Genotyping by apyrase-mediated allele-specific extension.
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.
Ahmadian
A
A
Center for Physics, Astronomy and Biotechnology, Department of Biotechnology, The Royal Institute of Technology (KTH), Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.
Gharizadeh
B
B
O'Meara
D
D
Odeberg
J
J
Lundeberg
J
J
D000483:Alleles; D001081:Apyrase / Q000378:metabolism*; D016000:Cluster Analysis; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics
D004247:DNA; D001081:Apyrase