Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples

Wang, Gang; Brennan, Cameron; Rook, Martha; Wolfe, Jia Liu; Leo, Christopher; Chin, Lynda; Pan, Hongjie; Liu, Weihua; Price, Brendan D.; Makrigiorgos, G. Mike

doi:10.1093/nar/gnh070

Cited by 56 publications

(52 citation statements)

References 29 publications

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“…14 In contrast to a previous observation 13 of almost complete failure of Phi29 to amplify DNA from FFPE sample, we have demonstrated similar amplification efficiencies in DNA from pairs of archival fresh-frozen and FFPE tumor and a similar average product length of DNA amplified from corresponding tissue specimens. …”

Section: Discussioncontrasting

confidence: 57%

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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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Section: Discussioncontrasting

confidence: 57%

“…8 -10 The utility of Phi29-based whole genome amplification has been shown for plasmid and bacteriophage DNA 11 as well as human DNA from whole blood, buccal swabs, buffy coats, and cultured cells. 6,[12][13][14] However, this technique results in biased gene representation.…”

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confidence: 99%

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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“…8 -14 The success of WGA itself also depends on sample quality. 9,16 Certain types of WGA such as multiple displacement amplification perform well with fresh or snap-frozen (intact) DNA, [17][18][19] but their yield 9 and accuracy 20,21 diminish on DNA fragmentation. WGA methods that can tolerate various degrees of sample degradation have been recently developed.…”

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confidence: 99%

DNA Degradation Test Predicts Success in Whole-Genome Amplification from Diverse Clinical Samples

Wang

Briggs

et al. 2007

The Journal of Molecular Diagnostics

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The need to apply modern technologies to analyze DNA from diverse clinical samples often stumbles on suboptimal sample quality. We developed a simple approach to assess DNA fragmentation in minute clinical samples of widely different origin and the likelihood of success of degradation-tolerant whole genome amplification (restriction and circularization-aided rolling circle amplification, RCA-RCA) and subsequent polymerase chain reaction (PCR). A multiplex PCR amplification of four glyceraldehyde-3-phosphate dehydrogenase amplicons of varying sizes was performed using genomic DNA from clinical samples, followed by size discrimination on agarose gel or fluorescent denaturing high-performance liquid chromatography (dHPLC). RCA-RCA followed by realtime PCR was also performed, for correlation. Even minimal quantities of longer PCR fragments (ϳ300 to 400 bp), visible via high-sensitivity fluorescent dHPLC or agarose gel, were essential for the success of RCA-RCA and subsequent PCR-based assays. dHPLC gave a more accurate correlation between DNA fragmentation and sample quality than agarose gel electrophoresis. Multiplex-PCR-dHPLC predicted correctly the likelihood of assay success in formalin-fixed, paraffin-embedded samples fixed under controlled conditions and of different ages, in laser capture microdissection samples, in tissue print micropeels, and plasma-circulating DNA. Estimates of the percent information retained relative to snap-frozen DNA are derived for real-time PCR analysis. The assay is rapid and convenient and can be used widely to characterize DNA from any clinical sample of unknown quality. (J Mol

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“…For quantification, the data were analyzed using the LightCycler analysis software (Roche Diagnostics, Mannheim, Germany). Relative quantification of target gene expression was evaluated using the comparative C T method (Wang et al, 2004). The ΔC T value was determined by subtracting the target C T of each sample from its respective GAPDH C T value.…”

Section: Real-time Quantitative Pcr Analysis (Qpcr)mentioning

confidence: 99%

HOX gene analysis in the osteogenic differentiation of human mesenchymal stem cells

et al. 2008

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Human bone marrow-derived mesenchymal stem cells (hMSCs) have the capacity to differentiate into osteoblasts during osteogenesis. Several studies attempted to identify osteogenesis-related genes in hMSCs. Although HOX genes are known to play a pivotal role in skeletogenesis, their function in the osteogenesis of hMSCs has not yet been investigated in detail. Our aim was to characterize the expression of 37 HOX genes by multiplex RT-PCR to identify the ones most probably involved in osteogenic differentiation. The results showed that the expression patterns of four HOX genes were altered during this process. In particular, the expression levels of HOXC13 and HOXD13 were dramatically changed. Real-time PCR and Western blot analysis were performed in order to further analyze the expression of HOXC13 and HOXD13. The qRT-PCR results showed that transcription of HOXC13 was up-regulated by up to forty times, whereas that of HOXD13 was down-regulated by approximately five times after osteogenic differentiation. The Western blot results for the HOXC13 and HOXD13 proteins also corresponded well with the real-time PCR result. These findings suggest that HOXC13 and HOXD13 might be involved in the osteogenic differentiation of hMSCs.

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Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples

Cited by 56 publications

References 29 publications

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

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

DNA Degradation Test Predicts Success in Whole-Genome Amplification from Diverse Clinical Samples

HOX gene analysis in the osteogenic differentiation of human mesenchymal stem cells

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