微流控芯片电泳-多重聚合酶链反应法同时测定多个单碱基多态性位点

Microchip Electrophoresis Coupled with Multiplex Polymerase Chain Reaction for Typing Multiple Single Nucleotide Polymorphisms Simultaneously

以微流控芯片电泳为检测平台,建立了多重PCR扩增法同时测定多个单碱基多态性(SNP)位点的方法。先通过PCR扩增得一段含所有待测SNP位点的长片段;用限制性内切酶消化成短片段,再将酶切反应产物与脱氧核糖核酸适配器(DNAadapter)相连;以连接产物为模板,分成两管,分别用n条等位基因特异性引物和一条通用引物进行n重PCR扩增;最后用微流控芯片电泳法分离PCR扩增产物,根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。以细胞色素P4502D6(CYP2D6)基因中的5个SNP位点(100C>T、1661G>C、1758G>T、2470T>C和2850C>T)为检测对象,考察了各等位基因特异性引物之间的相互影响和扩增反应的特异性,采用微流控芯片电泳法成功测定了20名健康中国人的CYP2D6基因中5个SNP位点的基因多态性,与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)测定结果完全一致。

A new method was established for typing multiple single nucleotides polymorphisms （SNPs） by multiplex polymerase chain reaction（PCR） on the platform of microchip electrophoresis. Firstly, a long target containing all SNPs of interest were preamplified to enhance the specificity; secondly, the preamplified deoxyribonucleic acid （DNA） fragments were digested by a restriction endonuclease to form sticky ends ; thirdly, the adapter was ligated to either end of the digested fragment; fourthly, taking the adapter-ligated fragments as templates, an n-plex allele-specific amplification was performed by n allele-specific primers and a universal primer in one tube; finally, the allele-specific amplification products were separated by chip electrophoresis and the types of SNPs can be easily obtained by the length of the products. Taking five SNPs of 100C 〉 T, 1661G 〉 C, 1758G 〉 T, 2470T 〉 C and 2850C 〉 T in the cytochrome P4502D6 （CYP2D6） gene as a researching object, the interactions among allele-specific primers and the specificity of 5-plex allele-specific amplifi- cation were studied. Five SNPs in the CYP2D6 gene in 20 healthy Mainland Chinese were successfully typed by microchip electrophoresis and the results coincide with those obtained by PCR-restriction fragment length polymorphism（PCR-RFLP）. The established method is accurate and can be used to type multiple SNPs simultaneously. In this method, n ＋ 1 primers （n allele-specific primers and a universal primer） were used for an n-plex PCR amplification, so that the interactions among allele-specific primers were reduced and the design of primers were simplified. The specificity was increased by both the special design of the adapter structure and the introduction of an artificial mismatch base in the 3＇-terminal region of allele-specific primer. By coupling microchip electrophoresis with the method for the readout, the cost for the SNP detection is greatly reduced as the small sampling amount and the simplified process of the condition optimization.