Competitive enzymatic reaction to control allele-specific extensions
2005 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 33, no 5, e48:1-e48:10 p.Article in journal (Refereed) Published
Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3'-positions. As reported previously [Ahmadian, A., Gharizadeh, B., O'Meara, D., Odeberg, J. and Lundeberg, J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 50 end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.
Place, publisher, year, edition, pages
2005. Vol. 33, no 5, e48:1-e48:10 p.
DNA polymerase; proteinase; 3' untranslated region; 5' untranslated region; allele; article; bioassay; controlled study; enzyme activity; enzyme degradation; enzyme mechanism; genetic regulation; genotype; human; kinetics; normal human; polymerase chain reaction; priority journal; scoring system; single nucleotide polymorphism; Meara
IdentifiersURN: urn:nbn:se:kth:diva-8908DOI: 10.1093/nar/gni048ISI: 000228080000003ScopusID: 2-s2.0-20144372764OAI: oai:DiVA.org:kth-8908DiVA: diva2:14391
QC 201007142005-12-092005-12-092011-09-01Bibliographically approved