C677T MUTATION OF METHYLENETETRAHYDROFOLATE REDUCTASE GENE DETERMINEDIN 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 met hylenetetrahydrofolate reductase gene. The mutation creates a Hinfl re cognition site detected by restriction cleavage of a 198-bp fragment a mplified in the polymerase chain reaction (PCR). Digested samples were subjected to capillary electrophoresis with laser-induced fluorescenc e detection (CE-LIF), with hydroxypropylmethylcellulose as the sieving matrix and SYBR Green I as the fluorescent dye. After amplification b ut before digestion, we added to the PCR mixture a fragment with the H infI 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, a nd (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.