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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
3
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pubmed:dateCreated |
2003-1-31
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pubmed:abstractText |
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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pubmed:commentsCorrections |
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Feb
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pubmed:issn |
1362-4962
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pubmed:author |
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pubmed:issnType |
Electronic
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pubmed:day |
1
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pubmed:volume |
31
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
e7
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pubmed:dateRevised |
2009-11-18
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pubmed:meshHeading |
pubmed-meshheading:12560510-Base Sequence,
pubmed-meshheading:12560510-DNA Polymerase I,
pubmed-meshheading:12560510-DNA Primers,
pubmed-meshheading:12560510-DNA-Directed DNA Polymerase,
pubmed-meshheading:12560510-Deoxyribonucleotides,
pubmed-meshheading:12560510-Exonucleases,
pubmed-meshheading:12560510-Genotype,
pubmed-meshheading:12560510-Polymorphism, Single Nucleotide,
pubmed-meshheading:12560510-Sequence Analysis, DNA,
pubmed-meshheading:12560510-Spectrometry, Mass, Matrix-Assisted Laser...,
pubmed-meshheading:12560510-Templates, Genetic,
pubmed-meshheading:12560510-Thermodynamics,
pubmed-meshheading:12560510-Thionucleotides
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pubmed:year |
2003
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pubmed:articleTitle |
Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays.
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pubmed:affiliation |
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
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pubmed:publicationType |
Journal Article,
Comparative Study,
Evaluation Studies
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