C677T MUTATION OF METHYLENETETRAHYDROFOLATE REDUCTASE GENE DETERMINEDIN BLOOD OR PLASMA BY MULTIPLE-INJECTION CAPILLARY ELECTROPHORESIS AND LASER-INDUCED FLUORESCENCE DETECTION
A. Ulvik et al., C677T MUTATION OF METHYLENETETRAHYDROFOLATE REDUCTASE GENE DETERMINEDIN BLOOD OR PLASMA BY MULTIPLE-INJECTION CAPILLARY ELECTROPHORESIS AND LASER-INDUCED FLUORESCENCE DETECTION, Clinical chemistry, 43(2), 1997, pp. 267-272
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.