Objective: To establish a new method for single nucleotide polymorphism(SNP) typing based on allele specific PCR: single-tube bi-directional amplification (SB-ASA), and study the influence on specific extension by introducing a mismatch at the third 3'terminal base of allele specific primers.
Methods: Two allele specific primers, with a mismatch introduced at the third 3'terminal base, were both included in PCR system; they extended in opposite directions and amplified two allele specific fragments different in size. The genotype was determined by observing the length of amplified fragments after agarose electrophoresis. The proper ranges of annealing temperature (Ta) under which primers can specifically extend were achieved by observing the amplification status at different temperatures.
Results: SB-ASA was successfully used to type 36 samples for four different kinds of SNPs. Typing results were completely consistent with those by directional sequencing. Proper Ta ranges of two primers were expanded respectively from 64-69 degrees centigrade to 46-66 degrees centigrade and from 60-62 degrees centigrade to 56-61 degrees centigrade by introducing a mismatch at the third 3'terminal base.
Conclusion: SB-ASA is a simple, rapid and efficient new method for SNP typing. During allele specific PCR reaction, specific primers with a mismatch at the third 3'terminal base have more power to identify two alleles.