Premium
Chemical gradient‐mediated melting curve analysis for genotyping of SNPs
Author(s) -
Russom Aman,
Irimia Daniel,
Toner Mehmet
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800729
Subject(s) - nucleic acid denaturation , melting curve analysis , duplex (building) , formamide , denaturation (fissile materials) , chemistry , dna , analytical chemistry (journal) , genotyping , temperature gradient , biophysics , materials science , chromatography , biology , biochemistry , genotype , polymerase chain reaction , base sequence , organic chemistry , nuclear chemistry , gene , physics , quantum mechanics
This report describes a microfluidic solid‐phase chemical gradient‐mediated melting curve analysis method for SNP analysis. The method is based on allele‐specific denaturation to discriminate mismatched (MM) from perfectly matched (PM) DNA duplexes upon exposure to linear chemical gradient. PM and MM DNA duplexes conjugated on beads are captured in a microfluidic gradient generator device designed with dams, keeping the beads trapped perpendicular to a gradient generating channel. Two denaturants, formamide and urea, were tested for their ability to destabilize the DNA duplex by competing with Watson–Crick pairing. Upon exposure to the chemical gradient, rapid denaturing profile was monitored in real time using fluorescence microscopy. The results show that the two duplexes exhibit different kinetics of denaturation profiles, enabling discrimination of MM from PM DNA duplexes to score SNP.