C677T mutation of methylenetetrahydrofolate reductase gene determined in blood or plasma by multiple-injection capillary electrophoresis and laser-induced fluorescence detection.
We constructed an assay to detect the common C677T mutation in the methylenetetrahydrofolate reductase gene. The mutation creates a Hinfl recognition site detected by restriction cleavage of a 198-bp fragment amplified in the polymerase chain reaction (PCR). Digested samples were subjected to capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), with hydroxypropylmethylcellulose as the sieving matrix and SYBR Green I as the fluorescent dye. After amplification but before digestion, we added to the PCR mixture a fragment with the HinfI recognition site and a 15-bp truncation at the 3' end. Using this procedure, we could (a) verify completeness of digestion and monitor injection, (b) assign genotypes on the basis of pattern recognition, and (c) develop a multiple-injection mode with simultaneous separation of as many as eight samples. A seminested PCR protocol in combination with CE-LIF allowed genotyping of plasma/serum samples 20 years old.
Ulvik
A
A
Department of Clinical Biology, Division of Pharmacology, University of Bergen, Haukeland Hospital, Norway.
Refsum
H
H
Kluijtmans
L A
LA
Ueland
P M
PM
D052160:Benzothiazoles; D004247:DNA / Q000097:blood*; D004252:DNA Mutational Analysis / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D003959:Diamines; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007834:Lasers; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D009154:Mutation; D009930:Organic Chemicals; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000097:blood / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011804:Quinolines
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases