SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays

Abstract

This study reports the development of a microarray-based allele-specific extension method for typing of single nucleotide polymorphisms (SNPs). The use of allele-specific primers has been employed previously to identify single base variations but it is acknowledged that certain mismatches are not refractory to extension. Here we have overcome this limitation by introducing apyrase, a nucleotide-degrading enzyme, to the extension reaction. We have shown previously that DNA polymerases exhibit slower reaction kinetics when extending a mismatched primer compared with a matched primer. This kinetic difference is exploited in the apyrase-mediated allele-specific extension (AMASE) assay, allowing incorporation of nucleotides when the reaction kinetics are fast but degrading the nucleotides before extension when the reaction kinetics are slow. Here we show that five homozygous variants (14% of the total number of variants) that were incorrectly scored in the absence of apyrase were correctly typed when apyrase was included in the extension reaction. AMASE was performed in situ on the oligonucleotide microarrays using fluorescent nucleotides to type 10 SNPs and two indels in 17 individuals generating approximately 200 genotypes. Cluster analysis of these data shows three distinct clusters with clear-cut boundaries. We conclude that SNP typing on oligonucleotide microarrays by AMASE is an efficient, rapid and accurate technique for large-scale genotyping.

Demonstration of Loss of Heterozygosity by Single-Nucleotide Polymorphism Microarray Analysis and Alterations in Strain Morphology in Candida albicans Strains during Infection

“…Cluster or scatter plot analysis has become the method of choice for genotype assignment and determination of cutoff values for the three allelic genotypes (13,27,33,34). Tight clusters indicate good genotype discrimination, and the larger the distance between the clusters the better is the discrimination between homozygous and heterozygous genotypes (27).…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…9). The designed microfluidic confined bead array was successfully demonstrated for apyrase assisted allele-specific primer extension [20,82]. This approach showed reliable discrimination of synthetic target DNA of 0.5 pM concentration that corresponded to an absolute detection sensitivity of approximately one attomole of target molecules in a 15 µL sample.…”

Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism

“…Over the past twenty years, many different methods have been developed for SNP genotyping by PCR, including hybridization 16 , allele-specific PCR 17 , primer extension 18 , oligonucleotide ligation 19 , direct DNA sequencing 20 and endonuclease cleavage after amplification of the subjected genomic region by PCR [21][22] . Recent technologies for SNP genotyping (Taq Man method [23][24] , Invader method [25][26] , MALDI-TOF method 27 , GeneChips 28 ) have the potential for high-throughput, but they require the purchase of expensive equipment.…”