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Multiple Displacement Amplification (multiple + displacement_amplification)
Selected AbstractsMultiple Displacement Amplification of Isolated DNA from Human Gallstones: Molecular Identification of Helicobacter DNA by Means of 16S rDNA-Based Pyrosequencing AnalysisHELICOBACTER, Issue 6 2005Isabelle Nilsson ABSTRACT Background., Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp. Materials and Methods., DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions. Results., Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori- specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones. Conclusions., We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens. [source] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environmentsENVIRONMENTAL MICROBIOLOGY, Issue 7 2005Juan M. Gonzalez Summary Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and ,29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations ,,10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples. [source] Multiple displacement amplification to create a long-lasting source of DNA for genetic studies,HUMAN MUTATION, Issue 7 2006Lovisa Lovmar Abstract In many situations there may not be sufficient DNA collected from patient or population cohorts to meet the requirements of genome-wide analysis of SNPs, genomic copy number polymorphisms, or acquired copy number alternations. When the amount of available DNA for genotype analysis is limited, high performance whole-genome amplification (WGA) represents a new development in genetic analysis. It is especially useful for analysis of DNA extracted from stored histology slides, tissue samples, buccal swabs, or blood stains collected on filter paper. The multiple displacement amplification (MDA) method, which relies on isothermal amplification using the DNA polymerase of the bacteriophage ,29, is a recently developed technique for high performance WGA. This review addresses new trends in the technical performance of MDA and its applications to genetic analyses. The main challenge of WGA methods is to obtain balanced and faithful replication of all chromosomal regions without the loss of or preferential amplification of any genomic loci or allele. In multiple comparisons to other WGA methods, MDA appears to be most reliable for genotyping, with the most favorable call rates, best genomic coverage, and lowest amplification bias. Hum Mutat 27(7), 603,614, 2006. © 2006 Wiley-Liss, Inc. [source] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to detect ultra low population of Ralstonia solanacearum (Smith 1896) Yabuchi et al. (1996)LETTERS IN APPLIED MICROBIOLOGY, Issue 5 2009A. Grover Abstract Aims:, To develop a reliable and sensitive protocol for detection of Ralstonia solanacearum using MDA-PCR (Multiple displacement amplification,PCR amplification). Methods and Results:, MDA-PCR technique was performed on pure cell lysates as well as soil samples. Pure cell lysate as well as that of soil DNA was used as template in MDA reaction. MDA of template DNA was carried out in the presence of sample buffer, reaction buffer and enzyme mix (, 29 DNA polymerase and random hexamers). The MDA amplified DNA was used for PCR amplification using R. solanacearum -specific PCR primers. MDA-PCR could detect as low as 1 colony forming unit (CFU ml,1) of bacteria within 8 h including DNA isolation. Conclusion:, MDA followed by standard PCR facilitated the detection of pathogen from very low count samples. The method is of great importance in managing the brown rot disease of potato. Significance and Impact of study:, The ultrasensitive detection technique developed in the present study is sensitive and speedy enough to be included into integrated wilt disease control programmes. [source] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environmentsENVIRONMENTAL MICROBIOLOGY, Issue 7 2005Juan M. Gonzalez Summary Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and ,29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations ,,10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples. [source] Multiple Displacement Amplification of Isolated DNA from Human Gallstones: Molecular Identification of Helicobacter DNA by Means of 16S rDNA-Based Pyrosequencing AnalysisHELICOBACTER, Issue 6 2005Isabelle Nilsson ABSTRACT Background., Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp. Materials and Methods., DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions. Results., Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori- specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones. Conclusions., We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens. [source] PGD for monogenic disorders: aspects of molecular biology,PRENATAL DIAGNOSIS, Issue 1 2009Claudia Spits Abstract Preimplantation genetic diagnosis (PGD) for monogenic diseases has known a considerable evolution since its first application in the early 1990s. Especially the technical aspects of the genetic diagnosis itself, the single-cell genetic analysis, has constantly evolved to reach levels of accuracy and efficiency nearing those of genetic diagnosis on regular DNA samples. In this review, we will focus on the molecular biological techniques that are currently in use in the most advanced centers for PGD for monogenic disorders, including multiplex polymerase chain reaction (PCR) and post-PCR diagnostic methods, whole genome amplification (WGA) and multiple displacement amplification (MDA). As it becomes more and more clear that when it comes to ethically difficult indications, PGD goes further than prenatal diagnosis (PND), we will also briefly discuss ethical issues. Copyright © 2008 John Wiley & Sons, Ltd. [source] Preimplantation genetic diagnosis of Morquio diseasePRENATAL DIAGNOSIS, Issue 10 2008Wafa Qubbaj Abstract Objectives Morquio syndrome is an autosomal recessive disease and mutations in the N -acetylgalactosamine 6-sulfate sulfatase (GALNS) gene cause Morquio type A disease. Preimplantation genetic diagnosis (PGD), an early form of prenatal diagnosis for couples at risk of transmitting inherited diseases, was applied to prevent transmission of this disease. Methods A couple with three affected children, having homozygous W159C (p. Trp 159 Cys) mutation in GALNS gene, underwent in vitro fertilization (IVF) treatment and PGD. Mutation analyses from the embryos were performed following whole genome amplification of single blastomeres using multiple displacement amplification (MDA). Results Three embryos were diagnosed as normal and two were transferred on day 4. The cycle resulted in a pregnancy and a live birth of a carrier male infant. Genetic haplotyping analysis of the infant and the leftover MDA samples enabled us to determine which embryo was implanted. The discrepancy in results was explained by allele dropout (ADO) of the mutant allele from the MDA product. Conclusions A feasible strategy for PGD of Morquio disease including whole genome amplification by MDA and the use of preimplantation genetic haplotyping is described. MDA product archiving will be useful for future investigations if needed. Copyright © 2008 John Wiley & Sons, Ltd. [source] Whole genome amplification from a single cell: a new era for preimplantation genetic diagnosisPRENATAL DIAGNOSIS, Issue 4 2007Serdar Coskun Abstract Preimplantation genetic diagnosis (PGD) is a technique used for determining the genetic status of a single cell biopsied from embryos or oocytes. Genetic analysis from a single cell is both rewarding and challenging, especially in PGD. The starting material is very limited and not replaceable, and the diagnosis has to be made in a very short time. Different whole genome amplification (WGA) techniques have been developed to specifically increase the DNA quantities originating from clinical samples with limited DNA contents. In this review, currently available WGA techniques are introduced and, among them, multiple displacement amplification (MDA) is discussed in detail. MDA generates abundant assay-ready DNA to perform broad panels of genetic assays through its ability to rapidly amplify genomes from single cells. The utilization of MDA for single-cell molecular analysis is expanding at a high rate, and MDA is expected to soon become an integral part of PGD. Copyright © 2007 John Wiley & Sons, Ltd. [source] Determination of the genetic status of cleavage-stage human embryos by microsatellite marker analysis following multiple displacement amplificationPRENATAL DIAGNOSIS, Issue 3 2007Pamela J. Renwick Abstract Objectives To analyse genotype information from cleavage-stage human embryos and assess the chromosomal status and feasibility of performing aneuploidy screening by microsatellite analysis. Methods DNA from 49 blastomeres from eight cleavage-stage human embryos was amplified using multiple displacement amplification, then tested for panels of 64 polymorphic microsatellite markers on seven different chromosomes, and for two non-polymorphic sequences on the X and Y chromosomes. Results There was an overall allele drop out (ADO) rate of 28%. Novel alleles in single cells were seen in 0.3% of amplifications, interpreted as either somatic microsatellite mutation events or ,slippage' of the MDA , 29 polymerase. Three-allele results for a single marker in a single cell were found in 0.07% of amplifications, interpreted as ,slippage' of the MDA , 29 polymerase. One apparent segmental duplication was found. Only one embryo with no normal cells was found, probably arising from the chaotic cleavage division following a triploid conception. Six embryos were mosaic, of which four had only one abnormal cell. Conclusions Abnormalities in human embryos may be present in only a single cell, leading to potentially false abnormal results at pre-implantation genetic diagnosis. ADO associated with MDA reduces the efficacy of this approach for detection of aneuploidy. Statistical analysis showed that, for ADO of 28%, seven informative markers would be required to give 95% confidence of detecting trisomic embryos. Copyright © 2007 John Wiley & Sons, Ltd. [source] Multiple genotyping in bovine pre-implantation embryos with whole genome amplificationANIMAL SCIENCE JOURNAL, Issue 5 2008Hiroki HIRAYAMA ABSTRACT This study examined genetic diagnosis using whole genome amplification (WGA) in bovine embryos. The first experiment was conducted to compare the WGA efficiency of primer extension preamplification-PCR (PEP-PCR) and multiple displacement amplification (MDA), and to optimize the DNA extraction method. The sensitivity of SRY -specific PCR from MDA products increased when DNA of fibroblasts was extracted by a NaOH treatment instead of the conventional method (heat treatment). The detectability of SRY from PEP-PCR products was lower than that in MDA regardless of the DNA extraction method (proteinase K or NaOH treatment). Sexing and genotyping were performed using MDA products from embryo biopsy. The accuracy of PCR-based and LAMP-based sexing was 100% regardless of the amounts of biopsy. Genotyping of CL16, BND3, SCD and F11 in MDA products from 10 to 20% of trophectoderm was successful 97, 97, 95 and 95% of the time, respectively, but reduced biopsy amount (<10% of trophectoderm) decreased the accuracies (33,83%). Microsatellite markers were analyzed using MDA products from 10 to 20% of trophectoderm. In eight out of 16 microsatellites, genotypes were not contradictory among the dam, sire and embryos. In the other eight microsatellites, the inconsistency rates were 17,83%. These results indicate that MDA is useful for multiple genetic diagnoses in bovine embryos. [source] | ||||||||||