PubMed2006
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.