Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH.
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 microg 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.
Guillaud-Bataille
Marine
M
Génomique Cellulaire des Cancers, CNRS UMR 8125, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France.
Valent
Alexander
A
Soularue
Pascal
P
Perot
Christine
C
Inda
Maria Mar
MM
Receveur
Aline
A
Smaïli
Sadek
S
Roest Crollius
Hugues
H
Bénard
Jean
J
Bernheim
Alain
A
Gidrol
Xavier
X
Danglot
Gisèle
G
D045744:Cell Line, Tumor; D022202:Chromosomes, Artificial, Bacterial; D004247:DNA / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction
D004273:DNA, Neoplasm; D004247:DNA