Evaluation of Phi29-based whole-genome amplification for microarray-based comparative genomic hybridisation.
For the optimal performance of high throughput genomic technologies sufficient yields of high-quality DNA are crucial. Following microdissection, most samples fail to produce sufficient quantities of DNA for genome-wide experiments. Various PCR-based amplification methods have been used, but these usually produce nonuniform representations of the genome. Bacteriophage Phi29 DNA polymerase random-primed DNA amplification is based on isothermal multiple displacement amplification. We sought to define the genome representation of this method in a bacterial artificial chromosome microarray comparative genomic hybridisation (aCGH) platform. Test genomic female DNA was amplified using Phi29 amplification at four different starting concentrations (0.5, 5, 10 and 50 ng). These products were combined with unamplified and amplified genomic female DNA as reference. In addition, 50 ng of DNA from five microdissected breast cancer frozen samples, were amplified using the same method. Three combinations were performed: unamplified test with unamplified reference, amplified test with unamplified reference and both amplified tumour and reference DNA. aCGH was performed with an in-house 16 K BAC platform (a resolution of approximately 100 Kb). Pearson's correlation tests and hierarchical clustering were performed to compare the profiles obtained. aCGH profiles obtained with amplified test and unamplified reference female genomic DNA showed copy number biases throughout the genome. These biases were more conspicuous with smaller amounts of starting material and mapped to regions of known copy number polymorphisms. When similar concentrations of test and reference DNA were amplified, the biases were significantly reduced, rendering accurate profiles. For the tumours, representative profiles were obtained when both test and reference DNA were amplified. Phi29 amplification induces copy number biases and unamplified material remains the gold standard for copy number analysis. For accurate results using Phi29 amplification, samples subjected to aCGH analysis should be combined with reference DNA amplified with the same method, using similar amounts of starting template.
Arriola
Edurne
E
The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, UK.
Lambros
Maryou B K
MB
Jones
Chris
C
Dexter
Tim
T
Mackay
Alan
A
Tan
David S P
DS
Tamber
Narinder
N
Fenwick
Kerry
K
Ashworth
Alan
A
Dowsett
Mitch
M
Reis-Filho
Jorge S
JS
D001943:Breast Neoplasms / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D005260:Female; D023281:Genomics / Q000379:methods*; D006801:Humans; D008297:Male; D042282:Microdissection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D014764:Viral Proteins
D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29