTTTTAAGTTAATAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAAGCATTTACATACT；

b374)ATAAATACAGATCCTCTTTTATATTATCTCGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGTGGATTTTGCTTC；

b375)AAAGGTTTGTACCGGTAGTTGTTGATACTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGCTAACTGTATGTT；

b376)GAGATTCCATAAACTAACAAGCACTTATCAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAATGAAAGGTTTGTA；

b377)AATTAACTATATTGTGCATTACCTGTTTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTACAAATCCTATTAGG；

b378)AAGAGGCTTACTTTCAGATCACTAGTTAGCGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTGGTAGCTCCAACT；

b379)TGGATTCTGGTCGCCACTGGAGGTTGCTTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGAGGCTTAATAATGT；

b380)TTTTTCATTTTGTTGAATGTCTCTTGAAAGGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTAGCAGAAAACACA；

b381)AAAGCTATTTCCTTGATACTGGACTGTCAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTAAATGTGTGGTGATG；

b382)ACCAGAAGCTTGTTTCCTGTACCAGGAATGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGCCCTGAAGTACAG；

b383)ACATTTTCTAATTAAGTTTAATTACATTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTATACAACAGAATAT；

b384)TTTGAAGTCATCTGGGCTGAGACAGGTGTGGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTGGCCATACAAAGT；

b385)GCTCTTCTCTTTTTGCAGTTCTTTTGGTCAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCTTTACAAGACTTT；

b386)GGTGGCTGAAATGCCTTCTGTGCAGCCGGAGTCTTCCTAAGACCGCTTGGCCTCCGACTTATGGGACTAACAGGT；

b387)TTGAATTACTTTCCAAAAGAGAAATTTCATGTCTTCCTAAGACCGCTTGGCCTCCGACTTGAGTCATCTGAGGAG；

b388)GAGCAGTCCTAGTGGATTCACTGACAGATAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTGTTGAACCAGACA；

b389)GCTTATTTTTCTCACATTCTTCCGTACTGGGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTCTTTGATCAGAG；

b390)GTGGTTTGAAATTATATTCCAGTCTTATAAGTCTTCCTAAGACCGCTTGGCCTCCGACTTCGTCAATAATTTATT；

the nucleotide sequence of the upstream universal primer of the 74 universal primer pairs is CACAGAACGACATGGCTACGA;

the nucleotide sequences of the downstream universal primers of the 74 universal primer pairs are b392) -b465 respectively):

b392)AGCCAAGGAGTTGCGGATTGCCGTTGTCTTCCTAAGACCGCTTGG；

b393)AGCCAAGGAGTTGCATCACTCACTTGTCTTCCTAAGACCGCTTGG；

b394)AGCCAAGGAGTTGCAGCTGACTCTTGTCTTCCTAAGACCGCTTGG；

b395)AGCCAAGGAGTTGTTCGCAGACATTGTCTTCCTAAGACCGCTTGG；

b396)AGCCAAGGAGTTGTTGTACCAATTTGTCTTCCTAAGACCGCTTGG；

b397)AGCCAAGGAGTTGACCACAATCGTTGTCTTCCTAAGACCGCTTGG；

b398)AGCCAAGGAGTTGGGAAGTCTGTTTGTCTTCCTAAGACCGCTTGG；

b399)AGCCAAGGAGTTGAGAGTGTGGATTGTCTTCCTAAGACCGCTTGG；

b400)AGCCAAGGAGTTGGCTTGTGGTGTTGTCTTCCTAAGACCGCTTGG；

b401)AGCCAAGGAGTTGTTGTCCTCTATTGTCTTCCTAAGACCGCTTGG；

b402)AGCCAAGGAGTTGATTCGCTAGGTTGTCTTCCTAAGACCGCTTGG；

b403)AGCCAAGGAGTTGCGATGACTACTTGTCTTCCTAAGACCGCTTGG；

b404)AGCCAAGGAGTTGACAGCTCAGCTTGTCTTCCTAAGACCGCTTGG；

b405)AGCCAAGGAGTTGTATCTAGGTTTTGTCTTCCTAAGACCGCTTGG；

b406)AGCCAAGGAGTTGGAGATGGCAATTGTCTTCCTAAGACCGCTTGG；

b407)AGCCAAGGAGTTGCGCAAGATCTTTGTCTTCCTAAGACCGCTTGG；

b408)AGCCAAGGAGTTGGCCGATAGCGTTGTCTTCCTAAGACCGCTTGG；

b409)AGCCAAGGAGTTGCCATCGTTGCTTGTCTTCCTAAGACCGCTTGG；

b410)AGCCAAGGAGTTGTGAACGATTATTGTCTTCCTAAGACCGCTTGG；

b411)AGCCAAGGAGTTGTAGAGCGAACTTGTCTTCCTAAGACCGCTTGG；

b412)AGCCAAGGAGTTGATGTGTGAGATTGTCTTCCTAAGACCGCTTGG；

b413)AGCCAAGGAGTTGATCCTAACAGTTGTCTTCCTAAGACCGCTTGG；

b414)AGCCAAGGAGTTGCGCGTCTGCGTTGTCTTCCTAAGACCGCTTGG；

b415)AGCCAAGGAGTTGGATGATCCTTTTGTCTTCCTAAGACCGCTTGG；

b416)AGCCAAGGAGTTGGCTCAACGCTTTGTCTTCCTAAGACCGCTTGG；

b417)AGCCAAGGAGTTGATGCATCTAATTGTCTTCCTAAGACCGCTTGG；

b418)AGCCAAGGAGTTGAGCTCTGGACTTGTCTTCCTAAGACCGCTTGG；

b419)AGCCAAGGAGTTGCTATCACGTGTTGTCTTCCTAAGACCGCTTGG；

b420)AGCCAAGGAGTTGGGACTAGTGGTTGTCTTCCTAAGACCGCTTGG；

b421)AGCCAAGGAGTTGGCCAAGTCCATTGTCTTCCTAAGACCGCTTGG；

b422)AGCCAAGGAGTTGCCTGTCAAGCTTGTCTTCCTAAGACCGCTTGG；

b423)AGCCAAGGAGTTGTAGAGGTCTTTTGTCTTCCTAAGACCGCTTGG；

b424)AGCCAAGGAGTTGTATGGCAACTTTGTCTTCCTAAGACCGCTTGG；

b425)AGCCAAGGAGTTGCTGCGTACATTTGTCTTCCTAAGACCGCTTGG；

b426)AGCCAAGGAGTTGATCTCATTAATTGTCTTCCTAAGACCGCTTGG；

b427)AGCCAAGGAGTTGAAGTGGCGCATTGTCTTCCTAAGACCGCTTGG；

b428)AGCCAAGGAGTTGGGCCTTAATGTTGTCTTCCTAAGACCGCTTGG；

b429)AGCCAAGGAGTTGTCTGAGGCGGTTGTCTTCCTAAGACCGCTTGG；

b430)AGCCAAGGAGTTGCGAGCCGATTTTGTCTTCCTAAGACCGCTTGG；

b431)AGCCAAGGAGTTGGATAACCGGCTTGTCTTCCTAAGACCGCTTGG；

b432)AGCCAAGGAGTTGTCAATATTCCTTGTCTTCCTAAGACCGCTTGG；

b433)AGCCAAGGAGTTGTCCGTTGAATTTGTCTTCCTAAGACCGCTTGG；

b434)AGCCAAGGAGTTGCAGTACAGTTTTGTCTTCCTAAGACCGCTTGG；

b435)AGCCAAGGAGTTGATTGAGGTACTTGTCTTCCTAAGACCGCTTGG；

b436)AGCCAAGGAGTTGATTAGAAGTCTTGTCTTCCTAAGACCGCTTGG；

b437)AGCCAAGGAGTTGCAACGCTTCATTGTCTTCCTAAGACCGCTTGG；

b438)AGCCAAGGAGTTGGGATCGCACGTTGTCTTCCTAAGACCGCTTGG；

b439)AGCCAAGGAGTTGTGCCTTCCGATTGTCTTCCTAAGACCGCTTGG；

b440)AGCCAAGGAGTTGGCGACATCGGTTGTCTTCCTAAGACCGCTTGG；

b441)AGCCAAGGAGTTGCATTCTAAGTTTGTCTTCCTAAGACCGCTTGG；

b442)AGCCAAGGAGTTGCAGGCTTGGATTGTCTTCCTAAGACCGCTTGG；

b443)AGCCAAGGAGTTGATCATCGTCTTTGTCTTCCTAAGACCGCTTGG；

b444)AGCCAAGGAGTTGGTCTTGTGAGTTGTCTTCCTAAGACCGCTTGG；

b445)AGCCAAGGAGTTGAGTAGGAACGTTGTCTTCCTAAGACCGCTTGG；

b446)AGCCAAGGAGTTGTCACAACCACTTGTCTTCCTAAGACCGCTTGG；

b447)AGCCAAGGAGTTGGCAGGCCTTCTTGTCTTCCTAAGACCGCTTGG；

b448)AGCCAAGGAGTTGTGGCAAGCTATTGTCTTCCTAAGACCGCTTGG；

b449)AGCCAAGGAGTTGGAGCATTGTCTTGTCTTCCTAAGACCGCTTGG；

b450)AGCCAAGGAGTTGTGTGATTAGCTTGTCTTCCTAAGACCGCTTGG；

b451)AGCCAAGGAGTTGCCTATGGACTTTGTCTTCCTAAGACCGCTTGG；

b452)AGCCAAGGAGTTGTAGGCGATAGTTGTCTTCCTAAGACCGCTTGG；

b453)AGCCAAGGAGTTGAGACCACGATTTGTCTTCCTAAGACCGCTTGG；

b454)AGCCAAGGAGTTGGTATTAGCCATTGTCTTCCTAAGACCGCTTGG；

b455)AGCCAAGGAGTTGCTCTGCACTGTTGTCTTCCTAAGACCGCTTGG；

b456)AGCCAAGGAGTTGACCAGCCTGATTGTCTTCCTAAGACCGCTTGG；

b457)AGCCAAGGAGTTGGCGTGAGTATTTGTCTTCCTAAGACCGCTTGG；

b458)AGCCAAGGAGTTGCGCGGAGCATTTGTCTTCCTAAGACCGCTTGG；

b459)AGCCAAGGAGTTGCAAGTTCACATTGTCTTCCTAAGACCGCTTGG；

b460)AGCCAAGGAGTTGAGCACCTCTCTTGTCTTCCTAAGACCGCTTGG；

b461)AGCCAAGGAGTTGTTACAGTGCATTGTCTTCCTAAGACCGCTTGG；

b462)AGCCAAGGAGTTGTTGCCTAGGCTTGTCTTCCTAAGACCGCTTGG；

b463)AGCCAAGGAGTTGGCTATGATGGTTGTCTTCCTAAGACCGCTTGG；

464)AGCCAAGGAGTTGAATTACCATGTTGTCTTCCTAAGACCGCTTGG；

465)AGCCAAGGAGTTGAGACATGGTGTTGTCTTCCTAAGACCGCTTGG。

9. use of the specific primer pair and the universal primer pair as claimed in any one of claims 1 to 8 for detecting mutations in human BRCA1/2 gene.

10. A method for detecting human BRCA1/2 gene mutation, comprising the following steps:

(1) carrying out PCR amplification by using the genome DNA of a sample to be detected as a template and adopting all the specific primer pairs of any one of claims 1 to 8 to obtain a PCR amplification product;

(2) purifying the PCR amplification product obtained in the step (1) to obtain a purified product 1;

(3) taking the purified product 1 obtained in the step (2) as a template, and respectively carrying out PCR amplification by adopting the universal primer pair of any one of claims 1 to 8 to obtain corresponding PCR amplification products;

(4) respectively taking the PCR amplification products obtained in the step (3), and purifying to obtain purified products 2;

(5) respectively taking the purified products 2 obtained in the step (4) for sequencing; and obtaining the information of BRCA1/2 gene mutation in the genome DNA of the sample to be detected according to the sequencing result.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a high-throughput detection kit for human BRCA1/2 gene mutation, in particular to a high-throughput detection kit for human BRCA1/2 gene mutation based on a BGISEQ-500 sequencing platform.

Background

Breast cancer is one of the most common malignancies in women. Research shows that hereditary breast cancer accounts for 5-10% of the total breast cancer. About 90% of patients suffer from BRCA1 gene or BRCA2 gene mutation, while the risk of breast cancer of carriers suffering from BRCA1 gene mutation or BRCA2 gene mutation is 60-85%, and the risk of ovarian cancer is 39-44% and 11-18% respectively. Another study demonstrated that 40% to 50% of hereditary breast cancers are caused by mutations in BRCA1 gene. The NCCN guidelines strongly suggest genetic breast/ovarian cancer detection in people with BRCA1/2 pathogenic mutations or more than 1 breast cancer close relatives among family members. Therefore, the development of a BRCA1/2 detection kit with strong pertinence, high sensitivity and universality, the realization of early diagnosis screening, prognosis monitoring and individual medication guidance of breast cancer/ovarian cancer, the key point for preventing the occurrence of the breast cancer/ovarian cancer or improving the survival rate and the survival quality of patients is realized, and the construction is urgent and has great significance.

The BRCA1 gene and BRCA2 gene are cancer suppressor genes. The functions of the BRCA1 gene or BRCA2 gene in DNA damage repair, homologous recombination and transcription regulation are particularly remarkable and important. The mutation can be distributed in any region of the whole exon, and is usually a frame shift mutation or a nonsense mutation, so that the truncated body protein is generated. The mutations in genes BRCA1 and BRCA2 have high penetrance. According to the consensus of experts in BRCA interpretation, sequence variations encoding early stop codons, i.e., nonsense or frameshift mutations occurring before amino acid 1855 of BRCA1 and before amino acid 3309 of BRCA2, were defined as high-risk pathogenic mutations (pathogenic probability > 0.99).

The global NGS market is expected to amount to $ 100 billion in 2020, with tumor detection accounting for 60% (i.e., $ 60 billion), with 3 billion dollars for breast cancer gene detection. At present, domestic liquid biopsy companies are hundreds of companies, but few companies develop BRCA1/2 detection kits. The market quotations of similar products are different (hundreds to thousands) at home, while the quotations of foreign products are higher, for example, the quotation is 31680 yuan for the Rifeda tumor susceptibility gene BRCA1/2 breast cancer risk assessment and detection of ThermoFisher. Therefore, the BRCA1/2 detection kit with low cost is independently developed, and the kit is matched with a sequencing platform, so that the market preemption is facilitated.

At present, few companies are used for developing breast cancer/ovarian cancer BRCA1/2 detection kits in China, most of the companies are based on the fact that samples are provided by customers to perform probe hybridization capture and Next Generation Sequencing (NGS) detection services, and the requirements of BRCA1/2 full exon region capture and high-throughput sequencing are met. Disadvantages of this detection method are the high cost of ordering probes and the potential inefficient capture of probes. Other detection methods for BRCA gene mutation mainly comprise PCR + Sanger sequencing, fluorescent quantitative PCR and multiplex PCR + NGS method. Sanger sequencing is highly accurate but low throughput; the fluorescent quantitative PCR method generally detects a plurality of known gene mutation information and has low detection sensitivity; a plurality of companies of BRCA1/2 detection kits developed based on a multiplex PCR + NGS method exist, detection time can be shortened and cost can be reduced by preparing a target sequencing library of the multiplex PCR, but the problems of poor uniformity and difficult amplification of certain gene loci exist.

Disclosure of Invention

The invention aims to detect the mutation of the human BRCA1/2 gene.

The invention firstly provides a kit for detecting human BRCA1/2 gene mutation, which comprises a plurality of specific primer pairs and at least one universal primer pair;

each specific primer pair can consist of an upstream specific primer and a downstream specific primer;

the upstream specific primer can comprise a DNA fragment A, an Ad153 adaptor sequence 1 (namely omega ring 1 in the embodiment) and a DNA fragment B from a5 'end to a 3' end in sequence; DNA fragment A and DNA fragment B are respectively identical to two segments on BRCA1 gene or BRCA2 gene, and the distance between the two segments on BRCA1 gene or BRCA2 gene can be 12-15 nucleotides;

the downstream specific primer can comprise a DNA fragment C, an Ad153 adaptor sequence 2 (namely omega ring 2 in the embodiment) and a DNA fragment D from the 5 'end to the 3' end in sequence; DNA fragment C and DNA fragment D are identical to two segments on BRCA1 gene or BRCA2 gene respectively and the distance between the two segments on BRCA1 gene or BRCA2 gene can be 12-15 nucleotides;

neither Ad153 linker sequence 1 nor Ad153 linker sequence 2 can bind to BRCA1 gene or BRCA2 gene;

the upstream specific primer and the downstream specific primer of each specific primer pair have different nucleotide sequences and can be combined to different positions of BRCA1 gene or BRCA2 gene; the length of the target region detected by each specific primer pair can be 81-146 bp; the target areas of all the specific primer pairs can cover the coding regions of the BRCA1 gene and the BRCA2 gene after being spliced;

each universal primer pair can consist of an upstream universal primer and a downstream universal primer;

the upstream universal primer can include a d153 linker sequence 3;

the downstream universal primer may comprise a sequencing adaptor sequence and a DNA fragment 3 from the 5 'end to the 3' end;

The DNA fragment A can be a single-stranded DNA molecule consisting of 25-30 nucleotides.

The Ad153 linker sequence 1 can be a single-stranded DNA molecule consisting of 28-32 nucleotides.

The DNA fragment B can be a single-stranded DNA molecule consisting of 12-15 nucleotides.

The DNA fragment can be a single-stranded DNA molecule consisting of 25-30 nucleotides.

The Ad153 linker sequence 2 can be a single-stranded DNA molecule consisting of 28-32 nucleotides.

The DNA fragment can be a single-stranded DNA molecule consisting of 12-15 nucleotides.

The nucleotide sequences of the Ad153 linker sequence 1 and the Ad153 linker sequence 2 may be different or the same.

The 5' end of the upstream universal primer can be subjected to phosphorylation modification.

The downstream universal primer can also comprise a specific segment; the specific segment is used for sliding recognition of a template when downstream universal primers are used for PCR amplification; the specific segment may be located downstream of the sequencing adaptor sequence and upstream of the DNA fragment 3.

The downstream universal primer can also comprise a Barcode marker sequence; the Barcode tag sequence is located downstream of the sequencing adapter sequence, upstream of the DNA fragment 3 or the specific segment.

The sequencing adaptor sequence in the downstream universal primer is added for sequencing. According to the difference of sequencing platform, the sequencing linker sequence composed of other nucleotide sequence can be replaced. In embodiments of the invention, the BGISEQ-500 sequencing platform is used for sequencing. Accordingly, the nucleotide sequence of the sequencing adapter sequence (from 5 'to 3') was AGCCAAGGAGTTG.

Any of the above upstream universal primers may specifically be a d153 linker sequence 3 with a phosphorylated 5' end.

Any one of the downstream universal primers above may be composed of the sequencing adaptor sequence, the Barcode tag sequence, the specific segment and the DNA fragment 3 in sequence from the 5 'end to the 3' end.

The nucleotide sequence (from 5 'to 3') of any of the Ad153 linker sequences 1 described above may be GCTCACAGAACGACATGGCTACGATCCGACTT.

The nucleotide sequence (from 5 'to 3') of any of the Ad153 linker sequences 2 described above may be GTCTTCCTAAGACCGCTTGGCCTCCGACTT.

The nucleotide sequence (5 'to 3') of any of the sequencing adapter sequences described above may be AGCCAAGGAGTTG.

The nucleotide sequence (5 'to 3') of any of the specific segments described above may be TT.

The nucleotide sequence (from 5 'to 3') of any of the foregoing Barcode marker sequences may be any of a1) -a 74): a1) CGGATTGCCG, respectively; a2) CATCACTCAC, respectively; a3) CAGCTGACTC, respectively; a4) TTCGCAGACA, respectively; a5) TTGTACCAAT, respectively; a6) ACCACAATCG, respectively; a7) GGAAGTCTGT, respectively; a8) AGAGTGTGGA, respectively; a9) GCTTGTGGTG, respectively; a10) TTGTCCTCTA, respectively; a11) ATTCGCTAGG, respectively; a12) CGATGACTAC, respectively; a13) ACAGCTCAGC, respectively; a14) TATCTAGGTT, respectively; a15) GAGATGGCAA, respectively; a16) CGCAAGATCT, respectively; a17) GCCGATAGCG, respectively; a18) CCATCGTTGC, respectively; a19) TGAACGATTA, respectively; a20) TAGAGCGAAC, respectively; a21) ATGTGTGAGA, respectively; a22) ATCCTAACAG, respectively; a23) CGCGTCTGCG, respectively; a24) GATGATCCTT, respectively; a25) GCTCAACGCT, respectively; a26) ATGCATCTAA, respectively; a27) AGCTCTGGAC, respectively; a28) CTATCACGTG, respectively; a29) GGACTAGTGG, respectively; a30) GCCAAGTCCA, respectively; a31) CCTGTCAAGC, respectively; a32) TAGAGGTCTT, respectively; a33) TATGGCAACT, respectively; a34) CTGCGTACAT, respectively; a35) ATCTCATTAA, respectively; a36) AAGTGGCGCA, respectively; a37) GGCCTTAATG, respectively; a38) TCTGAGGCGG, respectively; a39) CGAGCCGATT, respectively; a40) GATAACCGGC, respectively; a41) TCAATATTCC, respectively; a42) TCCGTTGAAT, respectively; a43) CAGTACAGTT, respectively; a44) ATTGAGGTAC, respectively; a45) ATTAGAAGTC, respectively; a46) CAACGCTTCA, respectively; a47) GGATCGCACG, respectively; a48) TGCCTTCCGA, respectively; a49) GCGACATCGG, respectively; a50) CATTCTAAGT, respectively; a51) CAGGCTTGGA, respectively; a52) ATCATCGTCT, respectively; a53) GTCTTGTGAG, respectively; a54) AGTAGGAACG, respectively; a55) TCACAACCAC, respectively; a56) GCAGGCCTTC, respectively; a57) TGGCAAGCTA, respectively; a58) GAGCATTGTC, respectively; a59) TGTGATTAGC, respectively; a60) CCTATGGACT, respectively; a61) TAGGCGATAG, respectively; a62) AGACCACGAT, respectively; a63) GTATTAGCCA, respectively; a64) CTCTGCACTG, respectively; a65) ACCAGCCTGA, respectively; a66) GCGTGAGTAT, respectively; a67) CGCGGAGCAT, respectively; a68) CAAGTTCACA, respectively; a69) AGCACCTCTC, respectively; a70) TTACAGTGCA, respectively; a71) TTGCCTAGGC, respectively; a72) GCTATGATGG, respectively; a73) AATTACCATG, respectively; a74) AGACATGGTG are provided.

The nucleotide sequence (from 5 'to 3') of any of the d153 linker sequences 3 (i.e., DNA fragment 1 in the examples) above can be CACAGAACGACATGGCTACGA.

The nucleotide sequence (5 'to 3') of any of the above DNA fragments 3 may be GTCTTCCTAAGACCGCTTGG.

Any one of the above upstream specific primers can be composed of the DNA segment A, the Ad153 adaptor sequence 1 and the DNA segment B from the 5 'end to the 3' end in sequence.

Any one of the downstream specific primers can be composed of the DNA fragment C, the Ad153 adaptor sequence 2 and the DNA fragment D from the 5 'end to the 3' end in sequence.

Any of the above kits may comprise 195 specific primer pairs and 74 universal primer pairs.

The nucleotide sequence (from 5 'to 3') of the upstream specific primers of the 195 specific primer pairs can be b1) -b 195):