WFRF:(Jacob B)
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Genotyping by apyrase-mediated allele-specific extension
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- Ahmadian, Afshin (author)
- KTH,Bioteknologi
- Gharizadeh, B. (author)
- O'Meara, D. (author)
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- (creator_code:org_t)
- Oxford University Press (OUP), 2001
- 2001
- English.
- In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 29:24
- Journal article (peer-reviewed)
Abstract
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- This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.
Keyword
- single-nucleotide polymorphisms
- refractory mutation system
- polymerase-chain-reaction
- dna analysis
- discrimination
- diagnostics
- assay
Publication and Content Type
- ref (subject category)
- art (subject category)
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