Whole genome amplification for CGH analysis: Linker‐adapter PCR as the method of choice for difficult and limited samples

Abstract

BackgroundComparative genomic hybridization (CGH) is a powerful method to investigate chromosomal imbalances in tumor cells. However, DNA quantity and quality can be limiting factors for successful CGH analysis. The aim of this study was to investigate the applicability of degenerate oligonucleotide‐primed PCR (DOP‐PCR) and a recently developed linker‐adapter‐mediated PCR (LA‐PCR) for whole genome amplification for use in CGH, especially for difficult source material.MethodsWe comparatively analyzed DNA of variable quality derived from different cell/tissue types. Additionally, dilution experiments down to the DNA content of a single cell were performed. FISH and/or classical cytogenetic analyses were used as controls.ResultsIn the case of high quality DNA samples, both methods were equally suitable for CGH. When analyzing very small amounts of these DNA samples (equivalent to one or a few human diploid cells), DOP‐PCR‐CGH, but not LA‐PCR‐CGH, frequently produced false‐positive signals (e.g., gains in 1p and 16p, and losses in chromosome 4q). In case of formalin‐fixed paraffin‐embedded tissues, success rates by LA‐PCR‐CGH were significantly higher as compared to DOP‐PCR‐CGH. DNA of minor quality frequently could be analyzed correctly by LA‐PCR‐CGH, but was prone to give false‐positive and/or false‐negative results by DOP‐PCR‐CGH.ConclusionsLA‐PCR is superior to DOP‐PCR for amplification of DNA for CGH analysis, especially in the case of very limited or partly degraded source material. © 2004 Wiley‐Liss, Inc.

Dynamics of chemosensitivity and chromosomal instability in recurrent glioblastoma

“…Preparation of genomic DNA was performed as described previously (Pirker et al, 2004). Comparative genomic hybridisation (CGH), fluorescence in situ hybridisation (FISH) and CDD banding were performed as described previously (Pirker et al, 2004).…”

“…Comparative genomic hybridisation (CGH), fluorescence in situ hybridisation (FISH) and CDD banding were performed as described previously (Pirker et al, 2004). For DNA amplification, linker -adapter PCR was used as described (Pirker et al, 2004). For the detection of the EGFR locus, the BAC clone RP11-339F13 supplied by Pieter De Jong (Oakland, CA, USA) was used.…”

Simultaneous blockade of the epidermal growth factor receptor/mammalian target of rapamycin pathway by epidermal growth factor receptor inhibitors and rapamycin results in reduced cell growth and survival in biliary tract cancer cells

“…Cytogenetic Analyses Preparation of genomic DNA, comparative genomic hybridization (CGH), and CDD banding were done as described previously (19). FISH was done using the EGFR/ CEN-7 FISH Probe Mix and the DakoCytomation Cytology FISH Accessory Kit according to the manufacturer's recommendations (DakoCytomation).…”

Getting it right: designing microarray (and not ‘microawry') comparative genomic hybridization studies for cancer research

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