Multiple Displacement Amplification for Generating an Unlimited Source of DNA for Genotyping in Nonhuman Primate Species

We evaluated a whole genome amplification method—multiple displacement amplification (MDA)—as a means to conserve valuable nonhuman primate samples. We tested 148 samples from a variety of species and sample sources, including blood, tissue, cell-lines, plucked hair and noninvasively collected semen. To evaluate genotyping success and accuracy of MDA, we used routine genotyping methods, including short tandem repeat (STR) analysis, denaturing gradient gel electrophoresis (DGGE), Alu repeat analysis, direct sequencing, and nucleotide detection by tag-array minisequencing. We compared genotyping results from MDA products to genotypes generated from the original (non-MD amplified) DNA samples. All genotyping methods showed good results with the MDA products as a DNA template, and for some samples MDA improved genotyping success. We show that the MDA procedure has the potential to provide a long-lasting source of DNA for genetic studies, which would be highly valuable for the primate research field, in which genetic resources are limited and for other species in which similar sampling constraints apply.     

Whole-metagenome amplification of a microbial community associated with scleractinian coral by multiple displacement amplification using phi29 polymerase

Limitations in obtaining sufficient specimens and difficulties in extracting high quality DNA from environmental samples have impeded understanding of the structure of microbial communities. In this study, multiple displacement amplification (MDA) using phi29 polymerase was applied to overcome these hindrances. Optimization of the reaction conditions for amplification of the bacterial genome and evaluation of the MDA product were performed using cyanobacterium Synechocystis sp. strain PCC6803. An 8-h MDA reaction yielded a sufficient quantity of DNA from an initial amount of 0.4 ng, which is equivalent to approximately 10(5) cells. Uniform amplification of genes randomly selected from the cyanobacterial genome was confirmed by real-time polymerase chain reaction. The metagenome from bacteria associated with scleractinian corals was used for whole-genome amplification using phi29 polymerase to analyse the microbial diversity. Unidentified bacteria with less than 93% identity to the closest 16S rDNA sequences deposited in DNA Data Bank of Japan were predominantly detected from the coral-associated bacterial community before and after the MDA procedures. Sequencing analysis indicated that alpha-Proteobacteria was the dominant group in Pocillopora damicornis. This study demonstrates that MDA techniques are efficient for genome wide investigation to understand the actual microbial diversity in limited bacterial samples.     

Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 10⁵ droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.     

Multiple displacement amplification for malaria parasite DNA

Multiple displacement amplification (MDA) using Phi29 has proved to be an efficient, high-fidelity method for whole genome amplification in many organisms. This project was designed to evaluate this approach for use with the malaria parasite Plasmodium falciparum. In particular, we were concerned that the AT richness and presence of contaminating human DNA could limit efficiency of MDA in this system. We amplified 60 DNA samples using phi29 and scored 14 microsatellites, 9 single-nucleotide polymorphisms (SNPs), and gene copy number at GTP-cyclohydrolase I both before and after MDA. We observed 100% concordance in 829 microsatellite genotypes and in 499 SNP genotypes. Furthermore, copy number estimates for the GTP-cyclohydrolase I gene were correlated (r(2) = 0.67) in pre- and postamplification samples. These data confirm that MDA permits scoring of a range of different types of polymorphisms in P. falciparum malaria and can be used to extend the life of valuable DNA stocks.     

SNP-based chromosomal copy number ascertainment following multiple displacement whole-genome amplification

Whole genome amplification by multiple displacement amplification (MDA) offers investigators using precious genomic DNA samples a high fidelity method for amplifying nanogram quantities of DNA several thousandfold. This becomes especially important for the modemrn day genomics researcher who more and more commonly is applying today's genome scanning technologies to patient cohort samples collected years ago that are irrecoverable and invariably in short supply. We present evidence here that MDA-prepared genomic DNA includes artifacts of chromosomal copy number that resemble copy number polymorphisms (CNPs) upon analysis of the DNA on the Affymetrix 10K GeneChip. The study of CNPs in both health and disease is a rapidly growing area of research, however our current understanding of the relevance of CNPs is incomplete. Our data indicate that utilization of whole genome-amplified samples for analysis heavily reliant on accurate copy number retention could be confounded if the genomic DNA sample was subjected to MDA. We recommend that small amounts of patient cohort DNA stocks be set aside and not subjected to whole genome amplification in order to facilitate the unbiased determination of chromosomal copy numbers when desired.     

Documenting ancient DNA quality via alpha satellite amplification and assessment of clone sequence diversity

C/G-->T/A nucleotide alterations have been shown to hamper the straightforward interpretation of mitochondrial DNA sequence data derived from ancient tissues. Attempting to characterise this finding with respect to nuclear DNA, we contrasted two established protocols: (i) an enzymatic repair of damaged DNA, thereby translating and closing nicks in the DNA, and (ii) the application of N-phenacylthiazolium bromide, which cleaves glucose-derived protein crosslinks, presumably derived from Maillard reactions. We used medieval human bones that were refractory to standard PCR procedures. Due to negligible presence of short tandem repeat loci and also mitochondrial sequences, the extracted ancient DNA needed a higher copy PCR system to yield amplification products. The chosen PCR target was specific alphoid repetitive DNA with an experimentally determined minimum of 1000 copies per haploid genome. Alphoid repeat segments were generated from both contemporary DNA and DNA extracts of two human skeletons dating from 450-600 AD (omitting uracil N-glycosylase pre-treatment of the extracted samples), and were subsequently cloned and sequenced. The sequences were evaluated for the number and type of nucleotide alterations noted after the different pre-treatments, and were compared to our alphoid consensus sequence generated from modern DNA. Both methods failed to reflect the expected 32% variability among single alphoid repeats (accounting for locus-specific differences and polymerase errors) as well as to display the actual 2.88 ratio of transitions to transversions. Our data obtained from high-copy-number nuclear DNA mirror the phenomenon of sequence deviations observed in mitochondrial DNA extracted from old specimens.     

Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice

Summary The entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare) was identified using clone banks that cover the chloroplast and mitochondrial genomes. The mitochondrial fragments that were homologous to chloroplast DNA were mapped and sequenced. The nucleotide sequences around the termini of integrated chloroplast sequences in the rice mtDNA revealed no common sequences or structures that might enhance the transfer of DNA. Sixteen chloroplast sequences, ranging from 32 bases to 6.8 kb in length, were found to be dispersed throughout the rice mitochondrial genome. The total length of these sequences is equal to approximately 6% (22 kb) of the rice mitochondrial genome and to 19% of the chloroplast genome. The transfer of segments of chloroplast DNA seems to have occurred at different times, both before and after the divergence of rice and maize. The mitochondrial genome appears to have been rearranged after the transfer of chloroplast sequences as a result of recombination at these sequences. The rice mitochondrial DNA contains nine intact tRNA genes and three tRNA pseudogenes derived from the chloroplast genome.     

Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments

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 phi 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 >or= 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.     

Brugia malayi: whole genome amplification for genomic characterization of filarial parasites

Genetic characterization of field isolates and clinical specimens of filarial nematodes is often limited by a shortage of DNA; therefore, we evaluated a multiple displacement amplification (MDA) based whole genome amplification method. The quality of amplified DNA was examined by conventional PCR, real-time PCR, and DNA hybridization. MDA of 5.0 ng of adult Brugia malayi DNA and one-fifteenth of the DNA isolated from a single microfilaria resulted in 6.3 and 4.2 μg of amplified DNA, respectively. Amplified DNA was equivalent to native genomic DNA for hybridization to B. malayi BAC library clones or to an oligonucleotide microarray with approximately 18,000 filarial DNA sequences. MDA is useful for whole genome amplification of filarial DNA from very small amounts of starting material. This technology will permit detailed studies of genetic diversity that were not previously feasible.     

Environmental whole-genome amplification to access microbial populations in contaminated sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. The reported SSU rRNA sequences and library clone end sequences are listed with their respective GenBank accession numbers, DQ 404590 to DQ 404652, DQ 404654 to DQ 404938, and DX 385314 to DX 389173.     

Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using 29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and “clusters of orthologous groups” (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.     

Adaptor-mediated amplification of minute amounts of severely fragmented ancient nucleic acids

It has been repeatedly shown that high copy number mitochondrial DNA sequences can be recovered from ancient samples. A significant increase in the volume of information available to researchers will be observed when the amplification of nuclear DNA becomes commonplace and reproducible. To this end we established a modification of the Rapid Amplification of cDNA Ends (RACE) procedure normally used for the generation of cDNA ends from adaptor-ligated expressed sequence tag libraries. The modifications were designed to specifically address the problems associated with the highly damaged nucleic acids extracted from palaeontological specimens. For this study we used 6 human samples dating to 450 AD and approximately 6.500 BP that were refractory to reliable amplification of single copy loci by PCR. Racemate contents (ratio of D/L enantiomers) of aspartic acid, alanine, and leucine also indicated that no amplifiable DNA is present in 5 of the 6 samples. The proposed technique allowed us (i) to amplify four X-chromosomal loci from 5 human specimens, and (ii) to correct allelic drop-out phenomena at the amelogenin locus in one individual; thus showing that the threshold of 80 x 10-3 for D/Lasp as a borderline for the presence/absence of amplifiable aDNA requires reassessment. Reliability of the proposed technique (i.e. amplification of DNA sequences endogenous to the find) was validated by the application of "ancient RACE" (aRACE) to prehistoric animal samples.     

Amplification of uncultured single-stranded DNA viruses from rice paddy soil

Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with phi29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.     

Automated sequencing of complete mitochondrial genomes from laser-capture microdissected samples

Mitochondrial DNA mutations have been related to both aging and a variety of diseases such as cancer. Due to the relatively small size of the genome (16 kb) and with the use of automated DNA sequencing, the entire genome can be sequenced from clinical specimens in days. We present a reliable approach to complete mitochondrial genome sequencing from laser-capture microdissected human clinical cancer specimens that overcome the inherent limitations of relatively small tissue samples and partial DNA degradation, which are unavoidable when laser-capture microdissection is used to attain pure populations of cells from heterogeneous tissues obtained from surgical procedures. The acquisition of sufficient template combined with a standard set of 18 pairs of PCR primers allows for the efficient amplification of the genome. Subsequent single-stranded amplification is performed using 36 sequencing primers, and samples are run on an ABI PRISM 3100 Genetic Analyzer. The use of this procedure should allow even investigators with little experience sequencing from clinical specimens success in complete mitochondrial genome sequencing.     

降香黄檀的DNA条形码鉴定研究

DNA条形码技术是利用基因组中一段短的标准序列进行物种的鉴定并探索其亲缘进化关系。本研究对采自海南不同地区降香黄檀五个居群24份样品的psbA-trnH,rbcL,核ITS及ITS2序列进行PCR扩增和测序,比较各序列扩增和测序效率。种间和种内变异,采用BLAST1和邻接 (NJ) 法构建系统聚类树方法评价不同序列的鉴定能力。结果表明ITS2在所研究的材料中具有最高的扩增和测序效率,而ITS扩增效率较低。ITS2完整序列在区分黄檀属不同种间差异具有较大优势。因此可利用ITS2从分子水平区分降香黄檀与其他混伪种。     

Comparative analysis of single-cell genome sequencing techniques toward the characterization of germline and somatic genomes in ciliated protists

Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates during sexual processes, and the extent of rearrangements varies dramatically among species, which lead to significant differences in genomic architectures. However, genomic sequences remain largely unknown for most ciliates due to the difficulty in culturing and in separating the germline from the somatic genome in a single cell. Single-cell whole genome amplification (WGA) has emerged as a powerful technology to characterize the genomic heterogeneity at the single-cell level. In this study, we compared two single-cell WGA, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in characterizing the germline and somatic genomes in ciliates with different genomic architectures. Our results showed that: 1) MALBAC exhibits strong amplification bias towards MAC genome while MDA shows bias towards MIC genome of ciliates with extensively fragmented MAC genome; 2) both MDA and MALBAC could amplify MAC genome more efficiently in ciliates with moderately fragmented MAC genome. Moreover, we found that more sample replicates could help to obtain more genomic data. Our work provides a reference for selecting the appropriate method to characterize germline and somatic genomes of ciliates.     

Assessment of MDA efficiency for genotyping using cloned embryo biopsies

The possibility to genotype embryos prior to implantation would have advantages for increasing the speed of selection of cattle. Reliable genotyping requires more DNA than can be obtained from biopsies of embryos, if they are to remain viable. Multiple displacement amplification (MDA) is a whole genome amplification technique used to increase the amount of DNA from biopsies for analysis. Reduced genome coverage resulting in Allele Drop Out (ADO) at heterozygous loci or missing genotypes are drawbacks of MDA.The present article describes the correlation between the input DNA quantity or embryo biopsy size and MDA success. Missing genotypes and ADO drastically increased when fewer than 30–40 cells or the genomic equivalents were used. However, embryo viability was found to be reduced if biopsied with more than 10 cells. Therefore, in vitro cell culture was investigated as a means to increase the number of cells available and the genotyping reliability.     

Evaluation of multiple displacement amplification in a 5 cM STR genome-wide scan

Multiple displacement amplification (MDA) has emerged as a promising new method of whole genome amplification (WGA) with the potential to generate virtually unlimited genome-equivalent DNA from only a small amount of seed DNA. To date, genome-wide high marker density assessments of MDA–DNA have focussed mainly upon suitability for single nucleotide polymorphism (SNP) genotyping applications. Suitability for short tandem repeat (STR) genotyping has not been investigated in great detail, despite their inherent instability during DNA replication, and the obvious challenge that this presents to WGA techniques. Here, we aimed to assess the applicability of MDA in STR genotyping by conducting a genome-wide scan of 768 STR markers for MDAs of 15 high quality genomic DNAs. We found that MDA genotyping call and accuracy rates were only marginally lower than for genomic DNA. Pooling of three replicate MDAs resulted in a small increase in both call rate and genotyping accuracy. We identified 34 STRs (4.4% of total markers) of which five essentially failed with MDA samples, and 29 of which showed elevated genotyping failures/discrepancies in the MDAs. We emphasise the importance of DNA and MDA quality checks, and the use of appropriate controls to identify problematic STR markers.     

Propylene glycol: a promising preservative for insects,comparable to ethanol,from trapping to DNA analysis

Recent advances in DNA analysis allow us to identify an unprecedented number of insect samples collected by mass sampling techniques such as insect traps. In these circumstances, a preservative that can be applied from trap to storage is necessary to prevent degradation of DNA before analysis and save on the cost of labor for collecting insects from traps. Propylene glycol has a prominent feature as a trap solution. We aimed to examine the DNA preservability of 98% propylene glycol at 2 weeks and more than 6 months after initial collection in comparison with 99.5% ethanol, which is commonly used for storage of specimens for genetic analysis. We compared amplification performance of PCR targeting a specific region of the mitochondrial cytochrome c oxidase subunit I (COI) gene in the orders Hymenoptera, Diptera, and Coleoptera using two extraction methods varying in extraction efficiency. Even after 6 months, more than 75% of samples were recognized to have succeeded in PCR amplification irrespective of preservatives by the extraction method with higher extraction efficiency. It suggested that mitochondrial DNA was preserved in both solutions. However, dim bands in the electrophoreses of PCR products increased with time in extracts by another method with lower extraction efficiency. In Diptera and Coleoptera, the rate of dim bands increased more rapidly for ethanol-preserved than for propylene glycol-preserved specimens, indicating higher DNA preservability of propylene glycol over time for these taxa. On the other hand, in Hymenoptera, the preservatives did not affect PCR amplification performance. Considering its safer characteristics and high DNA preservability in a wide range of taxa, propylene glycol can be a promising solution from trapping of insects to storage for genetic analysis.     

Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification

Major efforts are underway to systematically define the somatic and germline genetic variations causally associated with disease. Genome-wide genetic analysis of actual clinical samples is, however, limited by the paucity of genomic DNA available. Here we have tested the fidelity and genome representation of phi29 polymerase-based genome amplification (phi29MDA) using direct sequencing and high density oligonucleotide arrays probing >10,000 SNP alleles. Genome representation was comprehensive and estimated to be 99.82% complete, although six regions encompassing a maximum of 5.62 Mb failed to amplify. There was no degradation in the accuracy of SNP genotyping and, in direct sequencing experiments sampling 500,000 bp, the estimated error rate (9.5 x 10(-6)) was the same as in paired unamplified samples. The detection of cancer-associated loss of heterozygosity and copy number changes, including homozygous deletion and gene amplification, were similarly robust. These results suggest that phi29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.