Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array–CGH

Abstract

Structural genetic alterations in cancer often involve gene loss or gene amplification. With the advent of microarray approaches for the analysis of the genome, as exemplified by array–CGH (Comparative GenomicHybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density. However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array–CGH analysis. We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples. We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias. The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours. Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines. Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples. Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material.

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

“…Similar effects were reported with the mutant single-strand binding protein protein of Thermus thermophilus (Inoue et al, 2006). One report suggested that background synthesis from primerprimer interactions may also be reduced or eliminated by the modification of hexamer primers with the incorporation of one or two 5 0 -terminal universal bases (Lage et al, 2003). Recent work demonstrated that carefully avoiding trace levels of DNA contamination resulted in only 0.36-1.0% of sequences being nonspecific (Marcy et al, 2007a).…”

Array comparative genomic hybridisation (aCGH) analysis of premenopausal breast cancers from a nuclear fallout area and matched cases from Western New York

“…Links to genomic databases helped to identify candidate genes for further investigation. A small number of published studies applied aCGH technology to breast cancer cells, but only to a very limited extent (Pinkel et al, 1998;Albertson et al, 2000;Kauraniemi et al, 2001;Hyman et al, 2002;Pollack et al, 2002;Lage et al, 2003). Almost all of these utilised a significantly smaller number of specimens.…”

“…Almost all of these utilised a significantly smaller number of specimens. Some of them focused only on cell lines (Hyman et al, 2002;Lage et al, 2003) or on individual chromosomes (Pinkel et al, 1998;Albertson et al, 2000;Kauraniemi et al, 2001). We are not aware of any published reports on array or conventional CGH analysis of DNA abnormalities in irradiated cells.…”

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

“…[20][21][22] In all, 10 ng of BAC DNA (1 ml) was added to 9 ml of the sample buffer before the mixture was incubated at 951C for 3 min and then cooled on ice for 5 min. In all, 10 ml of f29 polymerase reaction mix (1 ml of f29 polymerase in 9 ml of reaction buffer) was added, and the mixture was then incubated at 301C for Z16 h. To inactivate the enzyme the mixture was heated for 10 min at 651C.…”