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

Abstract:

: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 micro M, 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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Di Giusto D,King GC

doi

10.1093/nar/gng007

keywords:

subject

Has Abstract

pub_date

2003-02-01 00:00:00

pages

e7

issue

3

eissn

0305-1048

issn

1362-4962

journal_volume

31

pub_type

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