Genotyping of two single nucleotide polymorphisms in 5,10-methylenetetrahydrofolate reductase by multiplex polymerase chain reaction and capillary electrophoresis.
Two single nucleotide polymorphisms (SNPs) of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, A1298C and C677T, were widely considered to be related with various neoplasia disorders. We established a simple and effective capillary electrophoresis (CE) method for detection of two SNPs in MTHFR gene simultaneously. DNA samples were amplified by multiplex PCR with universal fluorescence-labeled primer and analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE method was performed using 1.5% hydroxyethyl cellulose in 1× TBE buffer containing 1M urea. The PCR products after SSCP procedure were electrokinetically injected at -10 kV, 30s. Separation voltage was -6 kV and the temperature was set at 20°C. The optimal SSCP-CE method was applied to detect two polymorphisms in MTHFR gene of acute lymphoblastic leukemia (ALL) and attention-deficit/hyperactivity disorder (ADHD) patients. Genotyping results were evaluated in terms of relationships between outcomes for ADHD patients after ALL chemotherapy and ALL disease. The SSCP-CE method and multiplex PCR with universal fluorescence primer were used as the fast technique for screening two SNPs in MTHFR gene, A1298C and C677T. The genotyping data were coincident with DNA sequencing. This SSCP-CE method was found feasible for detecting mutation of MTHFR gene in populations.
Cheng
Hui-Ling
HL
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chiou
Shyh-Shin
SS
Liao
Yu-Mei
YM
Chen
Yen-Ling
YL
Wu
Shou-Mei
SM
D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2) / Q000235:genetics*; D001289:Attention Deficit Disorder with Hyperactivity / Q000150:complications / Q000201:enzymology / Q000235:genetics; D016022:Case-Control Studies; D016009:Chi-Square Distribution; D019075:Electrophoresis, Capillary / Q000379:methods*; D020022:Genetic Predisposition to Disease; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D018807:Polymorphism, Single-Stranded Conformational; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000150:complications / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D014508:Urea / Q000737:chemistry
D014508:Urea; D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2)