Evaluation of a new efficient procedure for single nucleotide polymorphism genotyping: tetra-primer amplification refractory mutation system polymerase chain reaction

Tetraprimer amplification refractory mutation systempolymerase chain reaction (ARMSPCR) is a new efficient method for singlenucleotide polymorphism (SNP) genotyping. To determine the optimal conditions for ARMSPCR we attempted to genotype ten SNPs. DNA was extracted from the peripheral blood of 168 unrelated healthy Japanese volunteers. Two problems inhibited uniform efficiency of the amplification of three bands. The first problem was the lower amplification efficiency of the shorter and allelespecific products compared with the largest product. This phenomenon was overcome by increasing the relative concentration of the inner primers. The second problem was nonspecific amplification of the shorter products. To reduce the amplification of these nonspecific bands, adjusting any one of the following PCR conditions was effective: i) reducing the ratio of the inner primer concentration relative to that of the outer primers; ii) increasing the annealing temperature for the initial 510 cycles; iii) hot start PCR. With these procedures all ten of the SNPs were successfully genotyped. Our present data may be useful in the further application of tetraprimer ARMSPCR to SNP genotyping.