Genomic DNA amplification by the multiple displacement amplification (MDA) method

2009 ◽  
Vol 37 (2) ◽  
pp. 450-453 ◽  
Author(s):  
Roger S. Lasken
Keyword(s):  
Environmental Samples ◽  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Genomic Analysis ◽  
Dna Amplification ◽  
Genomic Sequencing ◽  
Extensive Coverage ◽  
Multiple Displacement Amplification Reaction ◽  
Amplification Reaction

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.

scholarly journals Cyclic Olefin Copolymer Microfluidic Devices for Forensic Applications

Biosensors ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 85 ◽  
Author(s):  
Brigitte Bruijns ◽  
Andrea Veciana ◽  
Roald Tiggelaar ◽  
Han Gardeniers
Keyword(s):  
Chemical Resistance ◽  
Multiple Displacement Amplification ◽  
Visible Region ◽  
Dna Amplification ◽  
Microfluidic Devices ◽  
Cyclic Olefin Copolymer ◽  
Color Test ◽  
Cyclic Olefin ◽  
On Chip ◽  
Amplification Reaction

Microfluidic devices offer important benefits for forensic applications, in particular for fast tests at a crime scene. A large portion of forensic applications require microfluidic chip material to show compatibility with biochemical reactions (such as amplification reactions), and to have high transparency in the visible region and high chemical resistance. Also, preferably, manufacturing should be simple. The characteristic properties of cyclic olefin copolymer (COC) fulfills these requirements and offers new opportunities for the development of new forensic tests. In this work, the versatility of COC as material for lab-on-a-chip (LOC) systems in forensic applications has been explored by realizing two proof-of-principle devices. Chemical resistance and optical transparency were investigated for the development of an on-chip presumptive color test to indicate the presence of an illicit substance through applying absorption spectroscopy. Furthermore, the compatibility of COC with a DNA amplification reaction was verified by performing an on-chip multiple displacement amplification (MDA) reaction.

Whole genome amplification of buccal cell DNA: genotyping concordance before and after multiple displacement amplification

2005 ◽  
Vol 43 (2) ◽  
Author(s):  
Miles D. Thompson ◽  
Raffick A. R. Bowen ◽  
Betty Y. L. Wong ◽  
Joan Antal ◽  
Zhanqin Liu ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Buccal Cell ◽  
Whole Genome ◽  
Buccal Cells ◽  
Genome Amplification ◽  
Dna Yield ◽  
Epidemiological Surveys ◽  
Before And After

AbstractWhile buccal cells provide an easily accessible source of genomic DNA, the amount extracted may be insufficient for many studies. Whole genome amplification (WGA) using multiple displacement amplification (MDA) may optimize buccal cell genomic DNA yield. We compared the usefulness, in epidemiological surveys, of DNA derived from buccal cells collected by alcohol mouthwash and amplified by WGA protocol and standard protocols. Buccal cell collection kits were mailed to 300 randomly selected members of a large cohort study, and 189 subjects returned buccal cell samples. We determined: (i) which QIAamp

scholarly journals Whole-genome amplification in double-digest RADseq results in adequate libraries but fewer sequenced loci

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5089 ◽  
Author(s):  
Bruno A. S. de Medeiros ◽  
Brian D. Farrell
Keyword(s):  
Population Genetics ◽  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Sequence Data ◽  
Multiple Displacement Amplification ◽  
Whole Genome ◽  
Promising Technique ◽  
Genome Amplification ◽  
Template Dna

Whole-genome amplification by multiple displacement amplification (MDA) is a promising technique to enable the use of samples with only limited amount of DNA for the construction of RAD-seq libraries. Previous work has shown that, when the amount of DNA used in the MDA reaction is large, double-digest RAD-seq (ddRAD) libraries prepared with amplified genomic DNA result in data that are indistinguishable from libraries prepared directly from genomic DNA. Based on this observation, here we evaluate the quality of ddRAD libraries prepared from MDA-amplified genomic DNA when the amount of input genomic DNA and the coverage obtained for samples is variable. By simultaneously preparing libraries for five species of weevils (Coleoptera, Curculionidae), we also evaluate the likelihood that potential contaminants will be encountered in the assembled dataset. Overall, our results indicate that MDA may not be able to rescue all samples with small amounts of DNA, but it does produce ddRAD libraries adequate for studies of phylogeography and population genetics even when conditions are not optimal. We find that MDA makes it harder to predict the number of loci that will be obtained for a given sequencing effort, with some samples behaving like traditional libraries and others yielding fewer loci than expected. This seems to be caused both by stochastic and deterministic effects during amplification. Further, the reduction in loci is stronger in libraries with lower amounts of template DNA for the MDA reaction. Even though a few samples exhibit substantial levels of contamination in raw reads, the effect is very small in the final dataset, suggesting that filters imposed during dataset assembly are important in removing contamination. Importantly, samples with strong signs of contamination and biases in heterozygosity were also those with fewer loci shared in the final dataset, suggesting that stringent filtering of samples with significant amounts of missing data is important when assembling data derived from MDA-amplified genomic DNA. Overall, we find that the combination of MDA and ddRAD results in high-quality datasets for population genetics as long as the sequence data is properly filtered during assembly.

Application of PCR to the Detection of Adenoviruses in Polluted Waters

1993 ◽  
Vol 27 (3-4) ◽  
pp. 235-241 ◽  
Author(s):  
R. Girones ◽  
A. Allard ◽  
G. Wadell ◽  
J. Jofre
Keyword(s):  
Environmental Samples ◽  
Suspended Solids ◽  
Reliable Method ◽  
Dna Amplification ◽  
Human Adenovirus ◽  
Glycine Buffer ◽  
Viral Particles ◽  
One Step ◽  
Polluted Waters ◽  
Amplification Reaction

A method for the detection of adenovirus in environmental samples has been developed. We tested several systems for concentrating viral particles by adding adenoviruses 2 and 12 to different sewage samples. The method selected was as follows: centrifugation of the sample in order to pellet adenovirus viral particles and all suspended solids, elution of the pelleted viruses by treatment with 0.25N glycine buffer pH 9.5, removal of solids from the sample by a short centrifugation and ultracentrifugation of the resulting supernatant. Elution with glycine buffer avoided inhibitors and showed more sensitivity than ultrasonication or filtration through a low binding protein filter to retain bacteria and suspended solids. Sewage samples were treated by this selected method and recovered viral particles were analyzed both by a two-step DNA amplification reaction and by infecting Hep-2 cells. About 50% of the samples were positive in a two-step PCR and these data were confirmed by tissue culture amplification and one step PCR. Two pairs of primers (external and nested) from the hexon region were used, which are able to detect human adenovirus from all subgenera. Although more studies are needed, the two-step PCR developed appears to be a quick and reliable method for adenovirus detection in environmental samples.

scholarly journals Quantitative comparison of single-cell sequencing methods using hippocampal neurons

2014 ◽  
Author(s):  
Luwen Ning ◽  
Guan Wang ◽  
Zhoufang Li ◽  
Wen Hu ◽  
Qingming Hou ◽  
...  
Keyword(s):  
Single Cell ◽  
Copy Number ◽  
Hippocampal Neurons ◽  
Cancer Biology ◽  
Multiple Displacement Amplification ◽  
Genetic Diagnosis ◽  
Genomic Analysis ◽  
Dna Amplification ◽  
Copy Number Variations ◽  
Cell Genome

Single-cell genomic analysis has grown rapidly in recent years and will find widespread applications in various fields of biology, including cancer biology, development, immunology, pre-implantation genetic diagnosis, and neurobiology. In this study, we amplified genomic DNA from individual hippocampal neurons using one of three single-cell DNA amplification methods (multiple annealing and looping-based amplification cycles (MALBAC), multiple displacement amplification (MDA), and GenomePlex whole genome amplification (WGA4)). We then systematically evaluated the genome coverage, GC-bias, reproducibility, and copy number variations among individual neurons. Our results showed that single-cell genome sequencing results obtained from the MALBAC and WGA4 methods are highly reproducible and have a high success rate. Chromosome-level and subchromosomal-level copy number variations among individual neurons can be detected.

scholarly journals Genomic DNA Amplification from a Single Bacterium

2005 ◽  
Vol 71 (6) ◽  
pp. 3342-3347 ◽  
Author(s):  
Arumugham Raghunathan ◽  
Harley R. Ferguson ◽  
Carole J. Bornarth ◽  
Wanmin Song ◽  
Mark Driscoll ◽  
...  
Keyword(s):  
16S Rrna ◽  
Dna Polymerase ◽  
Genomic Dna ◽  
Multiple Displacement Amplification ◽  
Dna Amplification ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Single Flow ◽  
The 16S Rrna Gene ◽  
New Strategies

ABSTRACT Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using φ 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying nonculturable microorganisms.

scholarly journals Quantitative comparison of single-cell sequencing methods using hippocampal neurons

2014 ◽  
Author(s):  
Luwen Ning ◽  
Guan Wang ◽  
Zhoufang Li ◽  
Wen Hu ◽  
Qingming Hou ◽  
...  
Keyword(s):  
Single Cell ◽  
Copy Number ◽  
Hippocampal Neurons ◽  
Cancer Biology ◽  
Multiple Displacement Amplification ◽  
Genetic Diagnosis ◽  
Genomic Analysis ◽  
Dna Amplification ◽  
Copy Number Variations ◽  
Cell Genome

Single-cell genomic analysis has grown rapidly in recent years and will find widespread applications in various fields of biology, including cancer biology, development, immunology, pre-implantation genetic diagnosis, and neurobiology. In this study, we amplified genomic DNA from individual hippocampal neurons using one of three single-cell DNA amplification methods (multiple annealing and looping-based amplification cycles (MALBAC), multiple displacement amplification (MDA), and GenomePlex whole genome amplification (WGA4)). We then systematically evaluated the genome coverage, GC-bias, reproducibility, and copy number variations among individual neurons. Our results showed that single-cell genome sequencing results obtained from the MALBAC and WGA4 methods are highly reproducible and have a high success rate. Chromosome-level and subchromosomal-level copy number variations among individual neurons can be detected.

scholarly journals Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification

2015 ◽  
Vol 112 (38) ◽  
pp. 11923-11928 ◽  
Author(s):  
Yusi Fu ◽  
Chunmei Li ◽  
Sijia Lu ◽  
Wenxiong Zhou ◽  
Fuchou Tang ◽  
...  
Keyword(s):  
Single Cell ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Simultaneous Detection ◽  
Dna Amplification ◽  
Copy Number Variations ◽  
Whole Genome ◽  
Genome Amplification ◽  
High Uniformity

Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (105) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.

Multiple displacement amplification (MDA) of total genomic DNA from Meloidogyne spp. and comparison to crude DNA extracts in PCR of ITS1, 28S D2-D3 rDNA and Hsp90

Nematology ◽  
2005 ◽  
Vol 7 (2) ◽  
pp. 285-293 ◽  
Author(s):  
Andrea Skantar ◽  
Lynn Carta
Keyword(s):  
Internal Transcribed Spacer ◽  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Genetic Material ◽  
Single Copy ◽  
Ribosomal Genes ◽  
Meloidogyne Spp ◽  
Successful Amplification ◽  
Dna Template

AbstractBecause the quantity of nematode specimens available for molecular analysis is often limited, the number of analyses possible is constrained by the availability of DNA. Multiple displacement amplification (MDA) was assessed for whole genome amplification of crude DNA from several Meloidogyne species. MDA produced microgram quantities of template that resulted in successful amplification of the ribosomal internal transcribed spacer (ITS1) and 28S D2-D3 expansion regions, producing PCR results that were comparable to template generated by the single nematode smash method. MDA greatly improved degenerate primer PCR of single-copy Hsp90, a gene which is more sensitive than multi-copy ribosomal genes to limited DNA template. MDA should expand the number of molecular analyses possible for single nematodes. MDA will also be useful for archiving DNA from valuable specimens and provide a way for laboratories to share identical genetic material for nematode diagnosis.

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