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Detection of DNA Double-Strand Breaks and Chromosome Translocations Using Ligation-Mediated PCR and Inverse PCR

  • Sheetal Singh
  • Shy-Jen Shih
  • Andrew T. M. VaughanEmail author
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Part of the Methods in Molecular Biology book series (MIMB, volume 2102)

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.

Key words

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

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Sheetal Singh
    • 1
  • Shy-Jen Shih
    • 1
  • Andrew T. M. Vaughan
    • 1
    Email author
  1. 1.Department of Radiation OncologyUniversity of California at DavisSacramentoUSA

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