Use of the DNA fiber spreading technique to detect the effects of mutant p53 on DNA replication

Methods Mol Biol. 2013;962:147-55. doi: 10.1007/978-1-62703-236-0_12.

Abstract

DNA replication involves a coordinated progression through S phase, and disruption of these regulated steps may cause gene abnormalities, which may lead to cancer. Different stages of DNA replication can be detected immunofluorescently that would indicate how replication is progressing in a cell population or under specific conditions. We describe a method for labeling replicating DNA with two nucleotide analogs, and then detecting the sequential patterns of incorporation using fluorescently labeled antibodies on DNA spread onto a glass slide. Quantification of the different types of replication patterns produced by this method reveals how replication is achieved under different conditions by the predominance and lengths of elongating replication forks progressing from single or clustered origins, as well as the sites of termination from two converging forks.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line, Tumor
  • DNA / genetics
  • DNA Replication*
  • Humans
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism*
  • S Phase
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*

Substances

  • Mutant Proteins
  • Tumor Suppressor Protein p53
  • DNA