基于多重PCR-LDR技术建立近交系大鼠单核苷酸多态性遗传检测方案

Establishing a Genetic Detection Protocol of Single Nucleotide Polymorphisms Panels in Inbred Rats Based on Multiplex PCR-LDR

目的建立一套基于多重PCR-连接酶检测反应(ligase detection reaction,LDR)技术的近交系大鼠单核苷酸多态性(single nucleotide polymorphisms,SNP)检测方案。方法在5个品系的SPF级近交系大鼠1~20号常染色体和X染色体上共选取40个大鼠SNP位点,将SNP位点随机分为4组,构建基于多重PCR-LDR技术的近交系大鼠4组SNP位点基因检测方案。采用本方案检测国内另两家大鼠供应商的9个常用大鼠品系。最后,通过第三方实验室对不同DNA聚合酶的扩增效果进行比对,验证本方案的可行性。结果用所构建的近交系大鼠SNP遗传检测方案测试5个大鼠品系时,各样本的所有位点均得到了良好的扩增结果。采用本方案检测国内另两家大鼠供应商的9个常用大鼠品系时也得到了良好的扩增结果,40个SNP位点在每个近交系大鼠中均为纯合。用3种来源不同的DNA聚合酶同时检测相同大鼠DNA样本的结果显示,Multiplex PCR Kit、AmpliTaq Gold^(TM)360 DNA聚合酶、PlatinumⅡTaq热启动DNA聚合酶在第1~3组SNP位点均有扩增产物的电泳峰,其中PlatinumⅡTaq热启动DNA聚合酶在第4组SNP位点中少了一个扩增产物的电泳峰。另外,不同实验室间的比对结果显示,相同扩增体系的检测结果一致。结论基于多重PCR-LDR技术成功建立了一套覆盖所有常染色体与X染色体的大鼠SNP检测方案,该方法的稳定性和重复性俱佳。

Objective To establish a set of single nucleotide polymorphisms(SNP)detection protocol for inbred rats based on multiplex PCR-ligase detection reaction(LDR).Methods A total of 40 rats SNP sites were selected on chromosomes 1-20 and X of rats among 5 inbred strains of rats,and the 40 SNP sites were randomly divided into four groups.A genetic detection protocol for 4 groups of SNP in inbred rats based on multiplex PCR-LDR technology was constructed.9 commonly used rat strains from two other domestic rat suppliers were detected by this protocol.Finally,the feasibility of this protocol was verified by comparing the amplification effects of different DNA polymerases by a third-party laboratory.Results When using the constructed SNP detection protocol for inbred rats to test 5 rat strains,all sites in each sample obtained good amplification results.The 9 commonly used rat strains from two other rat suppliers in china were also well amplified by this SNP detection protocol,and 40 SNPs were homozygous in each Inbred strain.The results of detection of the same rat DNA samples with three different DNA polymerases showed that the Multiplex PCR Kit,AmpliTaq Gold 360 DNA polymerase and Platinum II Taq hot start DNA polymerase had electrophoretic peaks of amplification products at all SNP sites in groups 1 to 3,and Platinum II Taq hot start DNA polymerase had one less electrophoretic peak of the amplification products at the SNP sites in group 4.In addition,inter-laboratory comparisons showed consistent results for the same amplification system.Conclusion Based on multiplex PCR-LDR technology,this study successfully established a SNP detection protocol for rats covering all autosomes and X chromosomes with the excellent stability and repeatability.