Fast detection of single nucleotide polymorphisms (SNPs) by primer elongation with monitoring of supercritical-angle fluorescence.
We describe the rapid detection of single nucleotide polymorphisms (SNPs) by real-time observation of primer elongation. The enzymatic elongation of surface-bound primers is monitored by detecting the increase of surface-bound fluorescence caused by the incorporation of Cy5-labelled deoxycytidine 5'-triphosphate residues (Cy5-dCTPs) into the corresponding strand. In order to discriminate against the fluorescence from unbound Cy5-dCTPs, the detection volume was restricted to the surface by collecting supercritical-angle fluorescence. The efficiency of enzymatic double-stranded DNA synthesis is governed by the complementarity of the primer and template. An SNP in the sequence of the primer obstructs its elongation increasingly with decreased distance of the mismatch to the 3' end of the primer. By real-time fluorescence detection during primer elongation, SNPs can be detected within a few minutes, which is significantly faster than in experiments where the fluorescence is measured after completion of the reaction. We demonstrate the efficiency of the method by detecting an SNP in the ErbB2 gene that is involved in causing a higher risk of breast cancer.
Krieg
Alexander
A
Physikalisch Chemisches Institut, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Laib
Stephan
S
Ruckstuhl
Thomas
T
Seeger
Stefan
S
D020137:Base Pair Mismatch; D002232:Carbocyanines; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004252:DNA Mutational Analysis; D017931:DNA Primers; D005453:Fluorescence; D005456:Fluorescent Dyes; D018734:Genes, erbB-2 / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D013698:Templates, Genetic
D002232:Carbocyanines; D017931:DNA Primers; D005456:Fluorescent Dyes; C085321:cyanine dye 5; D004247:DNA