Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single‐substrate assays

Model single base extension (SBE) genotyping reactions with individual deoxy‐, dideoxy‐ and acyclonucleoside triphosphates are monitored by MALDI‐TOF mass spectrometry. Three non‐proofreading DNA polymerases display remarkably high misincorporation (up to 64% of correct incorporation) when extending primers with single substrates at saturating concentrations. Introduction of one phosphorothioate (PS) linkage into the primer 3′ terminus reduces misincorporation by these enzymes an average 1.4‐fold (range 0‐ to 3.5‐fold) versus correct incorporation. Combined use of 3′‐PS primers with strongly proofreading DNA polymerases yields order of magnitude improvements in SBE fidelity over those produced by the equivalent non‐proofreading enzymes. Errors are reduced to below MALDI‐TOF detectable levels in almost all cases. The Sp diastereomer of the 3′‐PS primer, which can be prepared in situ by incubation with proofreading polymerase, is stable to 3′‐exonuclease activity over periods longer than 16 h. Products of correct extension by T7 DNAP are retained over 30–60 min during idling turnover at a dNTP concentration of 2.5 µM, indicating that the assay can be applied over a broad range of substrate concentrations. These results suggest that the use of PS primers and proofreading polymerases will offer a simple and cost‐effective means to improve fidelity in a range of single‐substrate SBE assay formats.