Optimization of nonradioisotopic single strand conformation polymorphism analysis with a conventional minislab gel electrophoresis apparatus.
We have developed a means of nonradioisotopic single strand conformation polymorphism (nonRI-SSCP) analysis and applied it to the detection of a point mutation in the human tumor suppressor gene, p53. The method does not require any particular facilities or apparatus, such as a laboratory for radioactive materials, a large gel unit for sequencing, or a semiautomated electrophoresis system. This technique comprises amplification of DNA fragments by the PCR technique with specific oligonucleotide primers, denaturation, and electrophoresis on neutral polyacrylamide gels in a conventional minislab apparatus. The SSCP patterns on electrophoresis were detected with a commercially available silver stain method. We also evaluated various electrophoretic conditions for nonRI-SSCP analysis, such as the gel concentration and buffer components. A tris/glycine buffer system gave better resolution of SSCP bands. The SSCP patterns of different sized DNAs could be analyzed in a gradient polyacrylamide gel. Thus, nonRI-SSCP analysis with a conventional minislab gel electrophoresis apparatus can be satisfactorily substituted for a commonly used RI-SSCP technique.
Oto
M
M
Department of Biotechnology, Tokyo Technical College, Japan.
Miyake
S
S
Yuasa
Y
Y
D001483:Base Sequence; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D016622:Silver Staining / Q000379:methods; D013698:Templates, Genetic
D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded