Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR

Sheetal Singh, Shy Jen Shih, Andrew T.M. Vaughan

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allow detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages271-288
Number of pages18
DOIs
StatePublished - Jan 1 2020

Publication series

NameMethods in Molecular Biology
Volume2102
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Apoptosis
  • DNA
  • Double-strand break repair
  • IPCR
  • LM-PCR
  • MLL
  • Parallel sequencing
  • Translocation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Singh, S., Shih, S. J., & Vaughan, A. T. M. (2020). Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR. In Methods in Molecular Biology (pp. 271-288). (Methods in Molecular Biology; Vol. 2102). Humana Press Inc.. https://doi.org/10.1007/978-1-0716-0223-2_15