Abstract
We report the validation of a new assay for typing single nucleotide polymorphisms (SNPs) that takes advantage of the 3'-to-5' exonuclease proofreading activity of many DNA polymerases. The assay uses one or more primers labeled on the 3' nucleotide base, and can be implemented in a variety of formats including a one-step PCR reaction that allows SNP typing directly from genomic DNA samples. The detection of genotypes can be accomplished by means of fluorescence detection on assays that have been purified to remove excess primer, or by means of fluorescence polarization without any additional cleanup. We also demonstrate that the Exo-Proofreading SNP assay can be used on pooled samples to obtain allele frequency data.
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