High-performance multiplex SNP analysis of three hemochromatosis-related mutations with capillary array electrophoresis microplates.
An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population.
Department of Chemistry, University of California, Berkeley, California 94720, USA.
I
I
Medintz
W W
WW
Wong
L
L
Berti
L
L
Shiow
J
J
Tom
J
J
Scherer
G
G
Sensabaugh
R A
RA
Mathies
D000483:Alleles; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005787:Gene Frequency; D006432:Hemochromatosis / Q000235:genetics*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*