Multiple fluorescence-based PCR-SSCP analysis.
Multiple fluorescence-based polymerase chain reaction single-strand conformation polymorphism (MF-PCR-SSCP) was developed. The target sequence was amplified by PCR using forward and reverse primers labeled with two different fluorescent dyes at their 5' ends. The amplified products were then heat-denatured, mixed with internal standard DNA markers labeled with a third fluorescent dye and applied to a temperature-controlled gel in an automated DNA sequencer, with a gel-temperature-controlling system. Mutations were detected as positional shifts of two-colored peaks in the electrophoretogram. The image data were analyzed by the computer program GENESCAN 672. The peak positions were standardized to internal DNA size markers. MF-PCR-SSCP analysis of 7 human tumor cell lines with 7 different single base mutations of the human K-ras oncogene detected all mutations even under the same electrophoresis conditions. Complete loss of heterozygosity was detected in two cell lines simultaneously. A gel temperature at 20 degrees C and polyacrylamide concentration of 10% gave the best separation. MF-PCR-SSCP is superior to the current PCR-SSCP in several ways: it does not involve radioactivity, migration patterns are standardized to internal standard DNA markers, there is a strict temperature-controlling system and the higher percentage of the gel enables better separation with resultant 100% detection of mutations most likely under one set of electrophoresis conditions.
Iwahana
H
H
Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, University of Tokushima, Japan.
Yoshimoto
K
K
Mizusawa
N
N
Kudo
E
E
Itakura
M
M
D001483:Base Sequence; D001709:Biotechnology; D004247:DNA / Q000235:genetics / Q000592:standards; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods / Q000592:standards / Q000706:statistics & numerical data; D011905:Genes, ras; D005990:Glycerol; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D011110:Polymorphism, Genetic; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013696:Temperature; D014407:Tumor Cells, Cultured / Q000737:chemistry
D017931:DNA Primers; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D004247:DNA; D005990:Glycerol