Multiplex PCR liquid chromatography assay for detection of gene rearrangements:: application to RB1 gene -: art. no. e139

被引:48
作者
Dehainault, C
Laugé, A
Caux-Moncoutier, V
Pagès-Berhouet, S
Doz, F
Desjardins, L
Couturier, J
Gauthier-Villars, M
Stoppa-Lyonnet, D
Houdayer, C [1 ]
机构
[1] Inst Curie, Serv Genet Oncol, F-75248 Paris 05, France
[2] Inst Curie, Serv Oncol Pediat, F-75248 Paris 05, France
[3] Inst Curie, Serv Ophtalmol, F-75248 Paris 05, France
[4] Inst Curie, INSERM, U509, F-75248 Paris 05, France
关键词
D O I
10.1093/nar/gnh137
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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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页数:9
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