Fluorescence-based polymerase chain reaction-single-strand conformation polymorphism analysis of p53 gene by capillary electrophoresis.
Mutation of the p53 gene plays an important role in neoplastic progression in human tumorigenesis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) techniques are now available for the detection of point mutations. The original method using polyacrylamide gel electrophoresis is disadvantageous, particularly for clinical tests and for analysis of large numbers of samples. Therefore, using an automated capillary electrophoresis (CE) technique with a molecular-sieving polymer solution, we have devised a completely automatic fluorescence-based PCR-SSCP system (CE-FSSCP) for the differential detection of point mutations that dose not require SSCP with radioisotopes and polyacrylamide gels. The automatic CE-FSSCP system was developed for reproducible operations in the denaturation of double-stranded DNA and electrophoresis of single-stranded DNA. The detection system consists of a 100 W I2 lamp and photomultiplier. We performed CE-FSSCP with a 2% linear polyacrylamide polymer solution containing 5% glycerol. Four tissue specimens of lung tumors with mutations in exon 7 of the p53 gene were found to have mutant alleles; six-base-pair deletion at codons 247-248, a one-base-pair deletion at codon 260, a one-base-pair deletion at codon 244 and a GGC to CGC substitution at codon 244. We expect this technique to prove useful for the clinical DNA diagnosis of human cancers, determination of the therapeutic effect of anticancer agents and for the study of the molecular aspects of the mechanisms involved in the pathogenesis of human cancers.
Katsuragi
K
K
Diagnostics Division, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan.
Kitagishi
K
K
Chiba
W
W
Ikeda
S
S
Kinoshita
M
M
D000483:Alleles; D001331:Automation; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D004273:DNA, Neoplasm