Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA

Abstract

Sufficient quantity of genomic DNA can be 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/paraffinembedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalinfixed/paraffin-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 fineneedle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens. The availability of a simple and reliable method for the amplification of entire genomes from limited sources of DNA would lend itself to high-throughput genetic analyses in virtually all areas of translational research. High-throughput genomic profiling, for example cDNA microarray-based comparative genomic hybridization (array-CGH), 1 requires g quantities of genomic DNA. A particular challenge for translation of array-CGH methodology to clinical application is to link it to a robust up-front technology that allows reliable and unbiased amplification of limited sources of DNA, for example from fine-needle biopsies. Much effort has been invested in developing methods for whole genome amplification, for example polymerase chain reaction-based methods. [2][3][4] In particular, the pitfalls of substantial variation in the extent of amplification occurring between different markers, incomplete representation, and inadequate average DNA size have limited the use of most existing amplification methods, making them particularly unsuitable for genomic applications. 2-4Bacteriophage Phi29 DNA polymerase random-primed DNA amplification 5-7 is based on an isothermal strand displacement amplification reaction in which random hexamer primers anneal to the genomic template at multiple sites and Phi29 initiates replication at these sites on the denatured linear DNA. As synthesis proceeds, strand displacement of complementary DNA generates new single-stranded DNA available to be primed by additional primers. The subsequent strand displacement replication of this DNA leads to the formation of double-stranded DNA. The presence of an associated proofreading activity of Phi29 ensures a high sequence accuracy of the amplified DNA, indicating its suitability f...

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