CN102586457B - 一种用于鉴别近交系小鼠的snp分型方法 - Google Patents
一种用于鉴别近交系小鼠的snp分型方法 Download PDFInfo
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Abstract
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。
Description
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(singlenuclear 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:
(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℃,GC%为60.0;
(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℃至65℃,跨度为3℃,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℃。
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1.5-2h,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。
附图说明
图1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1
(1)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Primer3程序完成。
引物和探针序列列于表1和表2。
表1基因组PCR扩增引物序列
表2基因组LDR探针序列
(2)PCR-LDR方案
多重PCR-LDR反应过程中,退火温度是其主要的参数之一,选择不同的退火温度对连接效率影响很大。分析ABI377测序电泳结果,最终确定了最佳的退火温度为56℃。
PCR反应体系如表3所示:
表3PCR反应体系
PCR反应程序如下所示:
95℃,15min;94℃,30s,56℃,1min30s,72℃,1min,进行35个循环;72℃,延伸10min。
PCR产物4℃保存。产物初步采用2%的琼脂糖凝胶电泳检测扩增效果。
LDR反应体系如表4所示:
表4LDR反应体系
LDR反应程序:
94℃,2min;94℃30s,50℃2min,共30个循环;
20℃保温。
(3)对BALB/c、C3H、C57BL/6、FVB和DBA/2等五个近交系的鉴定
45个位点的多重分型方案分成四组,每组多重PCR-LDR结果表明,不同近交系小鼠DNA在目标SNP位点有所不同,35个SNP位点的分型结果与NCBI数据库中信息完全一致并补充了NCBI中缺少的信息。此外,DBA/1、FVB、CBA分别有1、3、6个SNP位点的基因型与NCBI上的信息不符。进一步测序证明,这些位点的基因型与LDR分型结果一致。因此本实验方案实现了常见近交系小鼠快速、高通量的基因分型,可用于遗传质量检测和品系鉴定。分型结果如表5所示:
表5五个近交系小鼠分型结果
Claims (3)
1.一种用于鉴别近交系小鼠的SNP分型方法,包括:
(1)从NCBI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℃,GC%为60.0;其中,引物序列为:
(1)a1,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110bp:
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)a10,产物长度173bp:
上游,5'-GCAAAACGAAACAGCAAACA-3';
下游,TCACTGTCCCCAATTTCCTC;
(11)a11,产物长度122bp:
上游,5'-CTGATTTGGCTTTGTGAGTAGC-3';
下游,ATTTATGCCAGTTGCCCAAT;
(12)a12,产物长度174bp:
上游,5'-AGAACTGAGGATACAGCCATGA-3';
下游,AGGATGCCCGTGATGAGTAT;
(13)b1,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110bp:
上游,5'-TGTGGGTCTTATCCATTGAGG-3';
下游,CCATTTCATGGTCTCAAACAGA;
(21)b9,产物长度112bp:
上游,5'-TGGACCCTCTTATCTCCTTTATG-3';
下游,TGAAAGCCTTGTCTGTTAGCAA;
(22)b10,产物长度105bp:
上游,5'-CCCTTTTTCCTATGATGATGTG-3';
下游,CCTTGCATAGAAGCCCATTG;
(23)b11,产物长度174bp:
上游,5'-TCCTCGCTTACACTCAGAGC-3';
下游,AGGAGAAGTGCTGGCAGAGA;
(24)b12,产物长度167bp:
上游,5'-CCCACAGGCACTAGAGAATGA-3';
下游,GTTTTCTGCTAGGATATTTGACACC;
(25)c1,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)c10,产物长度146bp:
上游,5'-AGCCCCAGCTTCTGACATAG-3';
下游,TGACAGGAATGGATGCTTCA;
(35)c11,产物长度108bp:
上游,5'-AAGAGCACCAGGAATGACCT-3';
下游,CAGTGTCTAGAAGTGGGGTTTTG;
(36)d1,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)d10,产物长度144bp:
上游,5'-CCAAAAGGAATCCTCAGCAG-3';
下游,AGCAAGAGATGCACCAGTGA;
(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;其中,探针序列为:
(1)a1,产物长度79-81bp:
通用探针:P-TTTTCTTGACTTTTAATTCTTTTTTTTTTTTTTTTTTTTT-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:TTTTTTTTTAAACTGTGCAAGAAGCAAGGTAT;
G配对特异性探针:TTTTTTTTTTTAAACTGTGCAAGAAGCAAGGTAA;
(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111-113bp:
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)a10,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)a11,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)a12,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)b1,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:TTTTTTATTATCAGTAATGTACAAGCAG;
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:TTTTTTTGGAAAACAATGGCGGCCGGCGAT;
G配对特异性探针:TTTTTTTTTGGAAAACAATGGCGGCCGGCGAC;
(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:TTTTTTTTTTTCAAGAGGTCCAGAGGTCCAGAGT;
G配对特异性探针:TTTTTTTTTTTTTCAAGAGGTCCAGAGGTCCAGAGC;
(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111-113bp:
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)b10,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)b11,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)b12,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)c1,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:TTTTTTTAGGAGCAATGTTCTGAAAG;
T配对特异性探针:TTTTTTTTTAGGAGCAATGTTCTGAAAA;
(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:TTTTTTTTTTTTTAAAATAGCAATCAGAAACAGAAG;
G配对特异性探针:TTTTTTTTTTTTTTTAAAATAGCAATCAGAAACAGAAC;
(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111-113bp:
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)c10,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)c11,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)d1,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:TTTTTTGCACAAAAATGCCTTCAATAAT;
G配对特异性探针:TTTTTTTTGCACAAAAATGCCTTCAATAAC;
(39)d4,产物长度91-93bp:
通用探针:P-AGGTCAACCCAGGATTCCATTTTTTTTTTTTTTTTTTTTTTTTTTT-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-TCAATAAGGTTAATAGGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-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)d10,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;
(3)进行多重PCR-LDR反应,退火温度从50℃至65℃,跨度为3℃,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1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℃。
3.根据权利要求1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PCR-LDR反应产物用标准6%变性测序PAGE胶进行电泳,电泳在ABI373A测序仪上进行,功率为30W,时间为1.5-2h,ABI377数据进一步用GenemapperID3.2来进行基因分型。
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CN104975105B (zh) * | 2015-07-31 | 2018-04-13 | 华南理工大学 | 用于小鼠近交系鉴定的snp标记、引物对及其应用 |
CN108588236B (zh) * | 2018-05-17 | 2021-02-26 | 江苏集萃药康生物科技股份有限公司 | 一种近交系遗传质量监控的snp快速检测方法和snp位点及其引物 |
CN109022592B (zh) * | 2018-08-27 | 2020-11-24 | 华南理工大学 | 用于四种常用品系大鼠鉴定的snp标记及其应用 |
CN109609659B (zh) * | 2018-12-21 | 2021-09-28 | 江苏集萃药康生物科技股份有限公司 | 一组用于CBA/CaJ近交系小鼠遗传质量监控的SNP位点及其引物组合和应用 |
CN109694914B (zh) * | 2018-12-28 | 2022-06-03 | 江苏集萃药康生物科技股份有限公司 | 用于监控dba近交系小鼠遗传质量的方法、引物组及其应用 |
CN109777869A (zh) * | 2019-02-02 | 2019-05-21 | 广东安科华南生物科技有限公司 | 一种同时检测人类mthfr及mtgg基因序列的试剂盒 |
CN110484627B (zh) * | 2019-07-30 | 2023-05-02 | 江苏集萃药康生物科技股份有限公司 | 用于监控a/j近交系小鼠遗传质量的方法、引物组及其应用 |
CN110305974B (zh) * | 2019-08-08 | 2023-06-23 | 广东省实验动物监测所 | 基于检测五个snp位点区分常见小鼠近交系的pcr分析引物及其分析方法 |
CN114507744B (zh) * | 2022-04-19 | 2022-07-12 | 斯贝福(北京)生物技术有限公司 | Snp标记在近交系小鼠品系鉴定中的应用、引物序列 |
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