Detection and Localization of Single Base Changes by Denaturing Gradient Gel Electrophoresis

Academic Article

Abstract

  • This chapter discusses the detection and localization of single base changes by denaturing gradient gel electrophoresis. For most purposes, single-stranded deoxyribonucleic acid (DNA) probes 140–1000 bases in length are used, which allows about half of this number of base pairs to be screened for base changes in a single gel lane. The chapter discusses the use of single-stranded RNA probes. It describes the development of two new methods for detecting and localizing single base changes in cloned and genomic DNA. In both procedures, a single-stranded, radioactively labeled probe of wild-type sequence is annealed to cloned or genomic DNA. If the DNA under test carries a single base change, a hybrid double-stranded species containing a mismatch is formed. In the first procedure, either a ribonucleic acid (RNA) or a DNA probe is used, and the mismatched duplex is separated from the perfectly paired, wild type duplex by electrophoresis in a denaturing gradient gel. In the second procedure, the probe is composed of single-stranded RNA and the mismatch is cleaved by ribonuclease; the resulting cleaved products are examined by polyacrylamide gel electrophoresis and autoradiography. It is estimated that each method will detect approximately 50% of all possible substitutions, insertions, or deletions in a probed region. Although there is some overlap in the base changes that are detected with the two procedures, it is likely that the use of both methods will be complementary, resulting in the detection of a very large fraction of all possible substitutions and they should be particularly useful in mapping and diagnosing small mutations that result in genetic disease. This method has led to the detection of neutral polymorphisms for genetic linkage studies. The chapter lists all the equipment needed to prepare and run the gels and also describes the hybridization procedure for RNA probes. © 1987, Academic Press, Inc.
  • Digital Object Identifier (doi)

    Author List

  • Myers RM; Maniatis T; Lerman LS
  • Start Page

  • 501
  • End Page

  • 527
  • Volume

  • 155
  • Issue

  • C