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Anal Biochem. 1993 Aug 15;213(1):19-22.

Optimization of nonradioisotopic single strand conformation polymorphism analysis with a conventional minislab gel electrophoresis apparatus.

Author information

1
Department of Biotechnology, Tokyo Technical College, Japan.

Abstract

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.

PMID:
8238876
DOI:
10.1006/abio.1993.1379
[Indexed for MEDLINE]

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