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

Sheetal Singh, Shyh Jen Shih, Andrew T M Vaughan

Research output: Contribution to journalArticle

1 Citation (Scopus)

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 allows 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)
Pages (from-to)399-415
Number of pages17
JournalMethods in molecular biology (Clifton, N.J.)
Volume1105
DOIs
StatePublished - 2014

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Double-Stranded DNA Breaks
Ligation
Chromosomes
DNA Breaks
Polymerase Chain Reaction
Gene Rearrangement
Masks
Genes
DNA

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR. / Singh, Sheetal; Shih, Shyh Jen; Vaughan, Andrew T M.

In: Methods in molecular biology (Clifton, N.J.), Vol. 1105, 2014, p. 399-415.

Research output: Contribution to journalArticle

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