F-SSCP: fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis.
A fluorescence-based method for polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis, F-SSCP, was developed in which the target sequence is amplified by the PCR using fluorescent primers. The amplified products are then heat-denatured and applied to a water-jacket controlled gel in an automated DNA sequencer. The separated strands are detected as laser-excited fluorescence at the bottom of the gel, and mutations are detected as shifts in the position of the peaks in the fluorogram. The system does not involve radioactivity, and the conditions of electrophoresis are more strictly controlled than in the previous system, which relied on ambient air-cooling to maintain the gel at a constant temperature. The nature of the output data allows direct quantitative interpretation, and so the relative abundance of each allele in a mixture of two or more alleles can easily be estimated. The application of F-SSCP for detection of mutations and loss of heterozygosities of p53 in tumor tissues is reported.
Makino
R
R
Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan.
Yazyu
H
H
Kishimoto
Y
Y
Sekiya
T
T
Hayashi
K
K
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D004273:DNA, Neoplasm / Q000032:analysis; D004277:DNA, Single-Stranded / Q000032:analysis; D005456:Fluorescent Dyes; D005470:Fluorometry; D016158:Genes, p53; D006801:Humans; D007834:Lasers; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D001749:Urinary Bladder Neoplasms / Q000737:chemistry
D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes