Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device
2003 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1014, no 1-2, 37-45 p.Article in journal (Refereed) Published
A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) is presented. The method is based on single-step primer extension using pyrosequencing chemistry to monitor nucleotide incorporations in real-time. The method takes advantage of the fact that the reaction kinetics differ between matched and mismatched primer-template configurations. We show here that monitoring the initial reaction in real time accurately scores SNPs by comparing the initial reaction kinetics between matched and mismatched configurations. Thus, no additional treatment is required to improve the sequence specificity of the extension, which has been the case for many allele-specific extension assays. The microfluidic approach was evaluated using four SNPs. Three of the SNPs included primer-template configurations that have been previously reported to be difficult to resolve by allele-specific primer extension. All SNPs investigated were successfully scored. Using the microfluidic device, the volume for the bioluminescent assay was reduced dramatically, thus offering a cost-effective and fast SNP analysis method.
Place, publisher, year, edition, pages
2003. Vol. 1014, no 1-2, 37-45 p.
Microfluidics, Nucleotide polymorphism, Nucleotides, Primer extension, Pyrosequencing
IdentifiersURN: urn:nbn:se:kth:diva-5009DOI: 10.1016/S0021-9673(03)01033-1ISI: 000185557200005OAI: oai:DiVA.org:kth-5009DiVA: diva2:7463
16th International Symposium on Microscale Separation and Analysis SAN DIEGO, CALIFORNIA, JAN 17-22, 2003
QC 20100929 NR 201408052005-04-012005-04-012012-02-17Bibliographically approved