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Nucleic Acids Research, 2001, Vol. 29, No. 24 e121
© 2001 Oxford University Press

Genotyping by apyrase-mediated allele-specific extension

Afshin Ahmadian, Baback Gharizadeh, Deirdre O’Meara, Jacob Odeberg and Joakim Lundeberg*

Center for Physics, Astronomy and Biotechnology, Department of Biotechnology, The Royal Institute of Technology (KTH), Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden

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.

* To whom correspondence should be addressed. Tel: +46 8 55378327; Fax: +46 8 55378481; Email: joakim.lundeberg{at}biochem.kth.se


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