專利名稱：一種用于鑒別近交系小鼠的snp分型方法
技術領域：
本發(fā)明屬于近交系小鼠品系鑒定與遺傳質量檢測領域，特別涉及一種用于鑒別近交系小鼠的SNP分型方法。
背景技術：
實驗動物的來源與純度對實驗結果具有重要影響，近交系小鼠遺傳質量監(jiān)測與品系鑒定的高通量基因分型平臺亟待建立。目前分子遺傳標記有很多種，但多數(shù)不適用于大規(guī)?；蚪M研究。通過C57BL/6完整基因組序列和其他近交系小鼠序列對比已發(fā)現(xiàn)了大量的SNP信息。SNP數(shù)量龐大，而且能降低基因分型成本，數(shù)以萬計的SNP在小鼠品系間被發(fā)現(xiàn)，作為遺傳標記用于小鼠的基因分型和QTL分析。二元性遺傳標記單核苷酸多態(tài)性位點(single nuclear polymorphism point,SNP)在染色體上的分布具有相對均一性且密度高，具有高通量，可自動化操作，費用相對低廉的特點，可應用在大規(guī)?；蚪M的遺傳質量檢測。

發(fā)明內容
本發(fā)明所要解決的技術問題是提供一種用于鑒別近交系小鼠的SNP分型方法，該方法僅需要45個SNP遺傳位標就可用于近交系小鼠的分型和鑒定，不僅能減少反應次數(shù)和時間，而且操作簡便迅速，能大大節(jié)約成本。本發(fā)明的一種用于鑒別近交系小鼠的SNP分型方法，包括(I)從NCB上選取分型所用遺傳位標，21條染色體上挑選2_3個SNP位點，分成四組；然后對位點用Primer3在線設計引物，引物大小為15_25bp，Tm值為60°C，6(%為60. O ；(2)將上述分成四組的SNP位點用軟件設計探針，產物長度為75_125bp，組內各探針長度依次相差2個bp以上；(3)進行多重PCR-LDR反應，退火溫度從50°C至65°C，跨度為3°C，根據(jù)瓊脂糖電泳和ABI377測序電泳結果，最終確定最佳的退火溫度；(4)以近交系為模板，在上述最佳退火溫度下進行四組多重PCR-LDR反應，分析 377分型數(shù)據(jù)，并與NCBI上的SNP位點信息比較，完成近交系小鼠遺傳質量監(jiān)測與品系鑒定。所述步驟(3)中的最佳的退火溫度為56°C。所述步驟(3)中的LDR連接產物用標準6 %變性測序PAGE膠進行電泳，電泳在ABI 373A測序儀上進行，功率為30W,時間為I. 5-2h，ABI377數(shù)據(jù)進一步用GenemapperID 3. 2 來進行基因分型。有益.效果本發(fā)明的SNP分型方案，僅需要45個SNP遺傳位標就可用于近交系小鼠的分型和鑒定，不僅能減少反應次數(shù)和時間，而且操作簡便迅速，能大大節(jié)約成本，對其分型和研究具有重要意義，為近交系小鼠的鑒定和分型提供了一種新的方法。


圖I為45個SNP位點在小鼠基因組上的分布；圖2為同一模板DBA/2的四組377峰圖；圖3為不同模板的第三組引物的377峰圖。
具體實施例方式下面結合具體實施例，進一步闡述本發(fā)明。應理解，這些實施例僅用于說明本發(fā)明而不用于限制本發(fā)明的范圍。此外應理解，在閱讀了本發(fā)明講授的內容之后，本領域技術人員可以對本發(fā)明作各種改動或修改，這些等價形式同樣落于本申請所附權利要求書所限定的范圍。實施例I(I)從 NCBI (National Center for Biotechnology Information)得到選取 SNP 位點的序列，然后對這些位點進行引物設計，主引物設計主要借助Primerf程序完成。引物和探針序列列于表I和表2。表I基因組PCR擴增引物序列
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]
權利要求
1.一種用于鑒別近交系小鼠的SNP分型方法，包括(1)從NCB上選取分型所用遺傳位標，21條染色體上挑選2-3個SNP位點，分成四組； 然后對位點用Primer3在線設計引物，引物大小為15_25bp，Tm值為60°C,GC%^ 60. O ；(2)將上述分成四組的SNP位點用軟件設計探針，產物長度為75-125bp，組內各探針長度依次相差2個bp以上；(3)進行多重PCR-LDR反應，退火溫度從50°C至65°C，跨度為3°C，根據(jù)瓊脂糖電泳和 ABI377測序電泳結果，最終確定最佳的退火溫度；(4)以近交系為模板，在上述最佳退火溫度下進行四組多重PCR-LDR反應，分析377分型數(shù)據(jù)，并與NCBI上的SNP位點信息比較，完成近交系小鼠遺傳質量監(jiān)測與品系鑒定。
2.根據(jù)權利要求I所述的一種用于鑒別近交系小鼠的SNP分型方法，其特征在于所述步驟(I)中的引物序列為(1)al，產物長度199bp上游，5' -CACATCTGGGGCTCATCTG-3'；下游，CTCATGGTTCTTGGCAGGAT ；(2)a2，產物長度125bp上游，5' -TCGATCTTTTGTAGCCTCTGG-3'；下游，CAACAGCCCTTGACTCTCGT ；(3)a3，產物長度165bp上游，5' -ACTTGCATCTCCCTGTGAGG-3'；下游，CCAGTGTTCGACTGGGATCT ；(4)a4，產物長度159bp上游，5' -AACATGTGGAAACTTTGAGACG-3'；下游，CTGACAGTCATGGGTGTTCCT ；(5)a5，產物長度IlObp上游，5' -GAAAAAGAAGCTCGCTGTCC-3'；下游，GCTGAGTTTAACATTTTCCCACTG ；(6)a6，產物長度158bp上游，5' -ATGAAACAGCATTGACTCTGG-3'；下游，AGCGGGTTAAATGACTTCACA ；(7)a7，產物長度173bp上游，5' -GCTAGAGGAGTCGGCTCTGA-3'；下游，ACGTAGCAAAGGCAGAAGGA ；(8)a8，產物長度112bp上游，5' -TGTGGTGGTGTGCATCTGTA-3'；下游，GACCTCTCATTGGCTTGGAG ；(9)a9，產物長度183bp上游，5' -TCAGTCTCCAAAGATCACCTACT-3'；下游，AAGTCAGTTACCACATGCAAGC ；(10)al0，產物長度173bp 上游,5' -GCAAAACGAAACAGCAAACA-3'； 下游，TCACTGTCCCCAATTTCCTC ；(11)all，產物長度122bp 上游，5' -CTGATTTGGCTTTGTGAGTAGC-3'； 下游，ATTTATGCCAGTTGCCCAAT ；(12)al2，產物長度174bp 上游，5' -AGAACTGAGGATACAGCCATGA-3'； 下游，AGGATGCCCGTGATGAGTAT ；(13)bl，產物長度225bp 上游，5' -GGAATAACAGCCAGTAGAGCTGA-3'； 下游，TCAATAGCTGGTCCTGGAGTG ；(14)b2，產物長度 147bp 上游，5' -AATGGGCAATACGAAGCAAG-3'； 下游，CATGTGCAGGGCAGATTTTA ；(15)b3，產物長度 158bp 上游，5' -GCCATGCACCTTGAAAAGTT-3'； 下游，TGCACTGGACAGAGGTTTTG ；(16)b4，產物長度 102bp 上游，5' -TGTTCACGCTAGTGCAGCTC-3'； 下游，ACCAGGAGCCTGACCTTACC ；(17)b5，產物長度 122bp 上游，5' -GGCCTGGCAGTACAAGAATC-3'； 下游，GAGCCTCTTGCTTGGTTGTC ；(18)b6，產物長度 148bp 上游，5' -GCATTTTGCTCACTCACCAC-3'； 下游，CCCCTCACACATGCCTAAGT ；(19)b7，產物長度 144bp 上游，5' -GGTGTTCTTCTGTTGCTGTGG-3'； 下游，CCACCACAAGTGGTCTCTCA ；(20)b8，產物長度 IlObp 上游，5' -TGTGGGTCTTATCCATTGAGG-3'； 下游，CCATTTCATGGTCTCAAACAGA ；(21)b9，產物長度112bp 上游，5' -TGGACCCTCTTATCTCCTTTATG-3'； 下游，TGAAAGCCTTGTCTGTTAGCAA ；(22)bl0，產物長度105bp 上游，5' -CCCTTTTTCCTATGATGATGTG-3'； 下游，CCTTGCATAGAAGCCCATTG ；(23)bll，產物長度174bp 上游，5' -TCCTCGCTTACACTCAGAGC-3'； 下游，AGGAGAAGTGCTGGCAGAGA ；(24)bl2，產物長度167bp 上游，5' -CCCACAGGCACTAGAGAATGA-3'； 下游，GTTTTCTGCTAGGATATTTGACACC ；(25)Cl，產物長度 138bp 上游，5' -TCCAGAAAGGAAGAGCACTGA-3'； 下游，AGCTCGTCTGTGGATGTGTG ；(26)c2，產物長度 145bp 上游，5' -TTTGCTGTTGGAAACTGTGC-3'； 下游，TTGACTGCCCTGGTTTTAGG ；(27)c3，產物長度 152bp 上游，5' -TTGCAGGCTATGTGTATTCCA-3'； 下游，TGTGCTTTCTGCTCCTTAGTTTC ；(28)c4，產物長度 144bp 上游，5' -CAATGATGCCAGGTTTCTCA-3'； 下游，TCTCAAATCTGTGCTTTCTGCT ；(29)c5，產物長度 133bp 上游，5' -GGATGCCAACCTCTGACCT-3'； 下游，ACATCTGCCATCTCCTGCTT ；(30)c6，產物長度 168bp 上游，5' -CTCCAGTCCCAAGGGAAAGT-3'； 下游，TCATACCCAACCCATTCAAGA ；(31)c7，產物長度 105bp 上游，5' -GCACCCAAAGGCTCTTGTAA-3'； 下游，GGCTTCTGAGTGGAAGATGC ；(32)c8，產物長度 131bp 上游，5' -GAAGCCAGAGGCTGTCTCAC-3'； 下游，TGTCATCACGCTGAATCTCC ；(33)c9，產物長度 182bp 上游，5' -GGTGTGCATGACTTTCTCTTCA-3'； 下游，CCGACTAAAAACACCATCTTTTCC ；(34)cl0，產物長度146bp 上游，5' -AGCCCCAGCTTCTGACATAG-3'； 下游，TGACAGGAATGGATGCTTCA ；(35)cll，產物長度 108bp 上游，5' -AAGAGCACCAGGAATGACCT-3'； 下游，CAGTGTCTAGAAGTGGGGTTTTG ；(36)dl，產物長度 145bp 上游，5' -GCCGACTTAGGAATTCTGTTTG-3'；下游，CCATCCCAGGAAGCTACAAC ；(37)d2，產物長度 133bp 上游，5' -CAGGGTGGAGCAAAGAACAC-3'；下游，ATGAGAAGGGCAAACACCAG ；(38)d3，產物長度 141bp 上游，5' -ATGCCATGCTCCTGAGAACT-3'；下游，TGCAGACCCTGGACTTGTCT ；(39)d4，產物長度 154bp 上游，5' -CCAGCCAGCCTAGCCTACTT-3'；下游，TTTGTGTGTGTGTGCCTGTG ；(40)d5，產物長度 200bp 上游，5' -GAGTCCATGCTATTTGATGTTTGAG-3'；下游，CACATTGGTTCACAGGACAA ；(41)d6，產物長度 204bp 上游，5' -GGCCTTACAGCTACCATTTCC-3'；下游，CACACTGGCTTCCTTCAACA ；(42)d7，產物長度 160bp 上游，5' -TGTGGACAGACAACTGAACCT-3'；下游，TGTGCCACACTAAGCCTACAG ；(43)d8，產物長度 155bp 上游，5' -TGTAAATGTGAGCCCCAACA-3'；下游，GCTTCCACAACCAAATGGATA ；(44)d9，產物長度 113bp 上游，5' -TGCAGGAGCTTGTAGAATGAAC-3'；下游，CAGCCTATAGTAGTTTGCATCCTG ；(45)dl0，產物長度144bp 上游，5' -CCAAAAGGAATCCTCAGCAG-3'；下游，AGCAAGAGATGCACCAGTGA。
3.根據(jù)權利要求I所述的一種用于鑒別近交系小鼠的SNP分型方法，其特征在于所述步驟(2)中的探針序列為(1)al，產物長度79-81bp 通用探針P-TTTTCTTGACTTTTAATTC TTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TCTTTCTTGCACTCTCTCTTGCTG ；G 配對特異性探針TTTCTTTCTTGCACTCTCTCTTGCTA ；(2)a2，產物長度83-85bp 通用探針P-CCTTTCATTCTGTGGCTTCTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針TTTTAATTTCCTGTGTCGTTTCCTTT ；T 配對特異性探針TTTTTTAATTTCCTGTGTCGTTTCCTTC ；(3)a3，產物長度87-89bp 通用探針P-CACACACACACACACAAAATTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針TTTTTGTCTGTGCAGTTCACCCCCCCCC ；G 配對特異性探針TTTTTTTGTCTGTGCAGTTCACCCCCCCCA ；(4)a4，產物長度91-93bp 通用探針P-CATGATAAAACCACTTTCATTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；G 配對特異性探針TTTTTTTTTCCTACAACACTGCACAATCAT ；T 配對特異性探針TTTTTTTTTTTCCTACAACACTGCACAATCAG ；(5)a5，產物長度95-97bp 通用探針P-TAACTGCAAAGCTGTGGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針I(yè)TITITITTAAACTGTGCAAGAAGCAAGGTAT ；G 配對特異性探針I(yè)TITITITITTAAACTGTGCAAGAAGCAAGGTAA ；(6)a6，產物長度99-101bp 通用探針P-CAAGCAAATGCAGCCTCTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ； C 配對特異性探針TTTTTTTTTTTTTATACATAAATTTAAATTTACG ；G 配對特異性探針TTTTTTTTTTTTTTTATACATAAATTTAAATTTACA ；(7)a7，產物長度103-105bp 通用探針P-AAAAAAACCCCAAAAGGAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；G 配對特異性探針TTTTTTTTTTTTTTGTACAGCGAGCCCGTTTCCCCC ；T 配對特異性探針TTTTTTTTTTTTTTTTGTACAGCGAGCCCGTTTCCCCA ；(8)a8，產物長度107-109bp 通用探針P-TTCCTGTCTTTTCCTAGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTTTTTTTTTTTTTTGGCCCATGAGCCCTAGAGATCT ；G 配對特異性探針TTTTTTTTTTTTTTTTTTGGCCCATGAGCCCTAGAGATCC ；(9)a9，產物長度lll-113bp 通用探針P-GTTGCCCTCCGTTCATAGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTTTTTTTTTTTTTTTAAATGACCTTGCCAATTGCCTTT ；T 配對特異性探針TTTTTTTTTTTTTTTTTTTAAATGACCTTGCCAATTGCCTTC ；(10)al0，產物長度115-117bp 通用探針P-GCCGGGACCTTGGGAAAACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針TTTTTTTTTTTTTTTTTTTCAAGGAGACTCGAGTTTACCTCT ；T 配對特異性探針TTTTTTTTTTTTTTTTTTTTTCAAGGAGACTCGAGTTTACCTCG ；(11)all，產物長度119-121bp 通用探針P-AAGATGATCCTGGTAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTTTTTTTTTTTTTTTTTTTAATTTTGTCATTGATAATTTCTG ；G 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTAATTTTGTCATTGATAATTTCTC ；.(12)al2，產物長度123-125bp .通用探針P-GCAACTTCAGAAACAAAGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ； A 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTAGAAATCTTTACTCACCATTTAC ；G 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTTTAGAAATCTTTACTCACCATTTAA ；.(13)bl，產物長度79-81bp 通用探針P-ACCTTTGTCACTTTCAATATTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TACCTTTGTCACTTTCAATA ；C 配對特異性探針TTTAGGGATTTATTATGCTTTCTACG ；.(14)b2，產物長度 83-85bp 通用探針P-TGGTGGGTCTTGAAGTGTGTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTATCTATATACTCGCTGGAGTAGT ；G 配對特異性探針TTTTTATCTATATACTCGCTGGAGTAGC ；.(15)b3，產物長度 87-89bp 通用探針P-ATGAGAGGTGTATTAGGTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針TITITTATTATCAGTAATGTACAAGCAG ；G 配對特異性探針TTTTTTTTATTATCAGTAATGTACAAGCAC ；.(16)b4，產物長度91-93bp 通用探針P-GCCTTGATGACCCAGAGCATTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TITITTTGGAAAACAATGGCGGCCGGCGAT ；G 配對特異性探針TITITITTTGGAAAACAATGGCGGCCGGCGAC ；.(17)b5，產物長度 95-97bp 通用探針P-GTTCTCCACCAATGTCCCATTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTTTTTTTTTAGCAAAGCCAACTCGTTCTCT ；G 配對特異性探針TTTTTTTTTTTTTAGCAAAGCCAACTCGTTCTCC ；.(18)b6，產物長度 99-101bp 通用探針p-GGCTGAGAGAACTGTGGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TITITITITTTCAAGAGGTCCAGAGGTCCAGAGT ；G 配對特異性探針TITITITITITTTCAAGAGGTCCAGAGGTCCAGAGC ；.(19)b7，產物長度 103-105bp 通用探針p-GTGTTAACAAACCACAGCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；G 配對特異性探針TTTTTTTTTTTTTGATTTTAGAAGCCATAAAGACCT ；T 配對特異性探針TTTTTTTTTTTTTTTGATTTTAGAAGCCATAAAGACCC ；(20)b8，產物長度 107-109bp 通用探針P-ATTATTCCTGAACATAATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTTTTTTTTTTTTTCTTTTAATCAGGGTGAATGCCAT ；G 配對特異性探針TTTTTTTTTTTTTTTTTTCTTTTAATCAGGGTGAATGCCAC ；(21)b9，產物長度lll-113bp 通用探針p-TTTAGACCTCTAAGACTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTTTTTTTTTTTTTTTTAGCAACAGTCTTTTTTATAATT ；C 配對特異性探針TTTTTTTTTTTTTTTTTTTTAGCAACAGTCTTTTTTATAATG ；(22)bl0，產物長度115-117bp 通用探針p-GAGTGGCACTGTCATCACATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM A 配對特異性探針TTTTTTTTTTTTTTTTTTTCGGGCTGCCTTTATCTCACTATT ；G 配對特異性探針TTTTTTTTTTTTTTTTTTTTTCGGGCTGCCTTTATCTCACTATC ；(23)bll，產物長度119-121bp 通用探針p-CTTTAAATTTTTGGTTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM A 配對特異性探針TTTTTTTTTTTTTTTTTTTTTCAGGACTGCAATGGTTTGAGCGT ；T 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTCAGGACTGCAATGGTTTGAGCGA ；(24)bl2，產物長度123-125bp 通用探針p-CCATGGCTATGAGTGTGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ； G 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTATCAACTGGAAACTCACCCTTGC ；T 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTTTATCAACTGGAAACTCACCCTTGA ；(25)Cl，產物長度 79-81bp 通用探針P-CAGGCTATGACTAGTTCATTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TATATCTCTCTTGGATTCAATAAT ；G 配對特異性探針TTTATATCTCTCTTGGATTCAATAAC ；(26)c2，產物長度 83-85bp 通用探針P-GTTGTCATCCAAAAAATAATTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針I(yè)TITITTAGGAGCAATGTTCTGAAAG ；T 配對特異性探針I(yè)TITITITTAGGAGCAATGTTCTGAAAA ；(27)c3，產物長度 87-89bp 通用探針P-TCCCCAAGGTGTCTAGACATTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針TTTTTTACTACATTAGATATTAAATGTG ；G 配對特異性探針TTTTTTTTACTACATTAGATATTAAATGTC ；(28)c4，產物長度 95-97bp 通用探針P-CATTAAACATAAGTTGTCATTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；G 配對特異性探針TTTTTTTTTACTAGACATGGTTGCTTTTTGCC ；T 配對特異性探針TTTTTTTTTTTACTAGACATGGTTGCTTTTTGCA ；(29)c5，產物長度 99-101bp 通用探針P-TTCCACTCGTCAGATATCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ； C 配對特異性探針TTTTTTTTTTTCATCCGCCCCGAGGGGGTTTCCG ；G 配對特異性探針TTTTTTTTTTTTTCATCCGCCCCGAGGGGGTTTCCC ；(30)c6，產物長度 103-105bp 通用探針p-GGAACTTCCTAAATCTGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針I(yè)TITITITITITTAAAATAGCAATCAGAAACAGAAG ；G 配對特異性探針I(yè)TITITITITITITTAAAATAGCAATCAGAAACAGAAC ；(31)c7，產物長度 107-109bp 通用探針p-CAACACCTTGGGGACAAAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；G 配對特異性探針TTTTTTTTTTTTTTTATGCAGAAATGTGTTTAAAACCC ；T 配對特異性探針TTTTTTTTTTTTTTTTTTATGCAGAAATGTGTTTAAAACCA ；(32)c8，產物長度 lll-113bp 通用探針p-GGTATGTGTGAGCTGAGGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTTTTTTTTTTTTTTTAGCCAGTGATTGTGTCTCCAAGT ；G 配對特異性探針TTTTTTTTTTTTTTTTTTTAGCCAGTGATTGTGTCTCCAAGC ；(33)c9，產物長度 115-117bp 通用探針p-AACTTTAGAGAGATAAAGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTTTTTTTTTTTTTTTTTGAGCACTGTGTGCTGTGGATTAT ；G 配對特異性探針TTTTTTTTTTTTTTTTTTTTTGAGCACTGTGTGCTGTGGATTAC ；(34)cl0，產物長度119-121bp 通用探針p-ATGTAACTATGTCAGAAGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針TTTTTTTTTTTTTTTTTTTTTCTCAATGTGGACCAGATACTATG ；T 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTCTCAATGTGGACCAGATACTATA ；(35)cll，產物長度123-125bp 通用探針p-TCTCTCTCTCTTCTAGGTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ； A 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTTTATTTTTTGATGAACATTTTTT ；G 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTTTGATGAACATTTTTC ；(36)dl，產物長度 79-81bp 通用探針P-GACTCTAAATGATTATAAATTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TCAGCGAAGCGGTGCAATGAGATT ；G 配對特異性探針TTTCAGCGAAGCGGTGCAATGAGATC ；(37)d2，產物長度 83-85bp 通用探針P-GCACTGAGCCTGTGTTCTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTAAGATGTCTCTTTCCACTTGAGT ；G 配對特異性探針TTTTTAAGATGTCTCTTTCCACTTGAGC ；(38)d3，產物長度 87-89bp 通用探針P-CTGAAGGCTAGGGCTAGGATTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TITITTGCACAAAAATGCCTTCAATAAT ；G 配對特異性探針TTTTTTTTGCACAAAAATGCCTTCAATAAC ；(39)d4，產物長度91-93bp 通用探針P-AGGTCAACCCAGGATTCCAITITITITITITITITITITITITITT-FAM ；C 配對特異性探針TTTTTTTCAAATGTATATGGTTGAGGCTAG ；T 配對特異性探針TTTTTTTTTCAAATGTATATGGTTGAGGCTAA ；(40)d5，產物長度 95-97bp 通用探針P-TCAATAAGGTTAATAGGCTITITITITITITITITITITITITITITT-FAM ；C 配對特異性探針TTTTTTTTTCTGGAATGTATGACATAAAATTG ；G 配對特異性探針TTTTTTTTTTTCTGGAATGTATGACATAAAATTC ；(41)d6，產物長度 99-101bp 通用探針p-CCAGTCTGGAGAGGGCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTTTTTTTTTGAAACTGATATATTCCCTGTCTT ；C 配對特異性探針TTTTTTTTTTTTTGAAACTGATATATTCCCTGTCTG ；(42)d7，產物長度 103-105bp 通用探針p-TCTCCACAGTCAACAGTAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針TTTTTTTTTTTTTTCCCTGCTCAGAGGGGCCCAGTG ；G 配對特異性探針TTTTTTTTTTTTTTTTCCCTGCTCAGAGGGGCCCAGTC ；(43)d8，產物長度 107-109bp 通用探針p-TCCCAAGAAGAATTTTATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針TTTTTTTTTTTTTTTGATATTCAGAATATTTTACTTAG ；G 配對特異性探針TTTTTTTTTTTTTTTTTGATATTCAGAATATTTTACTTAC ；(44)d9，產物長度 119-121bp 通用探針p-CCATCTTGAAATGCCCAGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；A 配對特異性探針TTTTTTTTTTTTTTTTTTTTTGCCTATAGTAGTTTGCATCCTGT ；G 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTGCCTATAGTAGTTTGCATCCTGC ；(45)dl0，產物長度123-125bp 通用探針p-CATGGCCGGTGGGTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM ；C 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTGATGGCTCCTTGGAATTTGACTG ；T 配對特異性探針TTTTTTTTTTTTTTTTTTTTTTTTTGATGGCTCCTTGGAATTTGACTA。
4.根據(jù)權利要求I所述的一種用于鑒別近交系小鼠的SNP分型方法，其特征在于所述步驟(3)中的最佳的退火溫度為56°C。
5.根據(jù)權利要求I所述的一種用于鑒別近交系小鼠的SNP分型方法，其特征在于所述步驟⑶中的LDR連接產物用標準6%變性測序PAGE膠進行電泳，電泳在ABI 373A測序儀上進行，功率為30W，時間為I. 5-2h，ABI377數(shù)據(jù)進一步用GenemapperID 3. 2來進行基因分型。
全文摘要
本發(fā)明涉及一種用于鑒別近交系小鼠的SNP分型方法，包括(1)遺傳標記的選擇，平均覆蓋21染色體的共45個SNP位點，并對這些位點設計引物和探針；(2)四組多重PCR，每組含10～12對PCR引物；(3)四組多重LDR，每組含10～12個位點特異性探針；(4)產物快速分離鑒定。本發(fā)明的SNP分型方案，僅需要45個SNP遺傳位標就可用于近交系小鼠的分型和鑒定，不僅能減少反應次數(shù)和時間，而且操作簡便迅速，能大大節(jié)約成本，對其分型和研究具有重要意義，為近交系小鼠的鑒定和分型提供了一種新的方法。
文檔編號C12Q1/68GK102586457SQ20121006714
公開日2012年7月18日 申請日期2012年3月14日 優(yōu)先權日2012年3月14日
發(fā)明者姜憲環(huán), 李凱, 蔡慧強, 謝建云, 謝雯, 鮑世民 申請人:東華大學