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Nucleic Acids Research 2004 32(13):e112; doi:10.1093/nar/gnh108
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Published online 29 July 2004

Nucleic Acids Research, Vol. 32 No. 13 © Oxford University Press 2004; all rights reserved

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH

Marine Guillaud-Bataille, Alexander Valent, Pascal Soularue2, Christine Perot, Maria-del-Mar Inda, Aline Receveur, Sadek Smaïli, Hugues Roest Crollius3, Jean Bénard1, Alain Bernheim, Xavier Gidrol2 and Gisèle Danglot*

Génomique Cellulaire des Cancers, CNRS UMR 8125 and 1 Service de Génétique, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France, 2 Service de Génomique Fonctionnelle, CEA and 3 Genoscope, 2 rue Gaston Crémieux, 91057 Evry Cedex, France

* To whom correspondence should be addressed. Tel: +33 1 42 11 54 21; Fax: +33 1 41 11 52 60; Email: gdanglot{at}igr.fr

Received May 27, 2004; Revised and Accepted July 9, 2004

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 µg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.


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