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Nucleic Acids Research 2004 32(18):e139; doi:10.1093/nar/gnh137
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Published online 11 October 2004

Nucleic Acids Research, Vol. 32 No. 18 © Oxford University Press 2004; all rights reserved

Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene

C. Dehainault1, A. Laugé1, V. Caux-Moncoutier1, S. Pagès-Berhouet1, F. Doz2, L. Desjardins3, J. Couturier1,4, M. Gauthier-Villars1, D. Stoppa-Lyonnet1,4 and C. Houdayer1,*

1 Service de Génétique Oncologique, 2 Service d'Oncologie Pédiatrique, 3 Service d'Ophtalmologie and 4 INSERM U509, Pathologie Moléculaire des Cancers, Institut Curie, Paris, France

* To whom correspondence should be addressed at Service de Génétique Oncologique, Institut Curie, 75248 Paris Cedex 05, France. Tel: +33 1 44 32 46 98; Fax: +33 1 44 32 45 09; Email: claude.houdayer{at}curie.net

Received July 27, 2004; Revised and Accepted September 22, 2004

Screening for large gene rearrangements is established as an important part of molecular medicine but is also challenging. A variety of robust methods can detect whole-gene deletions, but will fail to detect more subtle rearrangements that may involve a single exon. In this paper, we describe a new, versatile and robust method to assess exon copy number, called multiplex PCR/liquid chromatography assay (MP/LC). Multiple exons are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and quantitated by fluorescent detection using a post-column intercalation dye. The relative peak intensities for each target directly reflect exon copy number. This novel technique was used to screen a panel of 121 unrelated retinoblastoma patients who were tested previously using a reference strategy. MP/LC correctly scored all deletions and demonstrated a previously undetected RB1 duplication, the first to be described. MP/LC appears to be an easy, versatile, and cost-effective method, which is particularly relevant to denaturing HPLC (DHPLC) users since it broadens the spectrum of available applications on a DHPLC system.


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