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Pascal Simonet - One of the best experts on this subject based on the ideXlab platform.
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An efficient procedure for purification of the obligate intracellular Wolbachia pipientis and representative Amplification of its genome by Multiple Displacement Amplification
Applied and Environmental Microbiology, 2005Co-Authors: Patrick Mavingui, Van Tran Van, Estelle Labeyrie, Edwige Rancès, Fabrice Vavre, Pascal SimonetAbstract:procedure to representatively amplify Wolbachia genome by Multiple Displacement Amplification from limited infected host tissue (0.2 g or 2 x 107 cell line). We obtained sufficient amounts (8 to 10 µg) of DNA of suitable quality for genomic studies with which we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of wRi strain, an obligatory endosymbiont of Drosophila simulans, shared similar overall architecture with its relative strain wMel.
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Efficient Procedure for Purification of Obligate Intracellular Wolbachia pipientis and Representative Amplification of Its Genome by Multiple-Displacement Amplification
Applied and Environmental Microbiology, 2005Co-Authors: Patrick Mavingui, Van Tran Van, Estelle Labeyrie, Edwige Rancès, Fabrice Vavre, Pascal SimonetAbstract:Bacteria belonging to the genus Wolbachia are obligatorymicroendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis, for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi, have been fully completed and six other genome sequencing projects are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by Multiple-Displacement Amplification from limited infected host tissue (0.2 g or 2 \texttimes 107 cells). We obtained sufficient amounts (8 to 10 μg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans, shares a similar overall architecture with its relative strain wMel.
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Efficient procedure for purification of obligate intracellular Wolbachia pipientis and representative Amplification of its genome by Multiple-Displacement Amplification.
Applied and environmental microbiology, 2005Co-Authors: Patrick Mavingui, Estelle Labeyrie, Edwige Rancès, Fabrice Vavre, Van Tran Van, Pascal SimonetAbstract:Bacteria belonging to the genus Wolbachia are obligatorymicroendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis, for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi, have been fully completed and six other genome sequencing projects are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by Multiple-Displacement Amplification from limited infected host tissue (0.2 g or 2 × 107 cells). We obtained sufficient amounts (8 to 10 μg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans, shares a similar overall architecture with its relative strain wMel.
Kaye N. Ballantyne - One of the best experts on this subject based on the ideXlab platform.
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increasing Amplification success of forensic dna samples using Multiple Displacement Amplification
Forensic Science Medicine and Pathology, 2007Co-Authors: Kaye N. Ballantyne, Roland A H Van Oorschot, John R MitchellAbstract:Multiple Displacement Amplification (MDA) is capable of amplifying nanogram template amounts of DNA with high accuracy to generate micrograms of representative product. Although MDA is able to amplify small template amounts (<100 pg), increased levels of preferential Amplification and allelic dropout are observed. The use of molecular crowders (polyethylene glycol 400) can decrease the Amplification bias and increase Amplification success. We describe the use of standard and crowded MDA on low-concentration casework samples originating from blood, semen, saliva, hair, and trace DNA. While standard MDA produced no significant increases in STR genotyping success, the use of crowded MDA yielded an average increase of 15% in the number of alleles, with a significant decrease in the Amplification bias, resulting in clearer, more-complete profiles from forensically relevant samples.
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Increasing Amplification success of forensic DNA samples using Multiple Displacement Amplification
Forensic Science Medicine and Pathology, 2007Co-Authors: Kaye N. Ballantyne, Roland A. H. Oorschot, R. John MitchellAbstract:Multiple Displacement Amplification (MDA) is capable of amplifying nanogram template amounts of DNA with high accuracy to generate micrograms of representative product. Although MDA is able to amplify small template amounts (
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Decreasing Amplification bias associated with Multiple Displacement Amplification and short tandem repeat genotyping
Analytical biochemistry, 2007Co-Authors: Kaye N. Ballantyne, Roland A H Van Oorschot, Iman Muharam, Angela Van Daal, R. John MitchellAbstract:Abstract Although Multiple Displacement Amplification (MDA) is being used increasingly to amplify genomes, the Amplification bias generated by the ϕ29 polymerase can be a concern with genotyping applications. It has been noted that the bias is pronounced with small template amounts, particularly with single nucleotide polymorphism (SNP) and short tandem repeat (STR) genotyping. Bias may occur between loci, or between alleles within a locus, and may differ between sample donors at the same loci. Previous research has suggested that omitting denaturation of the template prior to Amplification can reduce the observed bias significantly. Comparison of the two methods (with and without denaturation) has found that nondenaturation of template reverses the direction of bias observed between allelic pairs following MDA. By combining two MDA reactions, one denatured and one nondenatured, the bias was found to be reduced significantly, aiding copy number analysis and subsequent genotyping.
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Molecular crowding increases the Amplification success of Multiple Displacement Amplification and short tandem repeat genotyping
Analytical biochemistry, 2006Co-Authors: Kaye N. Ballantyne, R. John Mitchell, Roland A H Van Oorschot, Irene KoukoulasAbstract:Multiple Displacement Amplification (MDA) can generate large quantities of genomic DNA product from small amounts of template. We investigated the ability of MDA to amplify samples containing very small amounts of target DNA (5 pg to 1 ng) in the presence of a second, larger DNA sample for downstream short tandem repeat (STR) Multiplex genotyping. We observed that STR Amplification success of the minor fraction was increased in these mixed samples when compared with standard PCR only or MDA containing only the single trace DNA sample. Increased numbers of alleles were detected, with less Amplification bias between loci than in single source samples undergoing the same protocol. To improve the STR genotyping accuracy, animal DNA was substituted for the additional human DNA, maintaining the increase in the number and quality of human-specific STR loci amplified. Polyethylene glycol 400, a commonly used crowding agent, was used as a replacement for the added genomic DNA in the MDA reaction and produced very similar results. Therefore, we suggest that additional DNA is acting as a molecular crowding agent during MDA. Performing MDA on trace amounts of DNA under crowded conditions results in greater numbers of alleles being amplified and more balanced Amplification occurring between alleles.
Patrick Mavingui - One of the best experts on this subject based on the ideXlab platform.
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An efficient procedure for purification of the obligate intracellular Wolbachia pipientis and representative Amplification of its genome by Multiple Displacement Amplification
Applied and Environmental Microbiology, 2005Co-Authors: Patrick Mavingui, Van Tran Van, Estelle Labeyrie, Edwige Rancès, Fabrice Vavre, Pascal SimonetAbstract:procedure to representatively amplify Wolbachia genome by Multiple Displacement Amplification from limited infected host tissue (0.2 g or 2 x 107 cell line). We obtained sufficient amounts (8 to 10 µg) of DNA of suitable quality for genomic studies with which we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of wRi strain, an obligatory endosymbiont of Drosophila simulans, shared similar overall architecture with its relative strain wMel.
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Efficient Procedure for Purification of Obligate Intracellular Wolbachia pipientis and Representative Amplification of Its Genome by Multiple-Displacement Amplification
Applied and Environmental Microbiology, 2005Co-Authors: Patrick Mavingui, Van Tran Van, Estelle Labeyrie, Edwige Rancès, Fabrice Vavre, Pascal SimonetAbstract:Bacteria belonging to the genus Wolbachia are obligatorymicroendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis, for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi, have been fully completed and six other genome sequencing projects are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by Multiple-Displacement Amplification from limited infected host tissue (0.2 g or 2 \texttimes 107 cells). We obtained sufficient amounts (8 to 10 μg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans, shares a similar overall architecture with its relative strain wMel.
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Efficient procedure for purification of obligate intracellular Wolbachia pipientis and representative Amplification of its genome by Multiple-Displacement Amplification.
Applied and environmental microbiology, 2005Co-Authors: Patrick Mavingui, Estelle Labeyrie, Edwige Rancès, Fabrice Vavre, Van Tran Van, Pascal SimonetAbstract:Bacteria belonging to the genus Wolbachia are obligatorymicroendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis, for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi, have been fully completed and six other genome sequencing projects are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by Multiple-Displacement Amplification from limited infected host tissue (0.2 g or 2 × 107 cells). We obtained sufficient amounts (8 to 10 μg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans, shares a similar overall architecture with its relative strain wMel.
Roger S. Lasken - One of the best experts on this subject based on the ideXlab platform.
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Multiple Displacement Amplification as an adjunct to pcr based detection of staphylococcus aureus in synovial fluid
BMC Research Notes, 2010Co-Authors: Sandeep Kathju, Roger S. Lasken, Latha Satish, Sandra Johnson, Paul Stoodley, Christopher J Post, Garth D EhrlichAbstract:Detection of bacterial nucleic acids in synovial fluid following total joint arthroplasty with suspected infection can be difficult; among other technical challenges, inhibitors in the specimens require extensive sample preparation and can diminish assay sensitivity even using polymerase chain reaction (PCR)-based methods. To address this problem a simple protocol for prior use of Multiple Displacement Amplification (MDA) as an adjunct to PCR was established and tested on both purified S. aureus DNA as well as on clinical samples known to contain S. aureus nucleic acids. A single round of MDA on purified nucleic acids resulted in a > 300 thousand-fold increase in template DNA on subsequent quantitative PCR (qPCR) analysis. MDA use on clinical samples resulted in at least a 100-fold increase in sensitivity on subsequent qPCR and required no sample preparation other than a simple alkali/heat lysis step. Mixed samples of S. aureus DNA with a 103 - 104-fold excess of human genomic DNA still allowed for MDA Amplification of the minor bacterial component to the threshold of detectability. MDA is a promising technique that may serve to significantly enhance the sensitivity of molecular assays in cases of suspected joint infection while simultaneously reducing the specimen handling required.
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Genomic DNA Amplification by the Multiple Displacement Amplification (MDA) method.
Biochemical Society transactions, 2009Co-Authors: Roger S. LaskenAbstract:Large amounts of DNA are frequently required for use in detection assays and genomic analysis. The limited availability of DNA can be a critical obstacle to meeting research and clinical needs. DNA Amplification methods are often required to generate sufficient material from small specimens or environmental samples with low DNA content. The MDA (Multiple Displacement Amplification) reaction is increasingly the method of choice for many applications because of its extensive coverage of the genome, the generation of extremely long DNA products compared with older whole genome Amplification methods and the high DNA yields, even from exceedingly low amounts of starting material. Remarkably, MDA enables genomic sequencing even from single microbial cells. Some of the uses of MDA and its strengths and limitations will be discussed.
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Something from (almost) nothing: the impact of Multiple Displacement Amplification on microbial ecology
The ISME Journal, 2008Co-Authors: Erik K Binga, Roger S. Lasken, Josh D NeufeldAbstract:Microbial ecology is a field that applies molecular techniques to analyze genes and communities associated with a plethora of unique environments on this planet. In the past, low biomass and the predominance of a few abundant community members have impeded the application of techniques such as PCR, microarray analysis and metagenomics to complex microbial populations. In the absence of suitable cultivation methods, it was not possible to obtain DNA samples from individual microorganisms. Recently, a method called Multiple Displacement Amplification (MDA) has been used to circumvent these limitations by amplifying DNA from microbial communities in low-biomass environments, individual cells from uncultivated microbial species and active organisms obtained through stable isotope probing incubations. This review describes the development and applications of MDA, discusses its strengths and limitations and highlights the impact of MDA on the field of microbial ecology. Whole genome Amplification via MDA has increased access to the genomic DNA of uncultivated microorganisms and low-biomass environments and represents a ‘power tool’ in the molecular toolbox of microbial ecologists.
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Single-cell genomic sequencing using Multiple Displacement Amplification.
Current opinion in microbiology, 2007Co-Authors: Roger S. LaskenAbstract:Single microbial cells can now be sequenced using DNA amplified by the Multiple Displacement Amplification (MDA) reaction. The few femtograms of DNA in a bacterium are amplified into micrograms of high molecular weight DNA suitable for DNA library construction and Sanger sequencing. The MDA-generated DNA also performs well when used directly as template for pyrosequencing by the 454 Life Sciences method. While MDA from single cells loses some of the genomic sequence, this approach will greatly accelerate the pace of sequencing from uncultured microbes. The genetically linked sequences from single cells are also a powerful tool to be used in guiding genomic assembly of shotgun sequences of Multiple organisms from environmental DNA extracts (metagenomic sequences).
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Nanoliter reactors improve Multiple Displacement Amplification of genomes from single cells.
PLoS genetics, 2007Co-Authors: Yann Marcy, Roger S. Lasken, Thomas Ishoey, Timothy B. Stockwell, Brian P. Walenz, Aaron L. Halpern, Karen Beeson, Susanne M. D. Goldberg, Stephen R. QuakeAbstract:Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome Amplification was assessed by qPCR and compared favourably to single-cell Amplifications performed in standard 50-μl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.
R. John Mitchell - One of the best experts on this subject based on the ideXlab platform.
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Increasing Amplification success of forensic DNA samples using Multiple Displacement Amplification
Forensic Science Medicine and Pathology, 2007Co-Authors: Kaye N. Ballantyne, Roland A. H. Oorschot, R. John MitchellAbstract:Multiple Displacement Amplification (MDA) is capable of amplifying nanogram template amounts of DNA with high accuracy to generate micrograms of representative product. Although MDA is able to amplify small template amounts (
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Decreasing Amplification bias associated with Multiple Displacement Amplification and short tandem repeat genotyping
Analytical biochemistry, 2007Co-Authors: Kaye N. Ballantyne, Roland A H Van Oorschot, Iman Muharam, Angela Van Daal, R. John MitchellAbstract:Abstract Although Multiple Displacement Amplification (MDA) is being used increasingly to amplify genomes, the Amplification bias generated by the ϕ29 polymerase can be a concern with genotyping applications. It has been noted that the bias is pronounced with small template amounts, particularly with single nucleotide polymorphism (SNP) and short tandem repeat (STR) genotyping. Bias may occur between loci, or between alleles within a locus, and may differ between sample donors at the same loci. Previous research has suggested that omitting denaturation of the template prior to Amplification can reduce the observed bias significantly. Comparison of the two methods (with and without denaturation) has found that nondenaturation of template reverses the direction of bias observed between allelic pairs following MDA. By combining two MDA reactions, one denatured and one nondenatured, the bias was found to be reduced significantly, aiding copy number analysis and subsequent genotyping.
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Molecular crowding increases the Amplification success of Multiple Displacement Amplification and short tandem repeat genotyping
Analytical biochemistry, 2006Co-Authors: Kaye N. Ballantyne, R. John Mitchell, Roland A H Van Oorschot, Irene KoukoulasAbstract:Multiple Displacement Amplification (MDA) can generate large quantities of genomic DNA product from small amounts of template. We investigated the ability of MDA to amplify samples containing very small amounts of target DNA (5 pg to 1 ng) in the presence of a second, larger DNA sample for downstream short tandem repeat (STR) Multiplex genotyping. We observed that STR Amplification success of the minor fraction was increased in these mixed samples when compared with standard PCR only or MDA containing only the single trace DNA sample. Increased numbers of alleles were detected, with less Amplification bias between loci than in single source samples undergoing the same protocol. To improve the STR genotyping accuracy, animal DNA was substituted for the additional human DNA, maintaining the increase in the number and quality of human-specific STR loci amplified. Polyethylene glycol 400, a commonly used crowding agent, was used as a replacement for the added genomic DNA in the MDA reaction and produced very similar results. Therefore, we suggest that additional DNA is acting as a molecular crowding agent during MDA. Performing MDA on trace amounts of DNA under crowded conditions results in greater numbers of alleles being amplified and more balanced Amplification occurring between alleles.