Journal
JOURNAL OF MOLECULAR DIAGNOSTICS
Volume 7, Issue 2, Pages 171-182Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/S1525-1578(10)60543-0
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Funding
- NCI NIH HHS [R01 CA097139, R01 CA092474, CA 97139, CA 92474] Funding Source: Medline
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Sufficient quantity of genomic DNA can he a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Pbi29 amplified DNA from matched pairs of fresh-frozen and formalinfixed/parafrin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fine-needle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens.
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