Methods in laboratory investigation. Rapid and simple detection of c-Ki-ras2 gene codon 12 mutations by nonradioisotopic single-strand conformation polymorphism analysis.
BACKGROUND
To achieve simple and rapid detection of codon 12 mutations of the c-Ki-ras2 gene, we developed a nonradioisotopic single-strand conformation polymorphism analysis using silver stain.


METHODS
After a conventional polymerase chain reaction, amplified DNA fragments were mixed with formamide, heated and subjected to electrophoresis for 1.5 hours using minigels of polyacrylamide. The gels were then silver stained and single-strand DNA fragments were visualized directly.


RESULTS
Electrophoresis and subsequent silver staining were completed within 2.5 hours. Plasmids carrying various codon 12 mutations of the c-Ki-ras2 gene were used as controls of Nonradioisotopic single-strand confirmation polymorphism analysis, and all six mutations were successfully separated from the normal allele in a single electrophoretic run. In the analysis of tumor cell lines and tumor samples, the results were identical with those of conventional dot blot hybridization using 32P-labeled oligonucleotide probes. Of 10 colorectal carcinoma tissues examined, 4 tumors were shown to carry codon 12 mutations of the c-Ki-ras2 gene.


CONCLUSIONS
We conclude that this novel technique is a rapid, simple and useful method that may replace conventional methods.
Sugano
K
K
Division of Clinical Laboratory Oncology, National Cancer Center Hospital, Tokyo, Japan.
Kyogoku
A
A
Fukayama
N
N
Ohkura
H
H
Shimosato
Y
Y
Sekiya
T
T
Hayashi
K
K
D000483:Alleles; D003062:Codon / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D015151:Immunoblotting; D008175:Lung Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D009154:Mutation / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D013274:Stomach Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology
D003062:Codon; D004277:DNA, Single-Stranded; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)