Δ-PCR, A Simple Method to Detect Translocations and Insertion/Deletion Mutations.
| Autor: | Lin MT; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA., Tseng LH, Rich RG, Hafez MJ, Harada S, Murphy KM, Eshleman JR, Gocke CD |
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| Jazyk: | angličtina |
| Zdroj: | The Journal of molecular diagnostics : JMD [J Mol Diagn] 2011 Jan; Vol. 13 (1), pp. 85-92. Date of Electronic Publication: 2010 Dec 23. |
| DOI: | 10.1016/j.jmoldx.2010.11.004 |
| Abstrakt: | PCR detection of chromosomal translocations and small insertion/deletion mutations is challenging when potential amplicon size varies greatly. Molecular diagnostic laboratories face such difficulties with the BCL2-IGH translocation in follicular lymphoma and with internal tandem duplication mutation of the FLT3 gene in leukemia, where breakpoints are widely distributed, mutations may be multiple, signal strength is low, and background noise is elevated. We developed a strategy, called Δ-PCR, that ensures PCR specificity and identifies individual breakpoints. Δ-PCR uses two forward primers (external and internal) and a reverse primer simultaneously. The internal primer functions as a probe with a defined distance Δ from the external primer. For follicular lymphoma, we prepared upstream, BCL2-specific primers for potential breakpoints to pair with a common, downstream VLJH primer. Multiplexed PCR amplicons are sized by capillary electrophoresis. Each of the upstream pairs has a defined interval separating them that uniquely identifies the breakpoint. The presence of two amplicons with a defined size difference confirms validity of the rearrangement and identity of the specific breakpoint, even if signal strength is low. By testing 40 follicular lymphoma and 12 control specimens from formalin-fixed, paraffin-embedded (FFPE) blocks, we showed that multiplex Δ-PCR is a simple, sensitive strategy to identify translocations with multiple breakpoints or partners. The strategy was also applied to detect minor leukemic clones with internal tandem duplication mutations and could have broader applications for other insertion/deletion and duplication mutations. (Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
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