C677T mutation of methylenetetrahydrofolate reductase gene determined in blood or plasma by multiple-injection capillary electrophoresis and laser-induced fluorescence detection.

نویسندگان

  • A Ulvik
  • H Refsum
  • L A Kluijtmans
  • P M Ueland
چکیده

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.

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Simultaneous determination of methylenetetrahydrofolate reductase C677T and factor V G1691A genotypes by mutagenically separated PCR and multiple-injection capillary electrophoresis.

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عنوان ژورنال:
  • Clinical chemistry

دوره 43 2  شماره 

صفحات  -

تاریخ انتشار 1997