C677T mutation of methylenetetrahydrofolate reductase gene determined in blood or plasma by multiple-injection capillary electrophoresis and laser-induced fluorescence detection.
نویسندگان
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.
منابع مشابه
C677T mutation of methylenetetrahydrofolate reductase gene determined in blood or plasma multiple-injection capillary electrophoresis and induced fluorescence detection
متن کامل
C677T mutation of methylenetetrahydrofolate reductase gene determined in blood or plasma multiple-injection capillary electrophoresis and induced fluorescence detection
متن کامل
Simultaneous determination of methylenetetrahydrofolate reductase C677T and factor V G1691A genotypes by mutagenically separated PCR and multiple-injection capillary electrophoresis.
The C677T mutation of the methylenetetrahydrofolate reductase gene and the G1691A (Leiden) mutation of the factor V gene are established risk factors for thromboembolic disease. We here present an assay for the simultaneous genotyping of these common genetic variants. The assay involves a strategy based on multiplex mutagenically separated PCR performed in a single tube containing six primers. ...
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BACKGROUND Plasma total homocysteine (tHcy) is a risk factor for cardiovascular disease. tHcy concentrations are partly determined by folate, cobalamin, and vitamin B(6) status, and methylenetetrahydrofolate reductase (MTHFR) and other flavoenzymes are important for the biotransformation of these vitamins. This motivates the investigation of the possible relationship between riboflavin status a...
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Introduction: Cleft lips and cleft palates are common congenital abnormalities in children. Various chromosomal loci have been suggested to be responsible the development of these abnormalities. The present study was carried out to investigate the association between the suspected genes (methylenetetrahydrofolate reductase [MTHFR] A1298C and C677T) that might contribute into the etiology of the...
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ورودعنوان ژورنال:
- Clinical chemistry
دوره 43 2 شماره
صفحات -
تاریخ انتشار 1997