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

Anal Biochem. 1993 Aug 15;213(1):19-22. doi: 10.1006/abio.1993.1379.

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.

Publication types

  • Comparative Study

MeSH terms

  • Base Sequence
  • DNA, Neoplasm / analysis*
  • DNA, Neoplasm / genetics
  • DNA, Single-Stranded / analysis*
  • Electrophoresis, Polyacrylamide Gel / methods
  • Genes, p53 / genetics
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Polymerase Chain Reaction / methods
  • Polymorphism, Genetic*
  • Sensitivity and Specificity
  • Silver Staining / methods
  • Templates, Genetic

Substances

  • DNA, Neoplasm
  • DNA, Single-Stranded