Chemical mismatch cleavage combined with capillary electrophoresis: detection of mutations exon 8 of the cystathionine beta-synthase gene.
Mutation detection by chemical mismatch cleavage (CMC) is based on the chemical modification and cleavage at the site of mismatched C or T in heteroduplexes, using hydroxylamine or osmium tetroxide (OsO4) as chemical probes. In the present study, we evaluated CMC in combination with capillary electrophoresis (CE) by determining the common T833C and G919A mutations in exon 8 of the cystathionine beta-synthase gene in heterozygous and homozygous samples. A 186-bp fragment encompassing exon 8 was amplified by PCR with both primers labeled with 5'-fluorescein. Labeled single strands of 40 and 61 nucleotides (nt) were formed from the coding strand of the T833C sample and non-coding strand from the G919A sample, respectively. These single-stranded DNA (ssDNA) products were analyzed under denaturing conditions by CE with short-chain linear polyacrylamide as the sieving matrix and were detected by laser-induced fluorescence (LIF), using a sensitive, one-channel sheath-flow detector. The CE-LIF format afforded relatively high resolution of ssDNA (down to 1 nt), precise size assessment of CMC products, sensitive detection with small sample requirements, and fast analysis. Thus, CMC combined with CE-LIF is suitable for screening of known mutations, giving expected CMC products, but will also detect unknown mutations, the locations of which are indicated by the fragment sizes.
Department of Pharmacology, University of Bergen, Norway. Jicun.Ren@farm.uib.no
J
J
Ren
A
A
Ulvik
H
H
Refsum
P M
PM
Ueland
D003541:Cystathionine beta-Synthase / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D019075:Electrophoresis, Capillary; D005091:Exons; D006720:Homozygote; D006801:Humans; D019811:Hydroxylamine; D009993:Osmium Tetroxide; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
D019811:Hydroxylamine; D004247:DNA; D003541:Cystathionine beta-Synthase; D009993:Osmium Tetroxide