一种基于适配器连接介导的等位基因特异性扩增法测定多重SNP

建立了一种基于DNA适配器连接介导的等位基因特异性扩增法测定多重SNP。以CYP2D6基因中的5个SNP位点（100C>T，1661G>C，1758G>T，2470T>C和2850C>T）为例，用PCR法预扩增得一段含所有待测SNP位点的长片段，然后用限制性内切酶将其消化成短片段，在连接酶的作用下与设计的DNA适配器（adapter）相连；该适配器的一端与限制性内切酶降解后留下的粘性末端相同，另一端带有一段公共序列。在两管中加入与适配器连接的片段作为PCR扩增模板，并分别加入SNP特异性引物和一种适配器特异性的通用引物进行PCR扩增，最后用凝胶电泳法分离PCR扩增产物。由于每管与SNP的两种特异性引物中的一种对应，可以根据每管中扩增片段的大小判断SNP的类型。通过凝胶电泳法可以一次分离与5种SNP类型相对应的引物特异性延伸反应产物；采用该法成功测定了20名健康中国人的CYP2D6基因中5个SNP位点的基因多态性，与限制性片段长度多态性法（RFLP）测定结果完全一致。该方法采用n+1种引物（n种SNP特异性引物和一种通用引物）进行n重PCR反应，极大提高了PCR反应的特异性，结果准确，可用于同时测定多个SNP位点。

Adapter-ligation Mediated Allele-specific Amplification (ALM-ASA) for Multiplex SNP Genotyping

To establish adapter-ligation mediated allele-specific amplification ("ALM-ASA" for short) for multiplex SNP genotyping, five SNPs, 100C>T, 1661G>C, 1758G>T, 2470T>C and 2850C>T in CYP2D6 gene were used as an example for evaluating the method. Firstly, a preamplification was carried out for producing a long target containing all SNPs of interest. Secondly, the preamplified DNA fragments were digested with a restriction endonuclease to form sticky ends. Thirdly, an adapter was ligated to either end of the digested fragment. One end of the adapter was designed as a sequence sticky to the ends of the enzymatically digested fragments, and the other end had a common sequence. Fourthly, an allele-specific amplification was performed by allele-specific primers and a universal primer in one tube by using the adapter-ligated fragments as templates. Finally, the allele-specific amplification products were separated by agarose gel electrophoresis. Because each tube corresponds to one kind of allele-specific primers, the genotype of an SNP can be easily discriminated by the length of the amplified products in each tube. The products of 5-plex allele-specific amplification can be separated by agarose gel electrophoresis. Five SNPs in the CYP2D6 gene were successfully typed for 20 healthy Mainland Chinese and the results were in agreement with those by RFLP. By ALM-ASA, n+1 primers (n SNP allele-specific primers and a universal primer) can be used for an n-plex PCR amplification; the specificity of PCR is thus enhanced significantly. It is concluded that ALM-ASA can be used for typing multiple SNPs simultaneously.