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Figure 1. Schematic presentation of the tetra-primer ARMS-PCR method. The single nucleotide polymorphism used here as an example is a G→A substitution, but the method can be used to type other types of single base substitutions. Two allele-specific amplicons are generated using two pairs of primers, one pair (indicated by pink and red arrows, respectively) producing an amplicon representing the G allele and the other pair (indicated by indigo and blue arrows, respectively) producing an amplicon representing the A allele. Allele specificity is conferred by a mismatch between the 3′-terminal base of an inner primer and the template. To enhance allelic specificity, a second deliberate mismatch (indicated by an asterisk) at position –2 from the 3′-terminus is also incorporated in the inner primers. The primers are 26 nt or longer, so as to minimize the difference in stability of primers annealed to the target and non-target alleles, ensuring that allele specificity results from differences in extension rate, rather than hybridisation rate. By positioning the two outer primers at different distances from the polymorphic nucleotide, the two allele-specific amplicons differ in length, allowing them to be discriminated by gel electrophoresis. (http://nar.oxfordjournals.org/content/29/17/e88/F1.expansion)
