ArticleNucleic acids research2003
Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays.
Article in Nucleic acids research, 2003. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed.
- Highly sensitive profiling somatic mutations of thyroid cancer by nucleotide-enrichment-based MALDI-TOF MS assay.Scientific reports · 2026Article
- One-Pot Detection of miRNA by Dual Rolling Circle Amplification at Ambient Temperature with High Specificity and Sensitivity.Biosensors · 2025Article
- Sensitive detection of low-abundance in-frame deletions in EGFR exon 19 using novel wild-type blockers in real-time PCR.Scientific reports · 2019Article
- A colorimetric strategy based on dynamic chemistry for direct detection of Trypanosomatid species.Scientific reports · 2019Article
- Efficient genome-wide genotyping strategies and data integration in crop plants.TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik · 2018Review
- Significance of stereochemistry of 3'-terminal phosphorothioate-modified primer in DNA polymerase-mediated chain extension.Molecular biotechnology · 2008Article
- Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples.Biochemical and biophysical research communications · 2006Article
- Creating advanced multifunctional biosensors with surface enzymatic transformations.Langmuir : the ACS journal of surfaces and colloids · 2006Article
- Superb nucleotide discrimination by a novel on/off switch for DNA polymerization and its applications.Molecular biotechnology · 2005Article
- Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays.Nucleic acids research · 2004Article
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2 authors.
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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.
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