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Genotyping by apyrase-mediated allele-specific extension
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
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2001 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 29, no 24Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2001. Vol. 29, no 24
Keyword [en]
single-nucleotide polymorphisms, refractory mutation system, polymerase-chain-reaction, dna analysis, discrimination, diagnostics, assay
URN: urn:nbn:se:kth:diva-21187ISI: 000172871800041OAI: diva2:339884
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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