Genotyping by apyrase-mediated allele-specific extension

Abstract

This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.

Figure 1

Allele-specific extension of the three variants of the SNP at codon 72 of the p53 gene. The SNP status was determined separately for each sample and extension primers (1 and 2) (as listed in Table 1) are shown at the top of the figure. The top panel shows the raw-data obtained using the bioluminometric assay without apyrase and the lower panel shows the raw-data with the use of apyrase in the allele-specific extension reaction. The arrows point out the signal of pyrophosphate, which was added to the reaction mixture in all samples prior to nucleotide addition to serve as a positive control as well as for peak calibration.

Figure 2

Cluster analysis of all samples without (upper chart) and with (lower chart) the use of apyrase in the allele-specific extension reactions. Allelic-fractions on the x-axis are calculated as wt/(wt + mut), where wt and mut correspond to the light signals from extension primers 1 and 2 respectively. The y-axis is a logarithmic scale of log(wt + mut).