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 language | English (US) |
|---|---|
| Pages (from-to) | 399-415 |
| Number of pages | 17 |
| Journal | Methods in molecular biology (Clifton, N.J.) |
| Volume | 1105 |
| DOIs | |
| State | Published - 2014 |
Fingerprint
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 journal › Article
}
TY - JOUR
T1 - Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.
AU - Singh, Sheetal
AU - Shih, Shyh Jen
AU - Vaughan, Andrew T M
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84908553258&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84908553258&partnerID=8YFLogxK
U2 - 10.1007/978-1-62703-739-6_30
DO - 10.1007/978-1-62703-739-6_30
M3 - Article
C2 - 24623244
AN - SCOPUS:84908553258
VL - 1105
SP - 399
EP - 415
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
SN - 1064-3745
ER -