Optimization of Nonradioisotopic Single Strand Conformation Polymorphism Analysis with a Conventional Minislab Gel Electrophoresis Apparatus
✍ Scribed by M. Oto; S. Miyake; Y. Yuasa
- Publisher
- Elsevier Science
- Year
- 1993
- Tongue
- English
- Weight
- 327 KB
- Volume
- 213
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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. O 1983 Academic Press, Inc.