Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device.
We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.
Russom
Aman
A
Department of Signals, Sensors and Systems, Microsystem Technology, Stockholm, Sweden.
Ahmadian
Afshin
A
Andersson
Helene
H
Nilsson
Peter
P
Stemme
Göran
G
D000483:Alleles; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004867:Equipment Design; D005456:Fluorescent Dyes; D016158:Genes, p53; D005821:Genetic Techniques / Q000295:instrumentation* / Q000706:statistics & numerical data; D014644:Genetic Variation; D006801:Humans; D008832:Microchemistry / Q000295:instrumentation* / Q000706:statistics & numerical data; D008904:Miniaturization; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
D017931:DNA Primers; D005456:Fluorescent Dyes