Figure 4

Schematic illustration of primer design for the tetra-primer ARMS PCR (redraw from Ye et al., 2001 [16]). The SNP R (G/A) is presented as an example, and the other types of SNPs can be applied in the same way. Four primers, one pair of inner allele-specific (AS) primers and one pair of outer standard primers, are required in a single PCR reaction. Two AS products, one for the G allele and the other for the A allele are amplified using two pairs of primers. The former consists of a G AS primer and an outer standard primer, and other latter contains an A AS primer and an outer standard primer. A mismatch (represented by *) is deliberately introduced at the third position from the 3' end of each of the two AS primers to increase allelic specificity (See Table 1). Two outer standard primers are designed in such a way that the amplicons of two alleles differ in sizes and can be resolved by agarose gel electrophoresis.