Assessment of multiple displacement amplification in molecular epidemiology

Well-characterized epidemiological resources are generated with great effort, yet associated patient DNA samples can be limiting. The efficacy of the whole genome amplification (WGA) method, termed multiple displacement amplification (MDA), was assessed for detecting heterozygous sequence variants, mutation scanning, and PCR for challenging segments. Fifteen common polymorphisms from 10 genes located on 8 chromosomes were genotyped by direct sequencing of 300 PCR products from 115 high-quality MDA-amplified DNA samples extracted by different methods. The GC content of these analyzed segments ranges from 30% to 69%. Genotyping results demonstrate 100% accuracy. For heterozygotes, the relative intensity of peaks generated by the two alleles is highly similar for genomic and MDA-amplified genomic DNA, independent of GC content. In contrast, one of four heterozygous loci was mistyped when lower quality MDA-amplified DNA samples were used. The results of single-stranded conformation polymorphism (SSCP)-type of mutation scanningfor seven MDA-amplified DNA samples in four genes were concordant with the genomic DNA samples. PCR on MDA-amplified DNA was routinely successful for challenging 10- and 12-kb segments with GC content ranging from 30% to 80%, demonstrating that rather long segments, which are difficult to amplify with PCR, are amplified well with MDA. These results suggest that MDA is an effective method of WGA with utility in molecular epidemiology. Quality control of the MDA-amplified DNA is critical for high performance.     

Contig assembly and microsynteny analysis using a bacterial artificial chromosome library for Epichloë festucae, a mutualistic fungal endophyte of grasses

We constructed and characterized a bacterial artificial chromosome (BAC) library for Epichlo? festucae, a genetically tractable fungal plant mutualist. The 6144 clone library with an average insert size of 87kb represents at least 18-fold coverage of the 29 Mb genome. We used the library to assemble a 110kb contig spanning the putative ornithine decarboxylase (odc) ortholog and subsequently expanded it to 228kb with a single walking step in each direction. Furthermore, we evaluated conservation of microsynteny between E. festucae and some model filamentous fungi by comparing sequence available from a 43kb region at the end of one BAC to publicly available fungal genome sequences. Orthologs to the 13 contiguous open reading frames (ORFs) identified in E. festucae are syntenic in Neurospora crassa and Magnaporthe grisea occurring in small sets of two, three or four colinear ORFs. This library is a valuable resource for research into traits important for the development and maintenance of a plant-fungus mutualistic symbiosis.     

Whole genome amplification: abundant supplies of DNA from precious samples or clinical specimens

Whole genome amplification methods are used to generate the large amounts of DNA that are required for genetic testing. Preparation of genomic DNA from clinical samples is a bottleneck in high-throughput genotyping and is frequently limited by the amount of specimen available. Precious DNA collections used for association and linkage analysis can be a nonrenewable resource available to only a limited number of laboratories. To address these needs, amplified DNA is now being used in a growing number of research and diagnostic applications. A new method, called multiple displacement amplification, dramatically improves the high-fidelity reproduction of genomic DNA, with 10–100 kb amplified DNA products providing uniform coverage of genes.     

Use of arbitrarily primed polymerase chain reaction to differentiate Trichophyton dermatophytes

Abstract Dermatophytes such as Trichophyton species are common human pathogens, the infection of which results in dermatophytosis (also known as ringworm). Several laboratory tests are used routinely for the diagnosis of dermatophytosis, but they are either slow or lacking specificity. Through examination of genomic DNA from Trichophyton dermatophytes and other fungi in arbitrarily primed PCR, it was shown that a random primer 5'-ACCCGACCTG-3' produced bands of 4.3 kb, 1.9 kb, 1.7 kb and 0.7 kb in T. rubrum DNA, bands of 2.5 kb, 1.9 kb and 0.8 kb in T. mentagrophytes var. interdigitale and T. mentagrophytes var. mentagrophytes DNA, and bands of 2.5 kb, 1.9 kb, 1.5 kb and 0.9 kb in T. tonsurans DNA. This primer amplified bands of different sizes in other fungal DNA. Therefore, based on the distinct band patterns observed in arbitrarily primed PCR using this primer, T. rubrum , T. mentagrophytes and T. tonsurans dermatophytes could be rapidly differentiated.     

Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples

Analysis of genomic DNA derived from cells and fresh or fixed tissues often requires whole genome amplification prior to microarray screening. Technical hurdles to this process are the introduction of amplification bias and/or the inhibitory effects of formalin fixation on DNA amplification. Here we demonstrate a balanced-PCR procedure that allows unbiased amplification of genomic DNA from fresh or modestly degraded paraffin-embedded DNA samples. Following digestion and ligation of a target and a control genome with distinct linkers, the two are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. We demonstrate genome-wide retention of allelic differences following balanced-PCR amplification of DNA from breast cancer and normal human cells and genomic profiling by array-CGH (cDNA arrays, 100 kb resolution) and by real-time PCR (single gene resolution). Comparison of balanced-PCR with multiple displacement amplification (MDA) demonstrates equivalent performance between the two when intact genomic DNA is used. When DNA from paraffin-embedded samples is used, balanced PCR overcomes problems associated with modest DNA degradation and produces unbiased amplification whereas MDA does not. Balanced-PCR allows amplification and recovery of modestly degraded genomic DNA for subsequent retrospective analysis of human tumors with known outcomes.     

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.     

稻曲病菌UV-2菌株细菌人工染色体文库构建及分析

【目的】稻曲病(Rice false smut)是由稻曲病菌[Villosiclava virens (Cooke) Tak.]引起的严重危害水稻的真菌病害。构建稻曲病菌UV-2的大片段DNA细菌人工染色体(Bacterial artificial chromosome, BAC)文库, 为致病相关基因的鉴定及在图位克隆、比较基因组学等方面的研究奠定基础。【方法】以幼嫩菌丝为材料制备大分子基因组DNA包埋块, 用Hind III部分酶解后经脉冲凝胶电泳筛选, 回收大片段DNA并与pIndigoBAC536-S 载体连接, 连接产物转化大肠杆菌菌株DH10B T1 Phage-Resistant 细胞后进行蓝白斑筛选, 白色菌落捡入384孔板置于?80 °C低温保存。【结果】成功构建UV-2菌株的高质量、高覆盖度的BAC文库, 该文库共含10 368个克隆, 平均插入片段为124.4 kb, 空载率小于1%, 约覆盖该菌基因组的36.8倍。【结论】克服了真菌大分子基因组DNA制备难控制的技术难题, 建立了首个稻曲病菌的BAC文库。该文库已作为一种公共基因组资源向研究者开放(http://GResource.hzau.edu.cn)。     

Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones.

The 264 bp mini-transposon Tn5supF was constructed to sequence DNAs cloned in phage lambda without extensive shotgun subcloning or primer walking. Unique sequences near each transposon end serve as primer binding sites, and a supF gene is used to select transposition to lambda. We describe here PCR methods that facilitate Tn5supF-based sequencing. In a first pass, insertions are mapped relative to the ends of the cloned fragment using pairs of primers specific for vector DNA next to the cloning site and for a Tn5supF end. Most insertions not mapped in this step are near the center of the cloned fragment or in the vector arms, and are then mapped relative to the two innermost insertions by 'crossover' PCR. This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. We routinely amplified more than 6 kb in direct PCR and 3 kb in crossover PCR; at the limit we amplified up to approximately 10 kb in direct PCR and approximately 6 kb in crossover PCR, but not reproducibly. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates. PCR products were purified by adsorption and then elution from glass slurry, and sequenced directly. Ladders of more than 400 bp were obtained from primer sites on each DNA strand; 2 kb was read from crossover PCR products, and showed that they were amplified with fidelity. In conclusion, direct and crossover PCR methods expedite transposon insertion mapping, and yield templates for accurate sequencing of both DNA strands.     

Mechanism of chimera formation during the Multiple Displacement Amplification reaction

Background  

Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them.     

Multiple displacement amplification enables large-scale clonal analysis following retroviral gene therapy

Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material.     

RAPD分析氮离子注入甜菊种子后的幼苗基因组DNA变异

应用ＲＡＰＤ 技术检测经低能氮离子注入甜菊纯系种子引起的幼苗基因组ＤＮＡ 变异。筛选出ＯＰＪ系列中的１５ 种引物对实验及对照基因组ＤＮＡ 进行了ＰＣＲ 扩增,共获扩增片段１０３ 条,分子量在０．３ － ３ｋｂ 之间,其中５ 种引物ＯＰＪ－ １ ,７ ,９,１１ ,１２ 扩增出差异片段１２ 条。结果表明,低能氮离子注入甜菊种子可引起体内基因组ＤＮＡ 发生突变;ＲＡＰＤ 技术是检测基因组ＤＮＡ 发生诱变的一种简便、有效方法。本文同时探讨了离子强度和Ｔａｇ ＤＮＡ 聚合酶用量对甜菊ＲＡＰＤ 分析结果的影响,以及氮离子注入诱变效应的可能机制。     

Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands

Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methanotrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methylocystis spp. were further confirmed by DNA stable-isotope probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifugation, (13)C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from (12)C DNA and subjected to multiple displacement amplification (MDA) to generate sufficient DNA for the preparation of a fosmid metagenomic library. Potential bias of MDA was detected by fingerprint analysis of 16S rRNA using denaturing gradient gel electrophoresis for low-template amplification (0.01 ng template). Sufficient template (1-5 ng) was used in MDA to circumvent this bias and chimeric artefacts were minimized by using an enzymatic treatment of MDA-generated DNA with S1 nuclease and DNA polymerase I. Screening of the metagenomic library revealed one fosmid containing methanol dehydrogenase and two fosmids containing 16S rRNA genes from these Methylocystis-related species as well as one fosmid containing a 16S rRNA gene related to that of Methylocella/Methylocapsa. Sequencing of the 14 kb methanol dehydrogenase-containing fosmid allowed the assembly of a gene cluster encoding polypeptides involved in bacterial methanol utilization (mxaFJGIRSAC). This combination of DNA stable-isotope probing, MDA and metagenomics provided access to genomic information of a relatively large DNA fragment of these thus far uncultivated, predominant and active methanotrophs in peatland soil.     

Cloning and characteristics of DNA sequences amplified in Djungarian hamster cells with multidrug resistance

A number of DNA clones containing the amplified DNA sequences were isolated from the genomic library of multidrug-resistant (MDR) Djungarian hamster cells using the DNAC0t 10-250 hybridization probe. Five independent nonoverlapping clones were obtained that covered more than 100 kb of the amplified genomic region. These clones were used as hybridization probes in blot-hybridization with DNA from 7 independently derived MDR Djungarian hamster cell lines selected for the resistance to colchicine or actinomycin D. Some clones contained the DNA sequences amplified in all of the cell lines tested while the others contained the cell line specific amplified sequences. Hybridization in situ was used to localize the amplified DNA in metaphase chromosomes of a MDR cell line that contained about 140 copies of these sequences. The approximate size of an amplicon calculated on the basis of the obtained data is about 1-2 X 10(3) kb.     

Single-cell genomic sequencing using Multiple Displacement Amplification

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).     

Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics

The concentrations of one-carbon substrates that fuel methylotrophic microbial communities in the ocean are limited and the specialized guilds of bacteria that use these molecules may exist at low relative abundance. As a result, these organisms are difficult to identify and are often missed with existing cultivation and gene retrieval methods. Here, we demonstrate a novel proof of concept: using environmentally-relevant substrate concentrations in stable-isotope probing (SIP) incubations to yield sufficient DNA for large-insert metagenomic analysis through multiple displacement amplification (MDA). A marine surface-water sample was labelled sufficiently by incubation with near in situ concentrations of methanol. Picogram quantities of labelled (13)C-DNA were purified from caesium chloride gradients, amplified with MDA to produce microgram amounts of high-molecular-weight DNA ( 10 000 clones. Denaturing gradient gel electrophoresis (DGGE) demonstrated minimal bias associated with the MDA step and implicated Methylophaga-like phylotypes with the marine metabolism of methanol. Polymerase chain reaction screening of 1500 clones revealed a methanol dehydrogenase (MDH) containing insert and shotgun sequencing of this insert resulted in the assembly of a 9-kb fragment of DNA encoding a cluster of enzymes involved in MDH biosynthesis, regulation and assembly. This novel combination of methodology enables future structure-function studies of microbial communities to achieve the long-desired goal of identifying active microbial populations using in situ conditions and performing a directed metagenomic analysis for these ecologically relevant microorganisms.     

Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: the nature and frequency of errors caused by Taq DNA polymerase

Using the polymerase chain reaction (PCR) with Taq DNA polymerase, we have amplified a 2.4-kb fragment of genomic DNA containing the adenine phosphoribosyltransferase (APRT) gene from patients with APRT deficiency. Several clones from each patient were sequenced after subcloning the PCR product into M13mp18. Selected regions of the amplified fragment were also sequenced directly. This enabled us to distinguish PCR-induced errors from endogenous mutations and polymorphisms in each clone. 44 PCR errors were found in a total of 57,94 kb of DNA sequenced from 25 clones from 7 patients. All the errors were due to the PCR process and not to subcloning, as shown by sequence analysis of 5 APRT-positive clones isolated from a phage genomic library.     

DNA amplification in multidrug, cross-resistant Chinese hamster ovary cells: molecular characterization and cytogenetic localization of the amplified DNA

Vincristine-resistant (VCR) Chinese hamster ovary (CHO) cells have been established by stepwise selection in increasing concentrations of vincristine. These cells exhibit multidrug cross-resistance to a number of drugs that have no structural or functional similarities. Cytogenetic analyses of resistant cells revealed the presence of double minutes and expanded chromosomal segments, thus implicating gene amplification as a possible mechanism of resistance. An amplified DNA segment isolated from other multidrug cross-resistant CHO cell lines (Roninson, I. B., H. T. Abelson, D. E. Housman, N. Howell, and A. Varshavsky, 1984, Nature (Lond.), 309:626-628) is also amplified in our VCR lines. This DNA segment was used as a probe to screen a cosmid library of VCR genomic DNA, and overlapping clones were retrieved. All of these segments, totaling approximately 45 kilobases (kb), were amplified in VCR cells. Using in situ hybridization, we localized the amplification domain to the long arm of CHO chromosome 1 or Z1. Northern hybridization analysis revealed that a 4.3-kb mRNA was encoded by this amplified DNA domain and was over-produced in the VCR cells. Suggestions for the involvement of these amplified DNA segments in the acquisition of multidrug cross-resistance in animal cells are also presented.     

云南腾冲热泉土壤微生物基因组文库的构建与分析

采用冻融、蛋白酶K、SDS-高盐-加热处理法联合的方法,直接从云南腾冲地区的一个弱碱性高温热泉沉积样品中提取和分离环境混合基因组DNA,产量为每克样品1~2μg DNA,用Promega试剂盒纯化后进行PstⅠ部分酶切处理,电泳回收3~8kb的片段后,构建了pSK( )为载体的基因组文库,共获得25000个阳性克隆,平均插入片段长度为4.6kb。通过随机DNA序列测定和基因注释,发现外源插入片段含有未见报道的序列。