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

Abstract

A new method was established for typing multiple single nucleotides polymorphisms (SNPs) using multiplex polymerase chain reaction (PCR) on the platform of microchip electrophoresis. First, a long target containing all SNPs of interest was pre-amplified to enhance the specificity; Second, the pre-amplified deoxyribonucleic acid (DNA) fragments were digested by a restriction endonuclease to form sticky ends; Third, the adapter was ligated to either end of the digested fragment; Fourth, 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 100 C > T, 1661 G > C, 1758 G > T, 2470 T > C, and 2850 C > T in the cytochrome P450 2D6 (CYP2D6) gene as a researching object, the interactions among the allele-specific primers and the specificity of 5-plex allele-specific amplification were studied. Five SNPs in the CYP2D6 gene in 20 healthy Mainland Chinese were successfully typed by microchip electrophoresis and the results agree with those 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 the allele-specific primers were reduced and the design of primers was 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 an allele-specific primer. By coupling microchip electrophoresis with the method for the read out, the cost for the SNP detection is greatly reduced as the small sampling amount and the simplified process for the optimization of conditions.