Use Of Whole Genome Amplification Research Articles

PD2-2-7: Two wrongs make a right: the use of whole genome amplification for pair-wise genome-wide copy number analysis of limited patient material

PD2-2-7: Two wrongs make a right: the use of whole genome amplification for pair-wise genome-wide copy number analysis of limited patient material

A test of the efficacy of whole‐genome amplification on DNA obtained from low‐yield samples

AbstractConservation and population genetic studies are sometimes hampered by insufficient quantities of high quality DNA. One potential way to overcome this problem is through the use of whole genome amplification (WGA) kits. We performed rolling circle WGA on DNA obtained from matched hair and tissue samples of North American red squirrels (Tamiasciurus hudsonicus). Following polymerase chain reaction (PCR) at four microsatellite loci, we compared genotyping success for DNA from different source tissues, both pre‐ and post‐WGA. Genotypes obtained with tissue were robust, whether or not DNA had been subjected to WGA. DNA extracted from hair produced results that were largely concordant with matched tissue samples, although amplification success was reduced and some allelic dropout was observed. WGA of hair samples resulted in a low genotyping success rate and an unacceptably high rate of allelic dropout and genotyping error. The problem was not rectified by conducting PCR of WGA hair samples in triplicate. Therefore, we conclude that WGA is only an effective method of enhancing template DNA quantity when the initial sample is from high‐yield material.

The use of whole genome amplification to study chromosomal changes in prostate cancer: insights into genome-wide signature of preneoplasia associated with cancer progression

BackgroundProstate cancer (CaP) is a disease with multifactorial etiology that includes both genetic and environmental components. The knowledge of the genetic basis of CaP has increased over the past years, mainly in the pathways that underlie tumourigenesis, progression and drug resistance. The vast majority of cases of CaP are adenocarcinomas that likely develop through a pre-malignant lesion and high-grade prostatic intraepithelial neoplasia (HPIN). Histologically, CaP is a heterogeneous disease consisting of multiple, discrete foci of invasive carcinoma and HPIN that are commonly interspersed with benign glands and stroma. This admixture with benign tissue can complicate genomic analyses in CaP. Specifically, when DNA is bulk-extracted the genetic information obtained represents an average for all of the cells within the sample.ResultsTo minimize this problem, we obtained DNA from individual foci of HPIN and CaP by laser capture microdissection (LCM). The small quantities of DNA thus obtained were then amplified by means of multiple-displacement amplification (MDA), for use in genomic DNA array comparative genomic hybridisation (gaCGH). Recurrent chromosome copy number abnormalities (CNAs) were observed in both HPIN and CaP. In HPIN, chromosomal imbalances involving chromosome 8 where common, whilst in CaP additional chromosomal changes involving chromosomes 6, 10, 13 and 16 where also frequently observed.ConclusionAn overall increase in chromosomal changes was seen in CaP compared to HPIN, suggesting a universal breakdown in chromosomal stability. The accumulation of CNAs, which occurs during this process is non-random and may indicate chromosomal regions important in tumourigenesis. It is therefore likely that the alterations in copy number are part of a programmed cycle of events that promote tumour development, progression and survival. The combination of LCM, MDA and gaCGH is ideally suited for the identification of CNAs from small cell clusters and may assist in the discovery of potential genomic markers for early diagnosis, or identify the location of tumour suppressor genes (TSG) or oncogenes previously unreported in HPIN and CaP.

Use of Whole Genome Amplification to Rescue DNA from Plasma Samples

While DNA of good quality and sufficient amount can be obtained easily from whole blood, buccal swabs, surgical specimens, or cell lines, these DNA-rich sources are not always available. This is particularly the case in studies for which biological specimens were collected when genotyping assays were not widely available. In those studies, serum or plasma is often the only source of DNA. Newly developed whole genome amplification (WGA) methods, based on phi29 polymerase, may play a significant role in recovering DNA in such instances. We tested a total of 528 plasma samples kept in storage at -40 degrees C for approximately 10 years for 8 single nucleotide polymorphisms (SNPs) using the 5' exonuclease (TaqMan) assay. These specimens yielded undetectable levels of DNA following extraction with an affinity column but produced an average 52.7 microg (standard deviation of 31.2 microg) of DNA when column-extracted DNA was used as a template for WGA. This increased the genotyping success rate from 54% to 93%. There were only 3 disagreements out of 364 paired genotyping results for pre- and post-WGA DNAs, indicating an error rate of 0.82%. These results are encouraging for expanding the use of poor DNA resources in genotyping studies.

Multiple displacement amplification of DNA from human colon and rectum biopsies: Bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis

Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis; the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens.

Use of whole genome amplification and comparative genomic hybridisation to detect chromosomal copy number alterations in cell line material and tumour tissue

We have established that whole genome amplification (WGA), in conjunction with genomic DNA array comparative genomic hybridisation (gaCGH) allows for the identification of genome-wide copy number abnormalities (CNAs) in DNA extracted from both cell line and patient material. To determine the fidelity and reproducibility of WGA to detect copy number imbalances using gaCGH, well characterized cell line genomic DNA was analysed. The gaCGH data obtained from non-amplified DNA and amplified DNA for the neuroblastoma cell line NUB7 and a paediatric medulloblastoma patient was almost identical. In addition, laser capture microdissection (LCM) of prostate tumour cells and subsequent WGA allowed for the detection of a number of CNAs that may not have been identified if DNA had been extracted in bulk from heterogeneous tissue. The results presented here demonstrate the use of WGA for generating sufficient DNA for gaCGH analysis without the introduction of significant sequence representation bias. The combination of amplification and gaCGH using DNA extracted from archival patient material has the potential for permitting the studying of DNA from small cancerous or pre-cancerous foci, which may help to identify potential genomic markers for early diagnosis.

The use of whole genome amplification in the study of human disease

The use of whole genome amplification in the study of human disease

Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA.

Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.