Improving degenerate oligonucleotide primed PCR-comparative genomic hybridization for analysis of DNA copy number changes in tumors

Huang, Qiang; Schantz, Stimson P.; Rao, Pulivarthi H.; Mo, Juan; McCormick, Steven A.; Chaganti, R. S. K.

doi:10.1002/1098-2264(200008)28:4<395::aid-gcc5>3.0.co;2-j

Cited by 53 publications

(30 citation statements)

References 15 publications

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“…We interpret these observations as indicative of a deterministic mechanism for amplification bias, which may be based on priming frequencies that are different, albeit reproducibly so, across the genome. Analogous observations have been reported in conventional CGH experiments, in which DOP-PCR-amplified DNA was used for both test and reference samples, resulting in reduced distortion of ratios (Voullaire et al 1999;Huang et al 2000). Thus, if amplification bias is not excessive, distortions in representation can be compensated for by using a reference genomic DNA amplified under identical conditions.…”

Section: Evaluation Of Amplification Bias Using Yeast Cdna Microarrayssupporting

confidence: 68%

Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array–CGH

Lage¹,

Leamon²,

Pejović³

et al. 2003

Genome Res.

233

210

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Structural genetic alterations in cancer often involve gene loss or gene amplification. With the advent of microarray approaches for the analysis of the genome, as exemplified by array–CGH (Comparative GenomicHybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density. However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array–CGH analysis. We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples. We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias. The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours. Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines. Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples. Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material.

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Section: Evaluation Of Amplification Bias Using Yeast Cdna Microarrayssupporting

confidence: 68%

Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array–CGH

Lage¹,

Leamon²,

Pejović³

et al. 2003

Genome Res.

233

210

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“…18 We have shown that the reproducible variation in regional amplification efficiency could be effectively normalized when test and reference genomes were amplified under identical conditions and compared by array-CGH. Analogous observations have been made in classical CGH experiments 30,31 and have been successfully extrapolated to the random-primed amplification of yeast and human genomic DNA using the strand-displacing Bacillus stearothermophilus polymerase.…”

Section: Discussionsupporting

confidence: 53%

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

Bredel

Juric

et al. 2005

The Journal of Molecular Diagnostics

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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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“…For either amplification method, DNA amounts from 300 ng to 20 pg resulted in reliable experiments, with identical results obtained from undiluted and diluted DNA samples. These data are in good agreement with previous studies in which the minimal DNA amounts for successful DOP-PCR-amplification and CGH were described to range between 12.5-50 pg (5,21,23). At lower amounts of target DNA, equivalent to one or a few diploid cells, we found LA-PCR to be clearly superior as compared to DOP-PCR.…”

Section: Discussionsupporting

confidence: 92%

“…Both, DOP-PCR and LA-PCR (also termed SCOMP) have been suggested to be suitable for analysis of very minor amounts of DNA, equivalent to one or a few cells (9,16,17,(21)(22)(23). We compared the sensitivity and efficiency of both amplification procedures in a series of DNA dilution experiments.…”

Section: Discussionmentioning

confidence: 99%

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

Pirker¹,

Raidl²,

Steiner³

et al. 2004

Cytometry Pt A

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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.

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Improving degenerate oligonucleotide primed PCR-comparative genomic hybridization for analysis of DNA copy number changes in tumors

Cited by 53 publications

References 15 publications

Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array–CGH

Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array–CGH

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

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

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