Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH.
Nucleic Acids Res
; 32(13): e112, 2004 Jul 29.
Article
em En
| MEDLINE
| ID: mdl-15284333
ABSTRACT
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.
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Base de dados:
MEDLINE
Assunto principal:
DNA
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Análise de Sequência com Séries de Oligonucleotídeos
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Cromossomos Artificiais Bacterianos
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Genômica
Limite:
Humans
Idioma:
En
Ano de publicação:
2004
Tipo de documento:
Article