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Identification of chromosomal breakpoints of cancer-specific translocations by rolling circle amplification and long-distance inverse PCR.

Abstract
We describe the use of rolling circle amplification and long-distance inverse polymerase chain reaction (LD-PCR) to identify chromosomal breakpoints and fusion genes in cancer cells carrying acquired translocations. This approach produced enough template for 100 inverse PCR reaction from as little as 20 ng of patient DNA, consequently enabling the use of up to 500 times less patient DNA compared to standard inverse PCR. The method is based on identifying restriction sites in a putative breakpoint area in a cancer-specific translocation, followed by circularization and amplification of the restriction DNA products by using T4 DNA ligase and Phi29 enzyme, respectively. The amplified DNA thus obtained is used as a template in long-distance inverse PCR to amplify and detect the precise breakpoint of the chromosomal rearrangements in question by sequencing of the obtained PCR products. We demonstrate the feasibility of this approach by identifying fusion genes TAF15-ZNF384 (brought about by a (12;17)(p13;q21) translocation) and BCR-ABL1 (produced by a (9:22)(q34;q11.2) translocation) in five leukemia samples. The application of rolling circle amplification before inverse PCR may be particularly useful in the search for chromosomal breakpoints and fusion genes brought about by new translocations when only minute amounts of DNA are available from the sampled malignant lesion.
AuthorsJim Thorsen, Francesca Micci, Sverre Heim
JournalCancer genetics (Cancer Genet) Vol. 204 Issue 8 Pg. 458-61 (Aug 2011) ISSN: 2210-7762 [Print] United States
PMID21962896 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2011 Elsevier Inc. All rights reserved.
Chemical References
  • DNA, Neoplasm
  • Oncogene Proteins, Fusion
Topics
  • Chromosome Aberrations
  • Chromosome Breakpoints
  • DNA, Neoplasm (analysis, genetics)
  • Humans
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive (genetics)
  • Oncogene Proteins, Fusion (genetics)
  • Polymerase Chain Reaction
  • Precursor Cell Lymphoblastic Leukemia-Lymphoma (genetics)
  • Translocation, Genetic

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