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

Analysis of genomic DNA derived from cells and fresh or fixed tissues often requires whole genome amplification prior to microarray screening. Technical hurdles to this process are the introduction of amplification bias and/or the inhibitory effects of formalin fixation on DNA amplification. Here we demonstrate a balanced-PCR procedure that allows unbiased amplification of genomic DNA from fresh or modestly degraded paraffin-embedded DNA samples. Following digestion and ligation of a target and a control genome with distinct linkers, the two are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. We demonstrate genome-wide retention of allelic differences following balanced-PCR amplification of DNA from breast cancer and normal human cells and genomic profiling by array-CGH (cDNA arrays, 100 kb resolution) and by real-time PCR (single gene resolution). Comparison of balanced-PCR with multiple displacement amplification (MDA) demonstrates equivalent performance between the two when intact genomic DNA is used. When DNA from paraffin-embedded samples is used, balanced PCR overcomes problems associated with modest DNA degradation and produces unbiased amplification whereas MDA does not. Balanced-PCR allows amplification and recovery of modestly degraded genomic DNA for subsequent retrospective analysis of human tumors with known outcomes.     

Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures

The availability of a near-complete (96%) collection of gene-deletion mutants in Saccharomyces cerevisiae greatly facilitates the systematic analyses of gene function in yeast. The unique 20 bp DNA 'barcodes' or 'tags' in each deletion strain enable the individual fitness of thousands of deletion mutants to be resolved from a single pooled culture. Here, we present protocols for the study of pooled cultures of tagged yeast deletion mutants with a tag microarray. This process involves five main steps: pooled growth, isolation of genomic DNA, PCR amplification of the barcodes, array hybridization and data analysis. Pooled deletion screening can be used to study gene function, uncover a compound's mode of action and identify drug targets. In addition to these applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, short interfering RNA vectors and molecular inversion probes.     

Hybridization of genomic DNA to microarrays: a challenge for the analysis of environmental samples

The use of DNA microarrays for detection and identification of bacteria and genes of interest from various environments (e.g. soil, sediment, water column...) is a major challenge for microbiologists working on functional diversity. So far, most of the genomic methods that have been described rely on the use of taxonomic markers (such as 16S rRNA) that can be easily amplified by PCR prior to hybridization on microarrays. However, taxonomical markers are not always informative on the functions present in these bacteria. Moreover, genes for which sequence database is limited or that lack any conserved regions will be difficult to amplify and thus to detect in unknown samples. Furthermore, PCR amplification often introduces biases that lead to inaccurate analysis of microbial communities. An alternative solution to overcome these strong limitations is to use genomic DNA (gDNA) as target for hybridisation, without prior PCR amplification. Though hybridization of gDNA is already used for comparative genome hybridization or sequencing by hybridization, yet to the high cost of tiling strategies and important data filtering, its adaptation for use in environmental research poses great challenges in terms of specificity, sensitivity and reproducibility of hybridization. Considering the very faint number of publications that have described hybridization of gDNA to microarrays for environmental applications, we confront in this review the different approaches that have been developed so far, and propose alternative strategies that may contribute to improve the development of microarrays for studying the microbial genetic structure and composition of samples of high environmental and ecological value.     

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).     

Array-MAPH: a methodology for the detection of locus copy-number changes in complex genomes

High-throughput genome-wide screening methods to detect subtle genomic imbalances are extremely important for diagnostic genetics and genomics. Here, we provide a detailed protocol for a microarray-based technique, applying the principle of multiplex amplifiable probe hybridization (MAPH). Methodology and software have been developed for designing unique PCR-amplifiable sequences (400-600 bp) covering any genomic region of interest. These sequences are amplified, cloned and spotted onto arrays (targets). A mixture of the same sequences (probes) is hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered and quantitatively amplified by PCR, labeled and hybridized to the array. The procedure can be completed in 4-5 working days, excluding microarray preparation. Unlike array-comparative genomic hybridization (array-CGH), test DNA of specifically reduced complexity is hybridized to an array of identical small amplifiable target sequences, resulting in increased hybridization specificity and higher potential for increasing resolution. Array-MAPH can be used for detection of small-scale copy-number changes in complex genomes, leading to genotype-phenotype correlations and the discovery of new genes.     

Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes.

Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes.     

A unique and universal molecular barcode array

Molecular barcode arrays allow the analysis of thousands of biological samples in parallel through the use of unique 20-base-pair (bp) DNA tags. Here we present a new barcode array, which is unique among microarrays in that it includes at least five replicates of every tag feature. The use of smaller dispersed replicate features dramatically improves performance versus a single larger feature and allows the correction of previously undetectable hybridization defects.     

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.     

Improvements of PCR-based identification targeting the DNA topoisomerase II gene to determine major species of the opportunistic fungi Candida and Aspergillus fumigatus

For the simple and rapid detection/identification of major pathogenic fungal species such as Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata and Aspergillus fumigatus, common primers for these species and specific primers for each species, designed on the basis on the genomic nucleotide sequences of the DNA topoisomerase II genes, were prepared and tested for their specificities in PCR amplifications. Twelve specific primers were pooled and designated PsVI. Genomic DNAs were amplified by the common primer pair, and followed by PCR amplification using PsVI. Using PsVI, six unique DNA fragments, all of which corresponded to a Candida or A. fumigatus species, were specifically and acceptably amplified from each template DNA even in the presence of other DNAs. Similarly, the results of identification of clinical samples based on the PCR amplification coincided with those of conventional identification techniques. The sensitivities of the direct PCR and the nested PCR using PsVI were found to be 1,000 and 50 yeast cells, respectively.     

Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria

We describe a fast and simple method for signature-tagged mutagenesis (STM) using defined oligonucleotides for tag construction into mini-Tn5 and PCR instead of hybridization for rapid screening of bacterial mutants in vivo. A collection of 12 unique 21-mers were synthesized as complementary DNA strands to tag bacterial mutants constructed by insertional mutagenesis using pUTmini-Tn5Km2 plasmids. Tags were tested in a combination of assays by PCR and compared to hybridization for specificity and for large-scale screening. Each defined tag has the same melting temperature, an invariable region to optimize PCRs and a variable region for specific amplification by PCR. A series of "suicide" plasmids carrying mini-Tn5s, each with a specific tag, were transferred into Pseudomonas aeruginosa, giving 12 libraries of mutants; groups of 12 mutants were pooled and arrayed into 96-well microplates, representing approximately one-sixth of the P. aeruginosa 5.9-Mb genome. This simple STM method can be adapted to any bacterial system and used for genome scanning in various growth conditions.     

Detection of Nitrosomonas spp. by polymerase chain reaction

Abstract A unique genomic DNA fragment was isolated from Nitrosomonas europaea ATCC 19718. Based on the sequence of this fragment, oligonucleotide primers for polymerase chain reaction amplification were prepared which amplify sequences of 775 and 658 bp. The predicted DNA fragments were both amplified from the genome of N. europaea and a Nitrosomonas spp. isolated from a local oxidation pond. The primers failed to amplify DNA from the genomes of the ammonia oxidiser Nitrosolobous multiformis , the nitrite oxidiser Nitrococcus mobilis as well as from the genomes of other unrelated heterotrophic bacteria. These DNA sequences could be amplified from 0.01 ng of N. europaea genomic DNA or from 100 intact cells, and it was possible to detect Nitrosomonas DNA in a DNA mixture extracted from water samples drawn from a local oxidation pond.     

Bisulfite-modified target DNA array for aberrant methylation analysis

Aberrant DNA methylation of CpG islands is among the earliest and most frequent alterations in cancer. It is of great importance to develop simple and high-throughput methods of methylation analysis for earlier cancer diagnosis or the detection of recurrence. In this study, bisulfite-modified target DNA arrays were prepared on positively charged nylon membrane with two different procedures: fixing PCR products and fixing genomic DNA. First, a bisulfite PCR product array was prepared through fixing PCR products amplified in bisulfite sequencing primers from the bisulfite-modified genomic DNA of different clinical samples on membrane. Furthermore, bisulfite-modified genomic DNA of the different samples was directly fixed on membrane to fabricate bisulfite genomic DNA arrays. The two kinds of arrays were hybridized by probes labeled with digoxigenin, and the hybridization signals were obtained through chemiluminescent detection. The methylation statuses of the IGFBP7 gene for breast tumor and normal tissue samples and for normal human blood cell samples were detected successfully by the two procedures. It was shown that the methods are reliable and sensitive and that they have high potential in screening molecular methylation markers from a large number of clinical samples.     

Development and comparison of two assay formats for parallel detection of four biothreat pathogens by using suspension microarrays

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics.     

Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis

A comprehensive genomic analysis of single cells is instrumental for numerous applications in tumor genetics, clinical diagnostics and forensic analyses. Here, we provide a protocol for single-cell isolation and whole genome amplification, which includes the following stages: preparation of single-cell suspensions from blood or bone marrow samples and cancer cell lines; their characterization on the basis of morphology, interphase fluorescent in situ hybridization pattern and antibody staining; isolation of single cells by either laser microdissection or micromanipulation; and unbiased amplification of single-cell genomes by either linker-adaptor PCR or GenomePlex library technology. This protocol provides a suitable template to screen for chromosomal copy number changes by conventional comparative genomic hybridization (CGH) or array CGH. Expected results include the generation of several micrograms of DNA from single cells, which can be used for CGH or other analyses, such as sequencing. Using linker-adaptor PCR or GenomePlex library technology, the protocol takes 72 or 30 h, respectively.     

Whole genome amplification from filamentous fungi using Phi29-mediated multiple displacement amplification

The availability of genomic DNA of sufficient quality and quantity is fundamental to molecular genetic analysis. Many filamentous fungi are slow growing or even unculturable and current DNA isolation methods are often unsatisfactory. We have used multiple displacement amplification (MDA) to amplify whole genomes for two fungal species, Penicillium paxilli and the slow growing endophyte of grasses Epichloe festucae. Up to 10 microg of high molecular weight DNA was routinely amplified from less than 10 ng of template DNA obtained using glass bead-mediated disruption of fungal spores or alkaline lysis of mycelium. PCR was possible from MDA-generated DNA and amplicons up to 10 kb were successfully amplified. RFLP analysis was successful, with bands of up to 5 kb routinely detected. Hybridization of MDA-amplified DNA to a cosmid library illustrated that the MDA product amplified from E. festucae is representative of the genome. MDA is a reliable method that could be applied to applications ranging from high-throughput screening of deletion mutants to genomic library construction.     

Comparison of sample preparation methods for ChIP-chip assays

A single chromatin immunoprecipitation (ChIP) sample does not provide enough DNA for hybridization to a genomic tiling array. A commonly used technique for amplifying the DNA obtained from ChIP assays is ligation-mediated PCR (LM-PCR). However; using this amplification method, we could not identify Oct4 binding sites on genomic tiling arrays representing 1% of the human genome (ENCODE arrays). In contrast, hybridization of a pool of 10 ChIP samples to the arrays produced reproducible binding patterns and low background signals. However the pooling method would greatly increase the number of ChIP reactions needed to analyze the entire human genome. Therefore, we have adapted the GenomePlex whole genome amplification (WGA) method for use in ChIP-chip assays; detailed ChIP and amplification protocols used for these analyses are provided as supplementary material. When applied to ENCODE arrays, the products prepared using this new method resulted in an Oct4 binding pattern similar to that from the pooled Oct4 ChIP samples. Importantly, the signal-to-noise ratio using the GenomePlex WGA method is superior to the LM-PCR amplification method.     

Mathematical modeling of 16S ribosomal DNA amplification reveals optimal conditions for the interrogation of complex microbial communities with phylogenetic microarrays

MOTIVATION: Many current studies of complex microbial communities rely on the isolation of community genomic DNA, amplification of 16S ribosomal RNA genes (rDNA) and subsequent examination of community structure through interrogation of the amplified 16S rDNA pool by high-throughput sequencing, phylogenetic microarrays or quantitative PCR. RESULTS: Here we describe the development of a mathematical model aimed to simulate multitemplate amplification of 16S ribosomal DNA sample and subsequent detection of these amplified 16S rDNA species by phylogenetic microarray. Using parameters estimated from the experimental results obtained in the analysis of intestinal microbial communities with Microbiota Array, we show that both species detection and the accuracy of species abundance estimates depended heavily on the number of PCR cycles used to amplify 16S rDNA. Both parameters initially improved with each additional PCR cycle and reached optimum between 15 and 20 cycles of amplification. The use of more than 20 cycles of PCR amplification and/or more than 50 ng of starting genomic DNA template was, however, detrimental to both the fraction of detected community members and the accuracy of abundance estimates. Overall, the outcomes of the model simulations matched well available experimental data. Our simulations also showed that species detection and the accuracy of abundance measurements correlated positively with the higher sample-wide PCR amplification rate, lower template-to-template PCR bias and lower number of species in the interrogated community. The developed model can be easily modified to simulate other multitemplate DNA mixtures as well as other microarray designs and PCR amplification protocols.     

Novel approach for deriving genome wide SNP analysis data from archived blood spots

ABSTRACT: BACKGROUND: The ability to transport and store DNA at room temperature in low volumes has the advantage of optimising cost, time and storage space. Blood spots on adapted filter papers are popular for this, with FTA (Flinders Technology Associates) Whatman[trade mark sign] technology being one of the most recent. Plant material, plasmids, viral particles, bacteria and animal blood have been stored and transported successfully using this technology, however the method of porcine DNA extraction from FTA Whatman[trade mark sign] cards is a relatively new approach, allowing nucleic acids to be ready for downstream applications such as PCR, whole genome amplification, sequencing and subsequent application to single nucleotide polymorphism microarrays has hitherto been under-explored. FINDINGS: DNA was extracted from FTA Whatman[trade mark sign] cards (following adaptations of the manufacturer's instructions), whole genome amplified and subsequently analysed to validate the integrity of the DNA for downstream SNP analysis. DNA was successfully extracted from 288/288 samples and amplified by WGA. Allele dropout post WGA, was observed in less than 2% of samples and there was no clear evidence of amplification bias nor contamination. Acceptable call rates on porcine SNP chips were also achieved using DNA extracted and amplified in this way. CONCLUSIONS: DNA extracted from FTA Whatman cards is of a high enough quality and quantity following whole genomic amplification to perform meaningful SNP chip studies.     

A PCR-based method for high stringency screening of DNA libraries.

A rapid method for cloning genomic DNA utilizing a PCR-based screening protocol is described. A murine genomic library in lambda phage was subdivided into 64 wells, each containing 1000 clones, and propagated in bacteria. Amplified phage from each of 8 wells across columns, and each of 8 wells down rows, were pooled. The pooled phage were screened for the presence of murine M-CSF DNA by PCR using specific oligonucleotide primers. A single well that contained an M-CSF genomic clone was identified by the synthesis of a PCR product of the correct size that hybridized to an internal M-CSF oligonucleotide probe. This well was subdivided into 64 wells, each containing approximately 30 individual phage, reamplified, and rescreened utilizing the same protocol. A positive well was then subdivided and amplified a third time starting with an average of 2 phage per well, and rescreened for M-CSF DNA by PCR. Phage from a PCR-positive well, now highly enriched for M-CSF DNA, were grown as individual plaques. PCR-screening of randomly picked plaques demonstrated that the majority contained an M-CSF genomic insert. This method obviates the more labor and time intensive method of plaque hybridization screening of DNA libraries, and is more stringent since three oligonucleotides (the two PCR primers, and the hybridization probe) are required to give a true positive signal. Similar methodology has also been used to clone a cDNA gene contained within a plasmid library.