Use Of Whole Genome Amplification Research Articles

Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing.

Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100-1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2-3 weeks would be a more typical turnaround time.

Erratum to: “The use of whole genome amplification for genomic evaluation of bovine embryos”

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The use of whole genome amplification for genomic evaluation of bovine embryos

The integration of high technologies into livestock production has been actively occurring in the last decade in the countries with a developed animal breeding. First of all, we are talking about reproductive technologies (IVF) and genomic technologies (general genomic evaluation of animal and genomic evaluation of breeding value). Combining reproductive and genomic technologies is a promising approach that allows receiving highquality breeding cattle in the shortest possible time. The basis of the proposed technology for accelerated reproduction of high-value breeding cattle is to obtain information about the genome of the embryo for genomic evaluation. The amount of genetic material that can be obtained for research is extremely limited, as it is necessary to preserve the viability of the embryo. The stage of the whole genome amplification was introduced to obtain a high quality of genetic material in a sufficient quantity. The main purpose of this work is to assess the possibility of using embryo biopsy specimens (bsp) for embryo genotyping using microarray chips and predicting the carrier status of lethal haplotypes at the embryo stage. We obtained 100 cattle embryos, of which 78 biopsy specimens were taken to analysis. For the biopsies obtained we performed the whole genome amplification. The quality and quantity of DNA for all the 78 samples after the whole genome amplification were satisfactory for further genotyping. The quality of the performed genotyping was satisfactory and allowed the assessment of lethal haplotype carriers (determining the sex of the animal and identification of the carrier status for sevenHolsteinlethal haplotypes). We tested 78 embryos. From the genotyping analysis, there was detected one carrier status for three lethal haplotypes, HH0 (Brachyspina), HH5, and HCD. The carrier status of HH0 and HH5 was confirmed by testing the casual mutation using PCR analysis. The carrier status for HCD has not been confirmed by casual mutation analysis. The situation in which an animal is an HCD carrier, but not the carrier of a casual mutation, can be explained. The putative ancestor of the haplotype is the bull HOCAN000000334489 WILLOWHOLME MARK ANTHONY (year of birth is 1975), but a casual mutation associated with this disease has arisen only in his descendant HOCAN000005457798 MAUGHLIN STORM (year of birth is 1991). The results obtained confirm the importance of testing the casual mutation in the animals that are carriers of lethal haplotypes according to the genotyping data.

Succession and persistence of microbial communities and antimicrobial resistance genes associated with International Space Station environmental surfaces

BackgroundThe International Space Station (ISS) is an ideal test bed for studying the effects of microbial persistence and succession on a closed system during long space flight. Culture-based analyses, targeted gene-based amplicon sequencing (bacteriome, mycobiome, and resistome), and shotgun metagenomics approaches have previously been performed on ISS environmental sample sets using whole genome amplification (WGA). However, this is the first study reporting on the metagenomes sampled from ISS environmental surfaces without the use of WGA. Metagenome sequences generated from eight defined ISS environmental locations in three consecutive flights were analyzed to assess the succession and persistence of microbial communities, their antimicrobial resistance (AMR) profiles, and virulence properties. Metagenomic sequences were produced from the samples treated with propidium monoazide (PMA) to measure intact microorganisms.ResultsThe intact microbial communities detected in Flight 1 and Flight 2 samples were significantly more similar to each other than to Flight 3 samples. Among 318 microbial species detected, 46 species constituting 18 genera were common in all flight samples. Risk group or biosafety level 2 microorganisms that persisted among all three flights were Acinetobacter baumannii, Haemophilus influenzae, Klebsiella pneumoniae, Salmonella enterica, Shigella sonnei, Staphylococcus aureus, Yersinia frederiksenii, and Aspergillus lentulus. Even though Rhodotorula and Pantoea dominated the ISS microbiome, Pantoea exhibited succession and persistence. K. pneumoniae persisted in one location (US Node 1) of all three flights and might have spread to six out of the eight locations sampled on Flight 3. The AMR signatures associated with β-lactam, cationic antimicrobial peptide, and vancomycin were detected. Prominent virulence factors were cobalt-zinc-cadmium resistance and multidrug-resistance efflux pumps.ConclusionsThere was an increase in AMR and virulence gene factors detected over the period sampled, and metagenome sequences of human pathogens persisted over time. Comparative analysis of the microbial compositions of ISS with Earth analogs revealed that the ISS environmental surfaces were different in microbial composition. Metagenomics coupled with PMA treatment would help future space missions to estimate problematic risk group microbial pathogens. Cataloging AMR/virulence characteristics, succession, accumulation, and persistence of microorganisms would facilitate the development of suitable countermeasures to reduce their presence in the closed built environment.

Genotyping DNA isolated from UV irradiated human bloodstains using whole genome amplification.

Analysis of DNA polymorphisms are the primary technique used for personal identification in forensic cases. However, DNA samples collected as evidence from crime scenes are usually degraded by environmental, physical, and chemical factors, which may interfere with the PCR analysis and, consequently, personal identification. Whole genome amplification (WGA) is a useful method to amplify genomic DNA from samples containing low quantity and poor quality of DNA, and it approach that shows promise to overcome the limited small fragments based upon random fragmentation by universal priming sites. In this study, we describe the use of WGA to genotype 15 short tandem repeat (STR) loci from dried blood samples irradiated with different types of ultraviolet (UV) light (UVA, UVB, and UVC). The result showed that UVC was the most damaging to DNA, followed by UVB and UVA. Samples exposed to UVA could be genotyped for all STR loci with or without WGA. For UVB and UVC irradiated blood samples, a greater number of STR loci could be genotyped after WGA. Although it hard to amplified a few higher molecular weight alleles, overall, the WGA method was useful in genotyping template DNA of poor quality but low quantity.

Implementing PGD/PGD-A in IVF clinics: considerations for the best laboratory approach and management.

For an IVF clinic that wishes to implement preimplantation genetic diagnosis for monogenic diseases (PGD) and for aneuploidy testing (PGD-A), a global improvement is required through all the steps of an IVF treatment and patient care. At present, CCS (Comprehensive Chromosome Screening)-based trophectoderm (TE) biopsy has been demonstrated as a safe, accurate and reproducible approach to conduct PGD-A and possibly also PGD from the same biopsy. Key challenges in PGD/PGD-A implementation cover genetic and reproductive counselling, selection of the most efficient approach for blastocyst biopsy as well as of the best performing molecular technique to conduct CCS and monogenic disease analysis. Three different approaches for TE biopsy can be compared. However, among them, the application of TE biopsy approaches, entailing the zona opening when the expanded blastocyst stage is reached, represent the only biopsy methods suited with a totally undisturbed embryo culture strategy (time lapse-based incubation in a single media). Moreover, contemporary CCS technologies show a different spectrum of capabilities and limits that potentially impact the clinical outcomes, the management and the applicability of the PGD-A itself. In general, CCS approaches that avoid the use of whole genome amplification (WGA) can provide higher reliability of results with lower costs and turnaround time of analysis. The future perspectives are focused on the scrupulous and rigorous clinical validations of novel CCS methods based on targeted approaches that avoid the use of WGA, such as targeted next-generation sequencing technology, to further improve the throughput of analysis and the overall cost-effectiveness of PGD/PGD-A.

Next-generation sequencing analysis of lung and colon carcinomas reveals a variety of genetic alterations.

The development of targeted therapies in cancer has accelerated the development of molecular diagnosis. This new cancer discipline is booming, with an increasing number of gene alterations to analyze in a growing number of patients. To deal with this fast-developing activity, current analysis techniques (Sanger sequencing, allelic discrimination and high resolution melting) take more and more time. In recent years, next generation sequencing (NGS) technologies have appeared and given new perspectives in oncology. In this study, we analyzed FFPE lung and colon carcinomas using the Truseq Cancer Panel, which analyzes the mutation hotspots of 48 genes. We also tested the use of whole-genome amplification before NGS analysis. NGS results were compared with the data obtained from routine diagnosis. All of the alterations routinely observed were identified by NGS. Moreover, NGS revealed mutations in the KRAS and EGFR genes in patients diagnosed as wild-type by routine techniques. NGS also identified concomitant mutations in EGFR and KRAS or BRAF mutations, and a 15-nt deletion in exon 19 of EGFR in colon carcinomas. The study of the other genes sequenced in the Panel revealed 14 genes altered by 27 different mutations and three SNP with a possible role in cancer susceptibility or in the response to treatment. In conclusion, this study showed that NGS analysis could be used for the analysis of gDNA extracted from FFPE tissues. However, given the high sensitivity of this technology, high-throughput clinical trials are needed to confirm its reliability for the molecular diagnosis of cancer.

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Approaches to genotyping individual miracidia of Schistosoma japonicum

Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have reemerged, and to characterize infrapopulations in individual hosts. The life stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4years), considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with the discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.

Whole genome and transcriptome amplification: practicable tools for sustainable tissue biobanking?

The use of whole genome amplification (WGA) and whole transcriptome amplification (WTA) techniques enables the enrichment of DNA and RNA from very small amounts of tissue. Here, we tested the suitability of WGA and WTA for tumor tissue biobanking. DNA and RNA from 13 standardized and seven non-standardized frozen and 12 formalin-fixed, paraffin-embedded (FFPE) clear cell renal cell carcinoma specimens (>9 years old) served to test the robustness of the WGA and WTA products by reidentifying von Hippel-Lindau (VHL) gene mutations known to exist in these samples. The enrichment of DNA and RNA from frozen tissue was up to 1,291-fold and 423-fold, respectively. The sizes and yields (10- to 73-fold) of the amplified DNA obtained from the 12 FFPE samples were generally lower. The quality of the RNA from the FFPE samples was too low to reliably perform WTA. Our results demonstrate that frozen tumor tissue is very suitable for WGA and WTA. All 20 VHL mutations were verified with WGA. Notably, we were able to show that 18 of the 20 (90 %) VHL mutations are also transcribed. In FFPE tumor tissue, 8 of 12 cases (67 %) showed the expected mutations after the first WGA. Accurate WTA with FFPE material is sophisticated and strongly depends on the modification and degradation status of the fixed tissue. We conclude that for sustainable tissue biobanking, the use of WGA and WTA is a unique opportunity to provide researchers with sufficient amounts of nucleic acids, preferably from limited frozen tissue material.

Accurate variant detection across non-amplified and whole genome amplified DNA using targeted next generation sequencing

BackgroundMany hypothesis-driven genetic studies require the ability to comprehensively and efficiently target specific regions of the genome to detect sequence variations. Often, sample availability is limited requiring the use of whole genome amplification (WGA). We evaluated a high-throughput microdroplet-based PCR approach in combination with next generation sequencing (NGS) to target 384 discrete exons from 373 genes involved in cancer. In our evaluation, we compared the performance of six non-amplified gDNA samples from two HapMap family trios. Three of these samples were also preamplified by WGA and evaluated. We tested sample pooling or multiplexing strategies at different stages of the tested targeted NGS (T-NGS) workflow.ResultsThe results demonstrated comparable sequence performance between non-amplified and preamplified samples and between different indexing strategies [sequence specificity of 66.0% ± 3.4%, uniformity (coverage at 0.2× of the mean) of 85.6% ± 0.6%]. The average genotype concordance maintained across all the samples was 99.5% ± 0.4%, regardless of sample type or pooling strategy. We did not detect any errors in the Mendelian patterns of inheritance of genotypes between the parents and offspring within each trio. We also demonstrated the ability to detect minor allele frequencies within the pooled samples that conform to predicted models.ConclusionOur described PCR-based sample multiplex approach and the ability to use WGA material for NGS may enable researchers to perform deep resequencing studies and explore variants at very low frequencies and cost.

Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples

Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification. In this study we investigate region dropout using a φ29 polymerase based WGA approach. DNA from 123 lung cancers specimens and corresponding normal tissue were used and evaluated by Sanger sequencing of the p53 exons 5–8. To enable comparative analysis of this scarce material, WGA samples were compared with unamplified material using a pooling strategy of the 123 samples. In addition, a more detailed analysis of exon 7 amplicons were performed followed by extensive cloning and Sanger sequencing. Interestingly, by comparing data from the pooled samples to the individually sequenced exon 7, we demonstrate that mutations are more easily recovered from WGA pools and this was also supported by simulations of different sequencing coverage. Overall this data indicate a limited random loss of genomic regions supporting the use of whole genome amplification for genomic analysis.

Use of Formalin-fixed Paraffin-embedded Tumor Tissue as a DNA Source in Molecular Epidemiological Studies of Pediatric CNS Tumors

Formalin-fixed paraffin-embedded tissue (FFPET) samples are a potential source of DNA for molecular epidemiological studies. However, the use of FFPET samples can be restricted by the yield and quality of DNA isolated. The aim of this study was to examine whether FFPET biopsies from pediatric central nervous system tumors were a feasible alternative to archival frozen tissue when characterizing common gene polymorphisms. DNA was isolated from 50 frozen pediatric central nervous system tumor biopsies and matched FFPET samples. Real-time polymerase chain reaction (PCR) was used to quantify DNA and characterize GSTT1, GSTM1, GSTP1, and MTHFR gene polymorphisms. The use of whole-genome amplification (WGA) to increase DNA yields was also investigated. The results showed that DNA isolated from FFPET samples was more fragmented and provided smaller yields than DNA isolated from frozen samples. Attempts to increase the DNA yield from FFPET using WGA were unsuccessful. DNA from FFPET samples was successfully genotyped for the GSTP1 Ile105Val and MTHFR 677 C>T polymorphisms in 98% of samples and was 100% concordant with the results from frozen tissue. However, DNA from FFPET performed poorly in real-time PCR assays for GSTM1 and GSTT1 deletion polymorphisms. Our investigations show that DNA extracted from FFPET is substantially fragmented and not readily amplified using WGA. In addition, careful validation of PCR assays should be carried out due to the variable amplification of fragmented FFPET DNA.

Abstract B31: Establishing the use of genomic profiling on rare prostate cancer disseminated tumor cells

Abstract Objective: Dissemination of prostate cancer tumor cells remains a critical challenge in understanding disease progression and effective long-term treatment of patients. Disseminated tumor cells (DTCs) from bone marrow are resistant to chemotherapy and radiation, and represent a potential long-lived source for lethal metastases. Quantification of disseminated and circulating tumor cells provides promise as a novel diagnostic in measuring tumor burden and assessing the risk of relapse. However, very limited knowledge exists about the biology of these rare cells, including the genomic aberrations associated with clinical outcome. Methods: DTCs are immunomagnetically enriched from bone marrow aspirates, then individually captured under the microscope based on immunofluorescent staining of EpCaM cell surface molecules. We have established use of whole genome amplification and high-density SNP-CGH arrays to genomically profile rare DTC populations (n=2-25 cells). Using Fused Lasso and Nexus Copy Number software, we quantify and characterize copy number alterations and loss-of-heterozygosity regions among samples isolated from 58 patients taken at the time of radical prostectomy and from 13 patients with advanced, metastatic or lethal prostate cancer. We classify DTC aberrations by tumor stage and compare to those aberrations identified in primary and metastatic tumors. Regions of interest are validated using a highly sensitive quantitative real-time PCR assay. In addition, we have completed extensive optimization of analysis parameters to handle the increased probe variation associated with low input DNA quantity and whole genome amplification. Results: DTCs isolated at the time of radical prostatectomy from early stage prostate cancer patients are generally heterogeneous and are marked with limited focal aberrations. Focal deletions of less than 100 kb are frequent events in these samples, as are focal gains of less than 500 kb, albeit less frequently. While many of these focal copy number changes are identified in regions of common copy number variation (CNV) within the population, a considerable number are rare and unique to DTCs. Cancer-specific alterations are observed in members of the cadherin family and transcription factors SIX3, SOX4, SOX17 and GATA6. Because most abberations are patient-specific, bioinformatic analysis is used to illuminate commonly altered biological pathways. In contrast, DTCs from patients with advanced stages of prostate cancer typically show frequent and large (>1 Mb) clonal amplifications and deletions, suggesting a high degree of genomic instability that is common among these patients. Aberrant regions that are uniquely identified in DTCs from patients with advanced, metastatic and castrationresistant prostate cancers include gain of 1q43–q44 and 1q31.3–q41. In addition, several regions were found to be recurrent among at least 25% of metastatic tumors and DTCs from advanced stages, including in order of prevalence: +8q, −8p, +9q, −13, −22, −16q, −7q, −6q13–q22.31, +3q24–q26.1, +11q13.2–13.4, −17p13.3–p12 and +2p16.3–p16.2. Conclusions: We have established methods and quality control measures for genome-wide profiling of rare cell populations. These methods will be functionally useful to gain a new understanding of the genomic aberrations that drive DTC dissemination, dormancy, and metastatic reactivation in prostate cancer. Citation Format: Jamie R. Schoenborn, Jing Xia, Sandy Larson, Lisha Brown, Colm Morrissey, Paul Lange, Peter S. Nelson, Robert L. Vessella, Min Fang. Establishing the use of genomic profiling on rare prostate cancer disseminated tumor cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B31.

Evaluation of circular DNA substrates for whole genome amplification prior to forensic analysis

Forensic biological evidence often contains low quantities of DNA or substantially degraded DNA which makes samples refractory to genotype analysis. One approach that shows promise to overcome the limited quantity of DNA is whole genome amplification (WGA). One WGA technique, termed rolling circle amplification (RCA), involves the amplification of circular DNA fragments and this study evaluates a single-stranded (ss) DNA ligase enzyme for generating circular DNA templates for RCA WGA. Fast, efficient ligation of several sizes of ssDNA templates was achieved. The enzyme also ligated double-stranded (ds) DNA templates, a novel activity not previously reported. Adapter sequences containing optimal terminal nucleotide ends for increased ligation efficiency were designed and ligation of adapters to template DNA was optimized. Increased amplification of DNA templates was observed following WGA; however, no amplification advantage for ssDNA ligase treatment of templates was evident compared to linear templates. A multi-step process to utilize ssDNA ligase prior to WGA was developed and short tandem repeat (STR) analysis of simulated low template (LT) and fragmented DNA was evaluated. The process resulted in the loss of template DNA and failed STR analysis whereas input of linear genomic DNA template directly into WGA prior to STR analysis improved STR genotyping results compared to non-WGA treated samples. Inclusion of an extreme thermostable single-stranded DNA binding protein (SSB) during WGA also increased DNA yields. While STR artifacts such as peak imbalance, drop-in, and dropout persisted, WGA shows potential for successful genetic profiling of LT and fragmented DNA samples. Further research and development is warranted prior to use of WGA in forensic casework.

Technical note: High fidelity of whole-genome amplified sheep (Ovis aries) deoxyribonucleic acid using a high-density single nucleotide polymorphism array-based genotyping platform1

Advances in high-throughput genotyping technologies have afforded researchers the opportunity to study ever-increasing numbers of SNP in animal genomes. However, many studies encounter difficulties in obtaining sufficient quantities of high-quality DNA for such analyses, particularly when the source biological material is limited or degraded. The recent development of in vitro whole-genome amplification approaches has permitted researchers to circumvent these challenges by increasing the amount of usable DNA in normally small-quantity samples. Here, we assess the performance of whole-genome amplification products generated from ovine genomic DNA using a high-throughput SNP genotyping platform, the newly developed Illumina ovineSNP50 BeadChip. Our results demonstrate a high genotype call rate for conventional genomic DNA and whole-genome amplified genomic DNA. The data also reveal an exceptionally high concordance rate ( > or = 99%) between the genotypes generated from whole-genome amplified products and their conventional genomic DNA counterparts. This study supports the use of whole-genome amplification as a viable solution for the analysis of high-density SNP genotypic data using compromised or limited starting material.

Multiply Primed Rolling-Circle Amplification Method for the Amplification of Circular DNA Viruses

The use of whole genome amplification and analysis of viruses is of increasing importance, as data generated using these methods are currently used for clinical diagnostics, epidemiological studies, phylogenetic analyses, and studies of genome organization and evolution. The best known amplification method for DNA is the polymerase chain reaction (PCR). This technique, however, has drawbacks: PCR produces relatively small amplicons and also requires prior knowledge of sequence data for the construction of the required consensus or degenerate primers. For circular DNA templates, it is possible to overcome these drawbacks by using the multiply primed rolling-circle amplification (RCA) technique, which mimics the rolling-circle mechanism that occurs in nature for replication of circular DNA molecules, e.g., plasmids. The RCA protocol described here is optimized for the amplification of circular DNA virus genomes.

Human platelet antigen typing of neonatal alloimmune thrombocytopenia patients using whole genome amplified DNA and a 5′‐nuclease assay

A serious constraint in the investigation of the human platelet antigen (HPA) status of potential neonatal alloimmune thrombocytopenia (NAIT) cases is the limited amount of DNA available from the neonates. Whole genome amplification (WGA) of these DNA samples could overcome this problem, but requires validation to ensure that it is sufficiently sensitive and accurate before its application in a clinical diagnostic setting. This study has validated the use of WGA DNA for HPA-1, -2, -3, -4, -5, and -15 genotyping with a panel of six controls and 13 previously HPA-typed samples from neonates together with parental DNA, using a 5'-nuclease (TaqMan) assay. WGA was performed using titrated amounts of genomic and WGA DNA template. HPA typing was performed on genomic and amplified DNA using a 5'-nuclease assay or polymerase chain reaction with sequence-specific primers (PCR-SSP). WGA DNA yields were in the suggested range of 400x to 800x, as assessed by spectrophotometry and gel analysis, and did not require further purification. HPA genotyping showed 100 percent concordance when using down to 5 ng of genomic or WGA template. This study demonstrates that WGA can be used for HPA typing using PCR-SSP or plate-based 5'-nuclease assays. The use of WGA for HPA typing in clinical samples from NAIT patients was validated with 100 percent concordance, and it is suggested that this technology can be used for other analyses where DNA amounts are limited.

Buccal Swabs and Treated Cards: Methodological Considerations for Molecular Epidemiologic Studies Examining Pediatric Populations

Self-collection of buccal cells provides a noninvasive method for obtaining biologic samples for genetic analyses in pediatric studies. Nevertheless, low yields, microbial contamination, and degradation of buccal samples present challenges for epidemiologic studies incorporating genetic investigations. The aims of this study were to compare the quality and yield of DNA extracted from buccal specimens with BuccalAmp swabs (Epicenter BioTechnologies, Madison, Wisconsin) or FTA cards (Whatman, Inc., Clifton, New Jersey) and to investigate the use of whole-genome amplification (WGA) for increasing DNA yields for single nucleotide polymorphism analyses. Buccal specimens were collected from 55 children with acute lymphoblastic leukemia and 52 control children without acute lymphoblastic leukemia in New South Wales, Australia, in 2003-2004. Real-time polymerase chain reaction was used to evaluate polymorphisms in the genes encoding the cytochrome p450 enzyme CYP3A4 (CYP3A4 A392G, also known as CYP3A4*1B) and the steroid xenobiotic receptor (SXR C25385T). Results showed that DNA could be isolated from buccal specimens collected by use of both methods and that yields could be substantially improved with WGA without introducing genotyping error. However, DNA quality was poorer in samples collected by BuccalAmp swabs, and the presence of polymerase chain reaction inhibitors in these samples reduced the sensitivity of quantitative real-time PCR analysis. These findings show that different methods for collecting buccal samples impact on the downstream success of genetic investigations and influence DNA quality after WGA.