MAMAs are used for SNP typing in many different pathogens. The detailed description of the
method is presented elsewhere [14]. In brief, MAMAs are based on allele-specific primers that
are SNP specific at the 3’end. A single base mismatch at the ante-penultimate (-3) position of
each allele-specific primer enhances the SNP discrimination capacity of these assays. One
allele-specific primer possesses an additional 15-20bp GC-clamp at the 5’end with a sequence
of CGGGG and the other allele primer has no additional sequence. The GC-clamp increases
the melting temperature (Tm) of the resulting PCR product by 3–5°C, a shift that is detectable
by fluorescent dye on a real-time PCR platform (Melt-MAMA) and it increases the size of the
PCR product, resulting in a size difference which can also be detected by 3% agarose gel electrophoresis (Agarose-MAMA).