专利名称：一种用于鉴别近交系小鼠的snp分型方法
技术领域：
本发明属于近交系小鼠品系鉴定与遗传质量检测领域，特别涉及一种用于鉴别近交系小鼠的SNP分型方法。
背景技术：
实验动物的来源与纯度对实验结果具有重要影响，近交系小鼠遗传质量监测与品系鉴定的高通量基因分型平台亟待建立。目前分子遗传标记有很多种，但多数不适用于大规模基因组研究。通过C57BL/6完整基因组序列和其他近交系小鼠序列对比已发现了大量的SNP信息。SNP数量庞大，而且能降低基因分型成本，数以万计的SNP在小鼠品系间被发现，作为遗传标记用于小鼠的基因分型和QTL分析。二元性遗传标记单核苷酸多态性位点(single nuclear polymorphism point,SNP)在染色体上的分布具有相对均一性且密度高，具有高通量，可自动化操作，费用相对低廉的特点，可应用在大规模基因组的遗传质量检测。
发明内容
本发明所要解决的技术问题是提供一种用于鉴别近交系小鼠的SNP分型方法，该方法仅需要45个SNP遗传位标就可用于近交系小鼠的分型和鉴定，不仅能减少反应次数和时间，而且操作简便迅速，能大大节约成本。本发明的一种用于鉴别近交系小鼠的SNP分型方法，包括(I)从NCB上选取分型所用遗传位标，21条染色体上挑选2_3个SNP位点，分成四组；然后对位点用Primer3在线设计引物，引物大小为15_25bp，Tm值为60°C，6(%为60. O ；(2)将上述分成四组的SNP位点用软件设计探针，产物长度为75_125bp，组内各探针长度依次相差2个bp以上；(3)进行多重PCR-LDR反应，退火温度从50°C至65°C，跨度为3°C，根据琼脂糖电泳和ABI377测序电泳结果，最终确定最佳的退火温度；(4)以近交系为模板，在上述最佳退火温度下进行四组多重PCR-LDR反应，分析 377分型数据，并与NCBI上的SNP位点信息比较，完成近交系小鼠遗传质量监测与品系鉴定。所述步骤(3)中的最佳的退火温度为56°C。所述步骤(3)中的LDR连接产物用标准6 %变性测序PAGE胶进行电泳，电泳在ABI 373A测序仪上进行，功率为30W,时间为I. 5-2h，ABI377数据进一步用GenemapperID 3. 2 来进行基因分型。有益.效果本发明的SNP分型方案，仅需要45个SNP遗传位标就可用于近交系小鼠的分型和鉴定，不仅能减少反应次数和时间，而且操作简便迅速，能大大节约成本，对其分型和研究具有重要意义，为近交系小鼠的鉴定和分型提供了一种新的方法。
图I为45个SNP位点在小鼠基因组上的分布；图2为同一模板DBA/2的四组377峰图；图3为不同模板的第三组引物的377峰图。
具体实施例方式下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。实施例I(I)从 NCBI (National Center for Biotechnology Information)得到选取 SNP 位点的序列，然后对这些位点进行引物设计，主引物设计主要借助Primerf程序完成。引物和探针序列列于表I和表2。表I基因组PCR扩增引物序列
GroupNo.Primer IDPrimer sequencePCRproduct (bp)Ialrsl3475716CACATCTGGGGCTCATCTG CTCATGGTTCTTGGCAGGAT199a2rsl3476538TCGATCTTTTGTAGCCTCTGG CAACAGCCCTTGACTCTCGT125a3rsl3476971ACTTGCATCTCCCTGTGAGG CCAGTGTTCGACTGGGATCT165a4rs27687437AACATGTGGAAACTTTGAGACG CTGACAGTCATGGGTGTTCCT159a5rs8271367GAAAAAGAAGCTCGCTGTCC GCTGAGTTTAACATTTTCCCACTG110a6rsl3478622ATGAAACAGCATTGACTCTGG AGCGGGTTAAATGACTTCACA158a7rs32390670GCTAGAGGAGTCGGCTCTGA ACGTAGC AAAGGCAGAAGGA173a8rs6373756TGTGGTGGTGTGCATCTGTA GACCTCTCATTGGCTTGGAG112a9rs30521859TCAGTCTCCAAAGATCACCTACT183
AAGTCAGTTACCACATGCAAGCalOrs29315008GCAAAACGAAACAGCAAACA TCACTGTCCCCAATTTCCTC173allrsl3476973CTGATTTGGCTTTGTGAGTAGC ATTTATGCCAGTTGCCCAAT122al2rsl3480113AGAACTGAGGATACAGCCATGA AGGATGCCCGTGATGAGTAT174IIblrsl3480972GGAATAACAGCCAGTAGAGCTGA TCAATAGCTGGTCCTGGAGTG225b2rs29171198AATGGGC AATACGAAGCAAG CATGTGCAGGGCAGATTTTA147b3rs29687299GCCATGCACCTTGAAAAGTT TGCACTGGACAGAGGTTTTG158b4rsl3482740TGTTCACGCTAGTGCAGCTC ACCAGGAGCCTGACCTTACC102b5rs4177540GGCCTGGCAGTACAAGAATC GAGCCTCTTGCTTGGTTGTC122b6rs2951B789GCATTTTGCTCACTCACCAC CCCCTCACACATGCCTAAGT148b7rs29581676GGTGTTCTTCTGTTGCTGTGG CCACCACAAGTGGTCTCTCA144b8rs31281387TGTGGGTCTTATCCATTGAGG CCATTTCATGGTCTCAAACAGA110b9rsl3483736TGGACCCTCTTATCTCCTTTATG TGAAAGCCTTGTCTGTTAGCAA112blOrsl3484115CCCTTTTTCCTATGATGATGTG CCTTGC ATAGAAGCCCATTG105bllrs8237229TCCTCGCTTACACTCAGAGC AGGAGAAGTGCTGGCAGAGA174bl2rsl3483732CCCACAGGCACTAGAGAATGA GTTTTCTGCTAGGATATTTGACACC167IIIclrsl3475728TCC.AGAAAGGAAGAGCACTGA AGCTCGTCTGTGGATGTGTG138c2rsl 3476543TTTGCTGTTGGAAACTGTGC TTGACTGCCCTGGTTTTAGG145c3rsl3477094TTGCAGGCTATGTGTATTCCA TGTGCTTTCTGCTCCTTAGTTTC152c4rs30307865CAATGATGCCAGGTTTCTCA TCTCAAATCTGTGCTTTCTGCT144c5rs8261944GGATGCCAACCTCTGACCT133[9200]
权利要求
1.一种用于鉴别近交系小鼠的SNP分型方法，包括(1)从NCB上选取分型所用遗传位标，21条染色体上挑选2-3个SNP位点，分成四组； 然后对位点用Primer3在线设计引物，引物大小为15_25bp，Tm值为60°C,GC%^ 60. O ；(2)将上述分成四组的SNP位点用软件设计探针，产物长度为75-125bp，组内各探针长度依次相差2个bp以上；(3)进行多重PCR-LDR反应，退火温度从50°C至65°C，跨度为3°C，根据琼脂糖电泳和 ABI377测序电泳结果，最终确定最佳的退火温度；(4)以近交系为模板，在上述最佳退火温度下进行四组多重PCR-LDR反应，分析377分型数据，并与NCBI上的SNP位点信息比较，完成近交系小鼠遗传质量监测与品系鉴定。
2.根据权利要求I所述的一种用于鉴别近交系小鼠的SNP分型方法，其特征在于所述步骤(I)中的引物序列为(1)al，产物长度199bp上游，5' -CACATCTGGGGCTCATCTG-3'；下游，CTCATGGTTCTTGGCAGGAT ；(2)a2，产物长度125bp上游，5' -TCGATCTTTTGTAGCCTCTGG-3'；下游，CAACAGCCCTTGACTCTCGT ；(3)a3，产物长度165bp上游，5' -ACTTGCATCTCCCTGTGAGG-3'；下游，CCAGTGTTCGACTGGGATCT ；(4)a4，产物长度159bp上游，5' -AACATGTGGAAACTTTGAGACG-3'；下游，CTGACAGTCATGGGTGTTCCT ；(5)a5，产物长度IlObp上游，5' -GAAAAAGAAGCTCGCTGTCC-3'；下游，GCTGAGTTTAACATTTTCCCACTG ；(6)a6，产物长度158bp上游，5' -ATGAAACAGCATTGACTCTGG-3'；下游，AGCGGGTTAAATGACTTCACA ；(7)a7，产物长度173bp上游，5' -GCTAGAGGAGTCGGCTCTGA-3'；下游，ACGTAGCAAAGGCAGAAGGA ；(8)a8，产物长度112bp上游，5' -TGTGGTGGTGTGCATCTGTA-3'；下游，GACCTCTCATTGGCTTGGAG ；(9)a9，产物长度183bp上游，5' -TCAGTCTCCAAAGATCACCTACT-3'；下游，AAGTCAGTTACCACATGCAAGC ；(10)al0，产物长度173bp 上游,5' -GCAAAACGAAACAGCAAACA-3'； 下游，TCACTGTCCCCAATTTCCTC ；(11)all，产物长度122bp 上游，5' -CTGATTTGGCTTTGTGAGTAGC-3'； 下游，ATTTATGCCAGTTGCCCAAT ；(12)al2，产物长度174bp 上游，5' -AGAACTGAGGATACAGCCATGA-3'； 下游，AGGATGCCCGTGATGAGTAT ；(13)bl，产物长度225bp 上游，5' -GGAATAACAGCCAGTAGAGCTGA-3'； 下游，TCAATAGCTGGTCCTGGAGTG ；(14)b2，产物长度 147bp 上游，5' -AATGGGCAATACGAAGCAAG-3'； 下游，CATGTGCAGGGCAGATTTTA ；(15)b3，产物长度 158bp 上游，5' -GCCATGCACCTTGAAAAGTT-3'； 下游，TGCACTGGACAGAGGTTTTG ；(16)b4，产物长度 102bp 上游，5' -TGTTCACGCTAGTGCAGCTC-3'； 下游，ACCAGGAGCCTGACCTTACC ；(17)b5，产物长度 122bp 上游，5' -GGCCTGGCAGTACAAGAATC-3'； 下游，GAGCCTCTTGCTTGGTTGTC ；(18)b6，产物长度 148bp 上游，5' -GCATTTTGCTCACTCACCAC-3'； 下游，CCCCTCACACATGCCTAAGT ；(19)b7，产物长度 144bp 上游，5' -GGTGTTCTTCTGTTGCTGTGG-3'； 下游，CCACCACAAGTGGTCTCTCA ；(20)b8，产物长度 IlObp 上游，5' -TGTGGGTCTTATCCATTGAGG-3'； 下游，CCATTTCATGGTCTCAAACAGA ；(21)b9，产物长度112bp 上游，5' -TGGACCCTCTTATCTCCTTTATG-3'； 下游，TGAAAGCCTTGTCTGTTAGCAA ；(22)bl0，产物长度105bp 上游，5' -CCCTTTTTCCTATGATGATGTG-3'； 下游，CCTTGCATAGAAGCCCATTG ；(23)bll，产物长度174bp 上游，5' -TCCTCGCTTACACTCAGAGC-3'； 下游，AGGAGAAGTGCTGGCAGAGA ；(24)bl2，产物长度167bp 上游，5' -CCCACAGGCACTAGAGAATGA-3'； 下游，GTTTTCTGCTAGGATATTTGACACC ；(25)Cl，产物长度 138bp 上游，5' -TCCAGAAAGGAAGAGCACTGA-3'； 下游，AGCTCGTCTGTGGATGTGTG ；(26)c2，产物长度 145bp 上游，5' -TTTGCTGTTGGAAACTGTGC-3'； 下游，TTGACTGCCCTGGTTTTAGG ；(27)c3，产物长度 152bp 上游，5' -TTGCAGGCTATGTGTATTCCA-3'； 下游，TGTGCTTTCTGCTCCTTAGTTTC ；(28)c4，产物长度 144bp 上游，5' -CAATGATGCCAGGTTTCTCA-3'； 下游，TCTCAAATCTGTGCTTTCTGCT ；(29)c5，产物长度 133bp 上游，5' -GGATGCCAACCTCTGACCT-3'； 下游，ACATCTGCCATCTCCTGCTT ；(30)c6，产物长度 168bp 上游，5' -CTCCAGTCCCAAGGGAAAGT-3'； 下游，TCATACCCAACCCATTCAAGA ；(31)c7，产物长度 105bp 上游，5' -GCACCCAAAGGCTCTTGTAA-3'； 下游，GGCTTCTGAGTGGAAGATGC ；(32)c8，产物长度 131bp 上游，5' -GAAGCCAGAGGCTGTCTCAC-3'； 下游，TGTCATCACGCTGAATCTCC ；(33)c9，产物长度 182bp 上游，5' -GGTGTGCATGACTTTCTCTTCA-3'； 下游，CCGACTAAAAACACCATCTTTTCC ；(34)cl0，产物长度146bp 上游，5' -AGCCCCAGCTTCTGACATAG-3'； 下游，TGACAGGAATGGATGCTTCA ；(35)cll，产物长度 108bp 上游，5' -AAGAGCACCAGGAATGACCT-3'； 下游，CAGTGTCTAGAAGTGGGGTTTTG ；(36)dl，产物长度 145bp 上游，5' -GCCGACTTAGGAATTCTGTTTG-3'；下游，CCATCCCAGGAAGCTACAAC ；(37)d2，产物长度 133bp 上游，5' -CAGGGTGGAGCAAAGAACAC-3'；下游，ATGAGAAGGGCAAACACCAG ；(38)d3，产物长度 141bp 上游，5' -ATGCCATGCTCCTGAGAACT-3'；下游，TGCAGACCCTGGACTTGTCT ；(39)d4，产物长度 154bp 上游，5' -CCAGCCAGCCTAGCCTACTT-3'；下游，TTTGTGTGTGTGTGCCTGTG ；(40)d5，产物长度 200bp 上游，5' -GAGTCCATGCTATTTGATGTTTGAG-3'；下游，CACATTGGTTCACAGGACAA ；(41)d6，产物长度 204bp 上游，5' -GGCCTTACAGCTACCATTTCC-3'；下游，CACACTGGCTTCCTTCAACA ；(42)d7，产物长度 160bp 上游，5' -TGTGGACAGACAACTGAACCT-3'；下游，TGTGCCACACTAAGCCTACAG ；(43)d8，产物长度 155bp 上游，5' -TGTAAATGTGAGCCCCAACA-3'；下游，GCTTCCACAACCAAATGGATA ；(44)d9，产物长度 113bp 上游，5' -TGCAGGAGCTTGTAGAATGAAC-3'；下游，CAGCCTATAGTAGTTTGCATCCTG ；(45)dl0，产物长度144bp 上游，5' -CCAAAAGGAATCCTCAGCAG-3'；下游，AGCAAGAGATGCACCAGTGA。
3.根据权利要求I所述的一种用于鉴别近交系小鼠的SNP分型方法，其特征在于所述步骤(2)中的探针序列为(1)al，产物长度79-81bp 通用探针P-TTTTCTTGACTTTTAATTC TTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TCTTTCTTGCACTCTCTCTTGCTG ；G 配对特异性探针TTTCTTTCTTGCACTCTCTCTTGCTA ；(2)a2，产物长度83-85bp 通用探针P-CCTTTCATTCTGTGGCTTCTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针TTTTAATTTCCTGTGTCGTTTCCTTT ；T 配对特异性探针TTTTTTAATTTCCTGTGTCGTTTCCTTC ；(3)a3，产物长度87-89bp 通用探针P-CACACACACACACACAAAATTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针TTTTTGTCTGTGCAGTTCACCCCCCCCC ；G 配对特异性探针TTTTTTTGTCTGTGCAGTTCACCCCCCCCA ；(4)a4，产物长度91-93bp 通用探针P-CATGATAAAACCACTTTCATTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；G 配对特异性探针TTTTTTTTTCCTACAACACTGCACAATCAT ；T 配对特异性探针TTTTTTTTTTTCCTACAACACTGCACAATCAG ；(5)a5，产物长度95-97bp 通用探针P-TAACTGCAAAGCTGTGGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针ITITITITTAAACTGTGCAAGAAGCAAGGTAT ；G 配对特异性探针ITITITITITTAAACTGTGCAAGAAGCAAGGTAA ；(6)a6，产物长度99-101bp 通用探针P-CAAGCAAATGCAGCCTCTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ； C 配对特异性探针TTTTTTTTTTTTTATACATAAATTTAAATTTACG ；G 配对特异性探针TTTTTTTTTTTTTTTATACATAAATTTAAATTTACA ；(7)a7，产物长度103-105bp 通用探针P-AAAAAAACCCCAAAAGGAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；G 配对特异性探针TTTTTTTTTTTTTTGTACAGCGAGCCCGTTTCCCCC ；T 配对特异性探针TTTTTTTTTTTTTTTTGTACAGCGAGCCCGTTTCCCCA ；(8)a8，产物长度107-109bp 通用探针P-TTCCTGTCTTTTCCTAGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTTTTTTTTTTTTTTGGCCCATGAGCCCTAGAGATCT ；G 配对特异性探针TTTTTTTTTTTTTTTTTTGGCCCATGAGCCCTAGAGATCC ；(9)a9，产物长度lll-113bp 通用探针P-GTTGCCCTCCGTTCATAGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTTTTTTTTTTTTTTTAAATGACCTTGCCAATTGCCTTT ；T 配对特异性探针TTTTTTTTTTTTTTTTTTTAAATGACCTTGCCAATTGCCTTC ；(10)al0，产物长度115-117bp 通用探针P-GCCGGGACCTTGGGAAAACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针TTTTTTTTTTTTTTTTTTTCAAGGAGACTCGAGTTTACCTCT ；T 配对特异性探针TTTTTTTTTTTTTTTTTTTTTCAAGGAGACTCGAGTTTACCTCG ；(11)all，产物长度119-121bp 通用探针P-AAGATGATCCTGGTAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTTTTTTTTTTTTTTTTTTTAATTTTGTCATTGATAATTTCTG ；G 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTAATTTTGTCATTGATAATTTCTC ；.(12)al2，产物长度123-125bp .通用探针P-GCAACTTCAGAAACAAAGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ； A 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTAGAAATCTTTACTCACCATTTAC ；G 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTTTAGAAATCTTTACTCACCATTTAA ；.(13)bl，产物长度79-81bp 通用探针P-ACCTTTGTCACTTTCAATATTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TACCTTTGTCACTTTCAATA ；C 配对特异性探针TTTAGGGATTTATTATGCTTTCTACG ；.(14)b2，产物长度 83-85bp 通用探针P-TGGTGGGTCTTGAAGTGTGTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTATCTATATACTCGCTGGAGTAGT ；G 配对特异性探针TTTTTATCTATATACTCGCTGGAGTAGC ；.(15)b3，产物长度 87-89bp 通用探针P-ATGAGAGGTGTATTAGGTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针TITITTATTATCAGTAATGTACAAGCAG ；G 配对特异性探针TTTTTTTTATTATCAGTAATGTACAAGCAC ；.(16)b4，产物长度91-93bp 通用探针P-GCCTTGATGACCCAGAGCATTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TITITTTGGAAAACAATGGCGGCCGGCGAT ；G 配对特异性探针TITITITTTGGAAAACAATGGCGGCCGGCGAC ；.(17)b5，产物长度 95-97bp 通用探针P-GTTCTCCACCAATGTCCCATTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTTTTTTTTTAGCAAAGCCAACTCGTTCTCT ；G 配对特异性探针TTTTTTTTTTTTTAGCAAAGCCAACTCGTTCTCC ；.(18)b6，产物长度 99-101bp 通用探针p-GGCTGAGAGAACTGTGGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TITITITITTTCAAGAGGTCCAGAGGTCCAGAGT ；G 配对特异性探针TITITITITITTTCAAGAGGTCCAGAGGTCCAGAGC ；.(19)b7，产物长度 103-105bp 通用探针p-GTGTTAACAAACCACAGCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；G 配对特异性探针TTTTTTTTTTTTTGATTTTAGAAGCCATAAAGACCT ；T 配对特异性探针TTTTTTTTTTTTTTTGATTTTAGAAGCCATAAAGACCC ；(20)b8，产物长度 107-109bp 通用探针P-ATTATTCCTGAACATAATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTTTTTTTTTTTTTCTTTTAATCAGGGTGAATGCCAT ；G 配对特异性探针TTTTTTTTTTTTTTTTTTCTTTTAATCAGGGTGAATGCCAC ；(21)b9，产物长度lll-113bp 通用探针p-TTTAGACCTCTAAGACTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTTTTTTTTTTTTTTTTAGCAACAGTCTTTTTTATAATT ；C 配对特异性探针TTTTTTTTTTTTTTTTTTTTAGCAACAGTCTTTTTTATAATG ；(22)bl0，产物长度115-117bp 通用探针p-GAGTGGCACTGTCATCACATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM A 配对特异性探针TTTTTTTTTTTTTTTTTTTCGGGCTGCCTTTATCTCACTATT ；G 配对特异性探针TTTTTTTTTTTTTTTTTTTTTCGGGCTGCCTTTATCTCACTATC ；(23)bll，产物长度119-121bp 通用探针p-CTTTAAATTTTTGGTTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM A 配对特异性探针TTTTTTTTTTTTTTTTTTTTTCAGGACTGCAATGGTTTGAGCGT ；T 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTCAGGACTGCAATGGTTTGAGCGA ；(24)bl2，产物长度123-125bp 通用探针p-CCATGGCTATGAGTGTGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ； G 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTATCAACTGGAAACTCACCCTTGC ；T 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTTTATCAACTGGAAACTCACCCTTGA ；(25)Cl，产物长度 79-81bp 通用探针P-CAGGCTATGACTAGTTCATTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TATATCTCTCTTGGATTCAATAAT ；G 配对特异性探针TTTATATCTCTCTTGGATTCAATAAC ；(26)c2，产物长度 83-85bp 通用探针P-GTTGTCATCCAAAAAATAATTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针ITITITTAGGAGCAATGTTCTGAAAG ；T 配对特异性探针ITITITITTAGGAGCAATGTTCTGAAAA ；(27)c3，产物长度 87-89bp 通用探针P-TCCCCAAGGTGTCTAGACATTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针TTTTTTACTACATTAGATATTAAATGTG ；G 配对特异性探针TTTTTTTTACTACATTAGATATTAAATGTC ；(28)c4，产物长度 95-97bp 通用探针P-CATTAAACATAAGTTGTCATTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；G 配对特异性探针TTTTTTTTTACTAGACATGGTTGCTTTTTGCC ；T 配对特异性探针TTTTTTTTTTTACTAGACATGGTTGCTTTTTGCA ；(29)c5，产物长度 99-101bp 通用探针P-TTCCACTCGTCAGATATCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ； C 配对特异性探针TTTTTTTTTTTCATCCGCCCCGAGGGGGTTTCCG ；G 配对特异性探针TTTTTTTTTTTTTCATCCGCCCCGAGGGGGTTTCCC ；(30)c6，产物长度 103-105bp 通用探针p-GGAACTTCCTAAATCTGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针ITITITITITITTAAAATAGCAATCAGAAACAGAAG ；G 配对特异性探针ITITITITITITITTAAAATAGCAATCAGAAACAGAAC ；(31)c7，产物长度 107-109bp 通用探针p-CAACACCTTGGGGACAAAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；G 配对特异性探针TTTTTTTTTTTTTTTATGCAGAAATGTGTTTAAAACCC ；T 配对特异性探针TTTTTTTTTTTTTTTTTTATGCAGAAATGTGTTTAAAACCA ；(32)c8，产物长度 lll-113bp 通用探针p-GGTATGTGTGAGCTGAGGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTTTTTTTTTTTTTTTAGCCAGTGATTGTGTCTCCAAGT ；G 配对特异性探针TTTTTTTTTTTTTTTTTTTAGCCAGTGATTGTGTCTCCAAGC ；(33)c9，产物长度 115-117bp 通用探针p-AACTTTAGAGAGATAAAGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTTTTTTTTTTTTTTTTTGAGCACTGTGTGCTGTGGATTAT ；G 配对特异性探针TTTTTTTTTTTTTTTTTTTTTGAGCACTGTGTGCTGTGGATTAC ；(34)cl0，产物长度119-121bp 通用探针p-ATGTAACTATGTCAGAAGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针TTTTTTTTTTTTTTTTTTTTTCTCAATGTGGACCAGATACTATG ；T 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTCTCAATGTGGACCAGATACTATA ；(35)cll，产物长度123-125bp 通用探针p-TCTCTCTCTCTTCTAGGTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ； A 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTTTATTTTTTGATGAACATTTTTT ；G 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTTTGATGAACATTTTTC ；(36)dl，产物长度 79-81bp 通用探针P-GACTCTAAATGATTATAAATTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TCAGCGAAGCGGTGCAATGAGATT ；G 配对特异性探针TTTCAGCGAAGCGGTGCAATGAGATC ；(37)d2，产物长度 83-85bp 通用探针P-GCACTGAGCCTGTGTTCTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTAAGATGTCTCTTTCCACTTGAGT ；G 配对特异性探针TTTTTAAGATGTCTCTTTCCACTTGAGC ；(38)d3，产物长度 87-89bp 通用探针P-CTGAAGGCTAGGGCTAGGATTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TITITTGCACAAAAATGCCTTCAATAAT ；G 配对特异性探针TTTTTTTTGCACAAAAATGCCTTCAATAAC ；(39)d4，产物长度91-93bp 通用探针P-AGGTCAACCCAGGATTCCAITITITITITITITITITITITITITT-FAM ；C 配对特异性探针TTTTTTTCAAATGTATATGGTTGAGGCTAG ；T 配对特异性探针TTTTTTTTTCAAATGTATATGGTTGAGGCTAA ；(40)d5，产物长度 95-97bp 通用探针P-TCAATAAGGTTAATAGGCTITITITITITITITITITITITITITITT-FAM ；C 配对特异性探针TTTTTTTTTCTGGAATGTATGACATAAAATTG ；G 配对特异性探针TTTTTTTTTTTCTGGAATGTATGACATAAAATTC ；(41)d6，产物长度 99-101bp 通用探针p-CCAGTCTGGAGAGGGCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTTTTTTTTTGAAACTGATATATTCCCTGTCTT ；C 配对特异性探针TTTTTTTTTTTTTGAAACTGATATATTCCCTGTCTG ；(42)d7，产物长度 103-105bp 通用探针p-TCTCCACAGTCAACAGTAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针TTTTTTTTTTTTTTCCCTGCTCAGAGGGGCCCAGTG ；G 配对特异性探针TTTTTTTTTTTTTTTTCCCTGCTCAGAGGGGCCCAGTC ；(43)d8，产物长度 107-109bp 通用探针p-TCCCAAGAAGAATTTTATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针TTTTTTTTTTTTTTTGATATTCAGAATATTTTACTTAG ；G 配对特异性探针TTTTTTTTTTTTTTTTTGATATTCAGAATATTTTACTTAC ；(44)d9，产物长度 119-121bp 通用探针p-CCATCTTGAAATGCCCAGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配对特异性探针TTTTTTTTTTTTTTTTTTTTTGCCTATAGTAGTTTGCATCCTGT ；G 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTGCCTATAGTAGTTTGCATCCTGC ；(45)dl0，产物长度123-125bp 通用探针p-CATGGCCGGTGGGTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTGATGGCTCCTTGGAATTTGACTG ；T 配对特异性探针TTTTTTTTTTTTTTTTTTTTTTTTTGATGGCTCCTTGGAATTTGACTA。
4.根据权利要求I所述的一种用于鉴别近交系小鼠的SNP分型方法，其特征在于所述步骤(3)中的最佳的退火温度为56°C。
5.根据权利要求I所述的一种用于鉴别近交系小鼠的SNP分型方法，其特征在于所述步骤⑶中的LDR连接产物用标准6%变性测序PAGE胶进行电泳，电泳在ABI 373A测序仪上进行，功率为30W，时间为I. 5-2h，ABI377数据进一步用GenemapperID 3. 2来进行基因分型。
全文摘要
本发明涉及一种用于鉴别近交系小鼠的SNP分型方法，包括(1)遗传标记的选择，平均覆盖21染色体的共45个SNP位点，并对这些位点设计引物和探针；(2)四组多重PCR，每组含10～12对PCR引物；(3)四组多重LDR，每组含10～12个位点特异性探针；(4)产物快速分离鉴定。本发明的SNP分型方案，仅需要45个SNP遗传位标就可用于近交系小鼠的分型和鉴定，不仅能减少反应次数和时间，而且操作简便迅速，能大大节约成本，对其分型和研究具有重要意义，为近交系小鼠的鉴定和分型提供了一种新的方法。
文档编号C12Q1/68GK102586457SQ20121006714
公开日2012年7月18日 申请日期2012年3月14日 优先权日2012年3月14日
发明者姜宪环, 李凯, 蔡慧强, 谢建云, 谢雯, 鲍世民 申请人:东华大学