An alternative to minisequencing strategies is allele-specific extension, which requires only a single detection reaction of one fluorophore. It is based on extension of allele-specific primers that differ at their 3' nucleotide defining the allelic variants of the SNPs and its success can primarily be attributed to the lowered intrinsic efficiency of DNA polymerases to extend mismatched primers.

A problem which has greatly hampered the development of such allele based discrimination methods is the extension of mismatched primers with several reports published on mismatches that are not refractory to allele-specific PCR amplification. Some mismatches such as G:T or C:A are clearly not refractory to extension and in independent studies it was observed that generally all T mismatches were well extended in addition to A:C and C:A mismatches. Allele-specific extension has recently been adapted to microarray formats for SNP analysis but either required the use of RNA for sufficient discrimination or reported a high error/no call rate for determination of homozygous nucleotide substitution.

We have described the addition of apyrase to allele-specific extension to increase the specificity of extension by minimizing extension of such mismatches in a bioluminometric assay. This Apyrase Mediated Allele-specific Extension (AMASE) exploits the fact that DNA polymerases exhibit slower reaction kinetics when extending a mismatched primer compared to a perfectly matched primer. Apyrase, a nucleotide degrading enzyme degrades the nucleotides before extension if the reaction kinetics are slow. We have recently describe genotyping on DNA microarrays based on this principle and we have shown increased specificity of allele-specific extension when apyrase is employed followed by application of this method to the typing of SNPs and mutation detection.

A novel improvement in the protocols has been introduced by replacing apyrase with proteinase K (PrASE) that has superior performance as compared to AMASE protocols.