Exo+ proofreading polymerases mediate genetic analysis and its application in biomedical studies1

Abstract

High-fidelity DNA polymerases mediate genotyping3´Terminal-labeled primer extension Terminal-labeled primer extension is a single nucleotide polymorphism (SNP) assay consisting of 3´ terminal-labeled, allele-specific primers and DNA polymerases with proofreading activity. Both 3´ terminal [ 3 H]-labeled and fluorescent-labeled primers have been successfully applied in genotyping analyses. The 3t erminal mismatched nucleotide that bears the signal to be detected was removed by the proofreading function, whereas the label was retained when the primer and template were perfectly matched. The terminal-labeled primer extension approach has several advantages over current SNP assays. The most significant advantage is that it greatly decreases false positive results by a direct consequence of the proofreading activity of Exo + polymerases. The second advantage of terminal-labeled primer extension is its high sensitivity.Terminally labeled primer extension harnesses the power of polymerase chain reaction (PCR) to improve the efficiency of genetic analysis [1][2][3][4][5][6][7] . SNP-triggered on/off switch Exo + polymerases together with 3´ phosphorothioate-modified mismatched primers work as an off switch in DNA polymerization. Phosphorothioate modification renders oligonucleotides nuclease-resistant, which blocks mismatch excision, a strategy widely used in antisense technology as well as in single base extension. For 3´ allele-specific primers with phosphorothioate modification, a perfectly matched primer turns on DNA polymerization, whereas mismatched primers turn it off, resulting in no product. This breakthrough observation of an on/off switch action has been repeatedly confirmed using either short artificial amplicons or natural genomic DNA templates. The off switch directly resulted from 3´exonu-clease activity and has been well supported by comparisons of a variety of DNA polymerases in both linear and expo- AbstractPolymerases with a proofreading function in their internal 3´ to 5´ exonuclease possess high fidelity for DNA replication both in vivo and in vitro. The obstacle facing Exo + polymerases for single nucleotide polymorphism (SNP) detection could be bypassed by using primer-3´-termini modification. This hypothesis has been well tested using three types of modified allele specific primers with: 3´ labeling, 3t o 5´exonuclease resistance, and 3´dehydroxylation. Accordingly, three new SNP assaying methods have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of Exo + polymerases. These new mutation detection assays are widely adaptable to a variety of platforms, including multi-well plate and microarray technologies. Application of Exo + polymerases to genetic analysis, including genotyping that is mostly relevant to pharmacogenetics, high-fidelity gene expression profiling, rare mutation detection and mutation load assay, will help to accelerate the pace of personalized medicine. In this review paper, we will first introduce three new assays that we have re...

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