Multiple Displacement Amplification Technique Research Articles

Bioreactor virome metagenomics sequencing using DNA spike-ins

With the emergence of Next Generation Sequencing, major advances were made with regard to identifying viruses in natural environments. However, bioinformatical research on viruses is still limited because of the low amounts of viral DNA that can be obtained for analysis. To overcome this limitation, DNA is often amplified with multiple displacement amplification (MDA), which may cause an unavoidable bias. Here, we describe a case study in which the virome of a bioreactor is sequenced using Ion Torrent technology. DNA-spiking of samples is compared with MDA-amplified samples. DNA for spiking was obtained by amplifying a bacterial 16S rRNA gene. After sequencing, the 16S rRNA gene reads were removed by mapping to the Silva database. Three samples were tested, a whole genome from Enterobacteria P1 Phage and two viral metagenomes from an infected bioreactor. For one sample, the new DNA-spiking protocol was compared with the MDA technique. When MDA was applied, the overall GC content of the reads showed a bias towards lower GC%, indicating a change in composition of the DNA sample. Assemblies using all available reads from both MDA and the DNA-spiked samples resulted in six viral genomes. All six genomes could be almost completely retrieved (97.9%–100%) when mapping the reads from the DNA-spiked sample to those six genomes. In contrast, 6.3%–77.7% of three viral genomes was covered by reads obtained using the MDA amplification method and only three were nearly fully covered (97.4%–100%). This case study shows that DNA-spiking could be a simple and inexpensive alternative with very low bias for sequencing of metagenomes for which low amounts of DNA are available.

Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples.

Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

Preimplantation genetic diagnosis of X-linked retinoschisis

The aim of this study was to perform preimplantation genetic diagnosis (PGD) for X-linked retinoschisis using multiple displacement amplification (MDA) for whole genome amplification and linked markers to the RS1 gene. The study evaluates the ability of MDA to amplify the whole genome directly from a single blastomere. MDA products were used for polymerase chain reaction analysis of two polymorphic markers flanking the RS1 gene and a new X/Y marker, X22, to sex embryos in an X-linked retinoschisis PGD programme. Two couples in whom the wives were carriers of the RS1 gene mutation (599G →A), previously identified in their families, were subjected to two PGD cycles each. The main outcome measure was the ability to analyse single blastomeres for X-linked retinoschisis using MDA. As a result, the development of an MDA—PGD protocol for X-linked retinoschisis allowed for the diagnosis of 20 embryos in the four PGD cycles performed. These were biopsied on day 3 of culture and analysed. Eight embryos were affected males and two embryos were female carriers. In summary, three healthy female and four healthy male embryos, and a female carrier embryo, were transferred 48 h after biopsy. One single pregnancy was achieved. This report shows that the MDA technique is useful for overcoming the problem of insufficient genomic DNA in PGD. It also allows the simultaneous amplification of different targets to perform diagnosis of any known gene defect and sexing test by standard methods and conditions.

Preimplantation genetic diagnosis of X-linked adrenoleukodystrophy with gender determination using multiple displacement amplification

To evaluate the use of multiple displacement amplification (MDA) for whole genome amplification in the preimplantation genetic diagnosis (PGD) of X-linked adrenoleukodystrophy. MDA was used to amplify the whole genome directly from a single blastomere. MDA products were used for polymerase chain reaction (PCR) analysis of two polymorphic markers flanking the ABCD1 gene and a new X/Y marker, X22, to sex embryos in an X-linked adrenoleukodystrophy PGD program. Fertility and gynecology private center in Alicante, Spain. A couple in which the wife is a carrier of the ABCD1 gene mutation (676A-->C) that was previously identified in her family. MDA of single blastomere and PCR tests for PGD. The ability to analyze single blastomeres for X-linked adrenoleukodystrophy using MDA. The development of an MDA-PGD protocol for X-linked adrenoleukodystrophy allowed for the diagnosis of five embryos. These were biopsied on day 3 of culture and analyzed. One embryo was an affected male and one embryo was a female carrier. Three healthy female embryos were transferred 48 hours after biopsy. Unfortunately, no pregnancy was achieved. The MDA technique is useful for overcoming the problem of insufficient genomic DNA in PGD and allows the simultaneous amplification of different targets to perform a diagnosis of any known gene defect and a sexing test by standard methods and conditions.

Multiple displacement amplification as an aid in checkerboard DNA–DNA hybridization

The study aimed to determine if multiple displacement amplification could be used to provide abundant target DNA and DNA probes for checkerboard DNA-DNA hybridization. Multiple displacement amplification was used to amplify 1 and 10 ng DNA from 16 individual bacterial species, DNA from single colonies, from a mixture of 20 bacterial species and oral biofilm samples, such as supragingival plaque, subgingival plaque, buccal swab and root canal samples. Samples in reaction buffer were heat-denatured at 95 degrees C for 3 min and cooled to 4 degrees C. Phi29 DNA polymerase was added and the mixture was incubated at 30 degrees C for 16-18 h. The quantity of the product was evaluated by the Picogreen assay. The amplified material was labeled with digoxigenin. The probes were compared with probes obtained from unamplified DNA using checkerboard DNA-DNA hybridization. Both amplified DNA and unamplified DNA were used as targets on the membrane. Amplified oral biofilm samples were compared to unamplified samples using checkerboard DNA-DNA hybridization. The DNA yield ranged from 4 to 11 microg. DNA-DNA hybridization showed that the amplified genome of each species used either as target or as probe provided signals equivalent to controls and that amplification of a mixture of species provided signals comparable to those provided by the unamplified source mixture. Amplified oral biofilm samples exhibited comparable proportions of bacterial DNA when compared to the original unamplified samples. The multiple displacement amplification technique is a simple and reliable method to uniformly amplify DNA for use in checkerboard DNA-DNA hybridization. It is also a useful tool in the amplification of clinical samples.

Preimplantation genetic diagnosis of Marfan syndrome using multiple displacement amplification

To evaluate the use of multiple displacement amplification (MDA) for whole-genome amplification in the preimplantation genetic diagnosis (PGD) of Marfan syndrome. Multiple displacement amplification was used to amplify the whole-genome directly from a single cell. The MDA product was used for polymerase chain reaction (PCR) analysis of five different loci. At this point MDA was used to develop a PGD-Marfan syndrome program. Fertility and gynecology private center in Alicante, Spain. A couple in which the husband is affected by Marfan syndrome and carries a novel mutation in the FBN-1 gene. The MDA of single cells and PCR tests for PGD. Allele drop-out (ADO), amplification efficiency rates, and the ability to detect Marfan syndrome using MDA. We report that isothermal whole-genome amplification from single cells allowed analysis of five different loci using standard conditions. The development of a MDA-PGD protocol for Marfan syndrome allowed for the diagnosis of seven embryos. These were biopsied on day 3 of culture and analyzed. Two healthy embryos were transferred 48 hours after culture, resulting in a singleton ongoing pregnancy and the birth of a healthy child. The MDA technique is useful for overcoming the problem of insufficient genomic DNA in PGD. The use of MDA as a universal step marks a new cycle for PGD as it allows for the diagnosis of any known gene defect by standard methods and conditions.

P-949: Quantity and quality of DNA by multiple displacement amplification (MDA) from single cells

The recently developed MDA technique for whole genome amplification (Dean et al., 2002, PNAS, 99:5261) is known to amplify high quality and quantities of DNA. The objective of this study was to determine an optimal time to obtain sufficient amount of DNA from single cells using MDA, allowing subsequent PCR amplification for accurate mutation analysis.

Sequence quality is maintained after multiple displacement amplification of non‐invasively obtained macaque semen DNA

Whole genome amplification protocols are revolutionizing the fields of molecular and conservation biology as they open the possibility of obtaining a large number of copies of a complete genome from minute amounts of sample. Multiple displacement amplification (MDA) is a whole genome amplification technique based on the properties of the phi29 DNA polymerase, which leads to a uniform representation of the genome with very low error rates. In this study we performed MDA on 28 macaque DNA samples extracted from blood or non-invasively collected semen from which we obtained mitochondrial control region sequences both before and after MDA. The length of the readable sequences was longer for the original samples than for the MDA products, but the number of unresolved positions was comparable both before and after MDA. We conclude that the MDA technique is useful for increasing the amount of DNA for sequencing mitochondrial regions in the case of non-invasively collected semen samples.

Estimation of the Rate of SNP Genotyping Errors From DNA Extracted From Different Tissues

High density single nucleotide polymorphism (SNP) genotyping panels provide an alternative to microsatellite markers for genome scans. However, genotype errors have a major impact on power to detect linkage or association and are difficult to detect for SNPs. We estimated error rates with the Affymetrix GeneChip SNP platform in samples from a family with a mixed set of monozygotic (MZ) and dizygotic (DZ) triplets using lymphocyte, buccal DNA and samples from whole genome amplification using the multiple displacement amplification (MDA) technique. The average call rate from 58,960 SNPs for five genomic samples was 99.48%. Comparison of results for the MZ twins showed only three discordant genotypes (concordance rate 99.995%). The mean concordance rate for comparisons of samples from lymphocyte and buccal DNA was 99.97%. Mendelian inconsistencies were identified in 46 SNPs with errors in one or more family members, a rate of 0.022%. Observed genotype concordance rates between parents, between parents and children, and among siblings were consistent with previously reported allele frequencies and Hardy-Weinberg equilibrium. Using the MDA technique, results for two samples had equivalent high accuracy to results with genomic samples. However, the SNP call rate for the remaining seven samples varied from 72.5% to 99.5%, with an average of 86.11%. Quality of the DNA sample following the MDA reaction appears to be the critical factor in SNP call rate for MDA samples. Our results demonstrate highly accurate and reproducible genotyping for the Affymetrix GeneChip Human Mapping Set in lymphocyte and buccal DNA samples.

Estimation of the Rate of SNP Genotyping Errors From DNA Extracted From Different Tissues

AbstractHigh density single nucleotide polymorphism (SNP) genotyping panels provide an alternative to microsatellite markers for genome scans. However, genotype errors have a major impact on power to detect linkage or association and are difficult to detect for SNPs. We estimated error rates with the Affymetrix GeneChip® SNP platform in samples from a family with a mixed set of monozygotic (MZ) and dizygotic (DZ) triplets using lymphocyte, buccal DNA and samples from whole genome amplification using the multiple displacement amplification (MDA) technique. The average call rate from 58,960 SNPs for five genomic samples was 99.48%. Comparison of results for the MZ twins showed only three discordant genotypes (concordance rate 99.995%). The mean concordance rate for comparisons of samples from lymphocyte and buccal DNA was 99.97%. Mendelian inconsistencies were identified in 46 SNPs with errors in one or more family members, a rate of 0.022%. Observed genotype concordance rates between parents, between parents and children, and among siblings were consistent with previously reported allele frequencies and Hardy–Weinberg equilibrium. Using the MDA technique, results for two samples had equivalent high accuracy to results with genomic samples. However, the SNP call rate for the remaining seven samples varied from 72.5% to 99.5%, with an average of 86.11%. Quality of the DNA sample following the MDA reaction appears to be the critical factor in SNP call rate for MDA samples. Our results demonstrate highly accurate and reproducible genotyping for the Affymetrix GeneChip® Human Mapping Set in lymphocyte and buccal DNA samples.

Use of multiple displacement amplification to amplify genomic DNA before sequencing of the α and β haemoglobin genes

Aims: To evaluate the technique of multiple displacement amplification (MDA) for whole genome amplification from small volume blood samples before sequencing in a clinical test to identify haemoglobin gene mutations....