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Model single base extension (SBE) genotyping reac- tions with individual deoxy-, dideoxy- and acyclo- nucleoside triphosphates are monitored by MALDI- TOF mass spectrometry. Three non-proofreading DNA polymerases display remarkably high misin- corporation (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 poly- merases 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¢-exo- nuclease 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 mM, indicating that the assay can be applied over a broad range of sub- strate concentrations. These results suggest that the use of PS primers and proofreading poly- merases will offer a simple and cost-effective means to improve fidelity in a range of single-substrate SBE assay formats.

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