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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共 34 条
  • [1] Gross rearrangement breakpoint database (GRaBD™)
    Abeysinghe, SS
    Stenson, PD
    Krawczak, M
    Cooper, DN
    [J]. HUMAN MUTATION, 2004, 23 (03) : 219 - 221
  • [2] Measurement of locus copy number by hybridisation with amplifiable probes
    Armour, JAL
    Sismani, C
    Patsalis, PC
    Cross, G
    [J]. NUCLEIC ACIDS RESEARCH, 2000, 28 (02) : 605 - 609
  • [3] Genomic rearrangements in the CFTR gene:: Extensive allelic heterogeneity and diverse mutational mechanisms
    Audrézet, M
    Chen, JM
    Raguénès, O
    Chuzhanova, N
    Giteau, K
    Le Maréchal, C
    Quéré, I
    Cooper, DN
    Férec, C
    [J]. HUMAN MUTATION, 2004, 23 (04) : 343 - 357
  • [4] Use of fluorescent DNA-intercalating dyes in the analysis of DNA via ion-pair reversed-phase denaturing high-performance liquid chromatography
    Bahrami, AR
    Dickman, MJ
    Matin, MM
    Ashby, JR
    Brown, PE
    Conroy, MJ
    Fowler, GJS
    Rose, JP
    Sheikh, QI
    Yeung, AT
    Hornby, DP
    [J]. ANALYTICAL BIOCHEMISTRY, 2002, 309 (02) : 248 - 252
  • [5] Real-time PCR-based gene dosage assay for detecting BRCA1 rearrangements in breast-ovarian cancer families
    Barrois, M
    Bièche, I
    Mazoyer, S
    Champème, MH
    Bressac-de Paillerets, B
    Lidereau, R
    [J]. CLINICAL GENETICS, 2004, 65 (02) : 131 - 136
  • [6] Rapid detection of noval BRCA1 rearrangements in high-risk breast-ovarian cancer families using multiplex PCR of short fluorescent fragments
    Casilli, F
    Di Rocco, ZC
    Gad, S
    Tournier, I
    Stoppa-Lyonnet, D
    Frebourg, T
    Tosi, M
    [J]. HUMAN MUTATION, 2002, 20 (03) : 218 - 226
  • [7] DENDUNNEN JT, 1989, AM J HUM GENET, V45, P835
  • [8] Significant contribution of large BRCA1 gene rearrangements in 120 French breast and ovarian cancer families
    Gad, S
    Caux-Moncoutier, V
    Pagès-Berhouet, S
    Gauthier-Villars, M
    Coupier, I
    Pujol, P
    Frénay, M
    Gilbert, B
    Maugard, C
    Bignon, YJ
    Chevrier, A
    Rossi, A
    Fricker, JP
    Nguyen, TD
    Demange, L
    Aurias, A
    Bensimon, A
    Stoppa-Lyonnet, D
    [J]. ONCOGENE, 2002, 21 (44) : 6841 - 6847
  • [9] Color bar coding the BRCAI gene on combed DNA:: A useful strategy for detecting large gene rearrangements
    Gad, S
    Aurias, A
    Puget, N
    Mairal, A
    Schurra, C
    Montagna, M
    Pages, S
    Caux, V
    Mazoyer, S
    Bensimon, A
    Stoppa-Lyonnet, D
    [J]. GENES CHROMOSOMES & CANCER, 2001, 31 (01) : 75 - 84
  • [10] HaywardLester A, 1996, BIOTECHNIQUES, V20, P250