Proofreading genotyping assays mediated by high fidelity exo+ DNA polymerases

DNA polymerases with 3′-5′ proofreading function mediate high fidelity DNA replication but their application for mutation detection was almost completely neglected before 1998. The obstacle facing the use of exo+ polymerases for mutation detection could be overcome by primer-3′-termini modification, which has been tested using allele-specific primers with 3′ labeling, 3′ exonuclease-resistance and 3′ dehydroxylation modifications. Accordingly, three new types of single nucleotide polymorphism (SNP) assays have been developed to carry out genome-wide genotyping making use of the fidelity advantage of exo+ polymerases. Such SNP assays might also provide a novel approach for re-sequencing and de novo sequencing. These new mutation detection assays are widely adaptable to a variety of platforms, including real-time PCR, multi-well plate and microarray technologies. Application of exo+ polymerases to genetic analysis could accelerate the pace of personalized medicine.