Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.7.1 (methylenetetrahydrofolate reductase)
 2,116 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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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PMID:C677T mutation of methylenetetrahydrofolate reductase gene determined in blood or plasma by multiple-injection capillary electrophoresis and laser-induced fluorescence detection. 902 28

A rapid analysis of single-strand conformation polymorphism (SSCP) by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detector was developed using a short-chain, linear polyacrylamide (PA) as sieving medium. Capillary filling of this low-viscosity medium and medium replacement were carried out by commercial capillary electrophoresis instruments. The approach was successfully applied to detect the C677T mutation of methylenetetrahydrofolate reductase gene. The influences of factors such as the concentration of polymers, voltage, temperature, and additives on the SSCP analysis were systematically investigated. Using 6% PA sieving medium and high electric field, four strands were resolved within 11 min in a DNA sample heterozygous for the C677T mutation, and a characteristic pattern was apparent for each of the three genotypes. When using multiple injection mode, the average analysis time per sample was reduced to about 4 min. In conclusion, our results indicate that CE-LIF may be an alternative to conventional SSCP analysis based on slab gel electrophoresis for the detection of genetic mutations. The technique is simple and rapid and is well suited to analysis of large numbers of clinical samples.
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PMID:Analysis of single-strand conformation polymorphism by capillary electrophoresis with laser-induced fluorescence detection using short-chain polyacrylamide as sieving medium. 902 71

Inverse-flow derivatization is a novel approach to obtain fluorescent DNA derivatives in DNA analysis based on CE with LIF detection. In the present work, we want to explore the feasibility of the application of this method into the mutation detection based on constant denaturant capillary electrophoresis (CDCE) and SSCP analysis. The DNA fragments were first amplified by PCR using a pair of common primers without fluorescent label, and then the mutations were determined by CDCE or SSCP analysis based on CE-LIF with inverse-flow derivatization of DNA fragments. The experimental conditions were investigated systematically, and different labeling modes including inverse-flow derivatization, on-column derivatization and fluorescent labeled primer technique were compared. The inverse-flow derivatization was successfully used in the detection of C677T mutation in the methylenetetrahydrofolate reductase gene by CDCE or SSCP analysis. Our preliminary results demonstrate that inverse-flow derivatization is very simple, inexpensive and sensitive and well suitable for the genetic analysis in clinical diagnosis.
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PMID:Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragments. 1694 50