Analysis of single-strand conformation polymorphisms by capillary electrophoresis with laser induced fluorescence detection.
Detection of point mutations in genomic DNA is important for diagnosis of inherited characteristics and genetic diseases. A point mutation in a specific region of DNA amplified by polymerase chain reaction (PCR) can be detected with single-strand conformation polymorphism (SSCP) analysis. Analysis of SSCP by laser-induced fluorescence capillary electrophoresis in entangled polymer solution (CE-LIF) has been developed in the present paper. K-ras genes including seven mutations were amplified with primer labeled with Texas Red at its 5' end. The labeled PCR products were dissociated to single strands by heating and separated with capillary gel electrophoresis and He-Ne laser-excited fluorescence detection. Our results suggest that all fragments having normal (Gly) and mutated (Ala, Arg, Cys, Ser, Val, Asp) sequences at codon 12 can be distinguished. Analysis of SSCPs with CE-LIF is well suited for clinical analysis of SSCPs because of its high sensitivity, resolution, reproducibility and speed.
Arakawa
H
H
School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Tsuji
A
A
Maeda
M
M
Kamahori
M
M
Kambara
H
H
D003062:Codon; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D011905:Genes, ras; D007834:Lasers; D009154:Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014966:Xanthenes
D003062:Codon; D005456:Fluorescent Dyes; D014966:Xanthenes; C034657:Texas red