Multiplex analysis of mutations in four genes using fluorescence scanning technology.
Multiplex analysis of genetic mutations using fluorescence scanning methodology is an accurate, efficient, and cost-effective approach to genotypic characterization. Fluorescence labeling during the synthesis of polymerase chain reaction primers allows the application of this technology to well-established protocols. We have simultaneously analyzed the four polymorphisms of factor V Leiden (G1691A), prothrombin G20210A, 5,10-methylenetetrahydrofolate reductase C677T, and cystathionine beta-synthase 844ins68. Three of these mutations have been associated with an increased risk of thrombosis. Following polymerase chain reaction with fluorescence-labeled primers, the polymerase chain reaction products were digested with an appropriate restriction enzyme (if necessary for detection of the mutation), diluted into one tube per sample for co-loading (multiplex loading), and analyzed with GeneScan software for fragment analysis following capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Foster City, CA, USA). Multiplex loading increased throughput without compromising precision.
Benson
J M
JM
Hematologic Diseases Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Ellingsen
D
D
Renshaw
M A
MA
Resler
A G
AG
Evatt
B L
BL
Hooper
W C
WC
D003541:Cystathionine beta-Synthase / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods; D005165:Factor V / Q000235:genetics*; D005456:Fluorescent Dyes; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D009154:Mutation; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011516:Prothrombin / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
D005456:Fluorescent Dyes; D005165:Factor V; D011516:Prothrombin; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D004262:DNA Restriction Enzymes; D003541:Cystathionine beta-Synthase