Whole genome amplification for CGH analysis: Linker-adapter PCR as the method of choice for difficult and limited samples.
BACKGROUND
Comparative 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.


METHODS
We 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.


RESULTS
In 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.


CONCLUSIONS
LA-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.
Pirker
Christine
C
Division of Applied & Experimental Oncology, Institute of Cancer Research, Vienna University, Vienna, Austria.
Raidl
Maria
M
Steiner
Elisabeth
E
Elbling
Leonilla
L
Holzmann
Klaus
K
Spiegl-Kreinecker
Sabine
S
Aubele
Michaela
M
Grasl-Kraupp
Bettina
B
Marosi
Christine
C
Micksche
Michael
M
Berger
Walter
W
D045744:Cell Line, Tumor; D002869:Chromosome Aberrations; D004273:DNA, Neoplasm / Q000032:analysis; D016678:Genome; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004273:DNA, Neoplasm