Direct sequencing of terminal regions of genomic P1 clones: A general strategy for the design of sequence-tagged site markers

A method for the preparation of P1 DNA is presented, which allows the direct sequencing of ends of inserts in genomic P1 clones using the Applied Biosystems 373A DNA Sequencer and the Dye Terminator sequencing methodology. We surveyed several common methods of DNA preparation including alkaline lysis, Triton-lysozyme lysis, CsCl density-gradient purification, and a commercial column matrix DNA purification kit manufactured by Qiagen. We found that a modified alkaline lysis preparation of P1 DNA was most successful for generating P1 DNA that could be sequenced directly. We also noted that the host bacterial strain from which the P1 DNA was purified dramatically affected the quality of sequencing templates. The bacterial strains NS3145 and NS3529, in which the Drosophila melanogaster and human P1 genomic libraries are harbored, routinely yielded poor-quality sequencing templates. However, the bacterial strain DH10B routinely yielded P1 DNA that was sequenced successfully. A bacterial mating scheme is presented that exploits γδ transposition events to allow the transfer of P1 clones from the library host strain to DH10B. Using either an SP6 or a T7 primer, an average of 350 base pairs of DNA sequence was obtained with an uncalled base frequency of ∼2%. About 4% of P1 end sequences generated corresponded to unique Drosophila loci present in the Genbank database. These single-pass DNA sequences were used to design sequence-tagged site markers for physical mapping studies in both humans and Drosophila.     

Analysis of DNA from a Beta procumbens chromosome fragment in sugar beet carrying a gene for nematode resistance

Summary We have begun to apply techniques for the preparation and anaylsis of large DNA segments from sugar beet (Beta vulgaris) addition lines carrying a mitotically stable chromosome fragment from B. procumbens that confers monogenic resistance to the nematode Heterodera schachtii, with a view towards isolating the resistance gene. DNA probes specific for this chromosome fragment were selected, and various methods for cloning genome-specific fragments, including probes from megabase DNA separated in pulsed-field slab gels, are compared. Probes that display high homology to B. procumbens have been used for hybridization of a representative genomic library and for initial step in mapping the chromosome fragment via pulsed-field gel electrophoresis after restriction with infrequently cutting enzymes. Our data indicate that DNA molecules from the entire chomosome fragment can be separated from protoplast DNA lysates.     

A rapid and efficient method for cloning genes of type II restriction-modification systems by use of a killer plasmid

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.     

A Rapid and Efficient Method for Cloning Genes of Type II Restriction-Modification Systems by Use of a Killer Plasmid

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.     

Statistical assessment of DNA extraction methodology for culture-independent analysis of microbial community associated with diverse environmental samples

Cost-effectiveness, quality, time-effectiveness and ease of the methodology are the most crucial factors in isolating quality DNA from wide variety of samples. Thus, research efforts focusing on the development of an efficient DNA extraction protocol is the need of the hour. The present study therefore, focuses on development of an efficient, rapid and free of inhibitory substances based methodology for extracting metagenomic DNA from diverse environmental samples viz. anaerobic biogas digesta, ruminant stomach, human feces, soil, and microbial starter cultures used for preparation of fermented food. PCR–DGGE based analysis and quality metagenomic library preparation, using DNA extraction methodology, validates the developed protocol. The developed protocol is cost effective, capable of isolating DNA from small sample size (100–1000 µl), time efficient (1.5–2.0 h protocol) and results in significantly higher DNA yield (4–8 times increased yield) when compared to previously available DNA extraction method and a commercial DNA extraction kit. The DNA extracted from the samples using different protocols was evaluated based on its ability to identify diverse microbial species using PCR–DGGE profiles targeting variable region within the 16S rRNA gene. The results of microbial community analysis revealed comparability of the developed protocol to commercial kits, in effectively identifying dominant representatives of the microbial community in different samples. Using the DNA extracted from the presented methodology, metagenomic libraries were prepared, which were found suitable for sequencing on Illumina platform.     

采用诱导环化酶制备Illumina Mate-paired DNA文库

新一代测序技术(NGS)的文库制备方法在基因组的拼装中起着重要作用。但是NGS技术制备的普通DNA文库片段只有500 bp左右,难以满足复杂基因组的从头(de novo)拼装要求。三代测序技术的读长可以达到20 kb,但是其高错误率及测序成本过高使得其又不易推广。因此二代测序的Mate-paired文库制备技术一直在基因组的de novo拼装中扮演着非常重要的角色。目前主流的NGS平台Illumina制备的Mate-paired文库的片段范围只有2~5 kb,为了得到更长的可用于Illumina平台测序的Mate-paired文库,本研究首次整合并优化了Illumina和Roche/454两种测序平台的Mate-paired文库制备技术,采用诱导环化酶来提高基因组长片段DNA的环化效率,成功建立了20 kb Mate-paired文库制备技术,并已将该技术应用于人类基因组20 kb Mate-paired文库制备。该技术为Illumina平台制备长片段Mate-paired库提供了方法指导。     

Sequencing historical specimens: successful preparation of small specimens with low amounts of degraded DNA

Despite advances that allow DNA sequencing of old museum specimens, sequencing small‐bodied, historical specimens can be challenging and unreliable as many contain only small amounts of fragmented DNA. Dependable methods to sequence such specimens are especially critical if the specimens are unique. We attempt to sequence small‐bodied (3–6 mm) historical specimens (including nomenclatural types) of beetles that have been housed, dried, in museums for 58–159 years, and for which few or no suitable replacement specimens exist. To better understand ideal approaches of sample preparation and produce preparation guidelines, we compared different library preparation protocols using low amounts of input DNA (1–10 ng). We also explored low‐cost optimizations designed to improve library preparation efficiency and sequencing success of historical specimens with minimal DNA, such as enzymatic repair of DNA. We report successful sample preparation and sequencing for all historical specimens despite our low‐input DNA approach. We provide a list of guidelines related to DNA repair, bead handling, reducing adapter dimers and library amplification. We present these guidelines to facilitate more economical use of valuable DNA and enable more consistent results in projects that aim to sequence challenging, irreplaceable historical specimens.     

利用SMART方法快速克隆异黄酮代谢途径多基因的研究

采用改进的酸酚法提取高质量的大豆叶片RNA,利用SMART思想和方法构建大豆叶片全长cDNA文库,直接以一级库液稀释液为模版进行PCR,快速克隆得到异黄酮代谢途径相关的5个基因。与传统的从DNA、RNA出发克隆基因,以及构建文库再进行基因筛选的克隆方法相比,该方法得到的基因均为全长基因,适用于快速、简便的进行多基因全长克隆。     

Differences in the probability of cloning specific DNA between primary and amplified libraries: theoretical considerations.

We present a theoretical study of the probability of isolating a particular clone from a DNA library. There are differences in this probability between primary and amplified libraries even if the desired clone represents the same fraction of both libraries. As the result, we must screen severalfold more phage or bacteria in an amplified library than in a primary library.     

Optimization of the genotyping‐by‐sequencing strategy for population genomic analysis in conifers

Flexibility and low cost make genotyping‐by‐sequencing (GBS) an ideal tool for population genomic studies of nonmodel species. However, to utilize the potential of the method fully, many parameters affecting library quality and single nucleotide polymorphism (SNP) discovery require optimization, especially for conifer genomes with a high repetitive DNA content. In this study, we explored strategies for effective GBS analysis in pine species. We constructed GBS libraries using HpaII, PstI and EcoRI‐MseI digestions with different multiplexing levels and examined the effect of restriction enzymes on library complexity and the impact of sequencing depth and size selection of restriction fragments on sequence coverage bias. We tested and compared UNEAK, Stacks and GATK pipelines for the GBS data, and then developed a reference‐free SNP calling strategy for haploid pine genomes. Our GBS procedure proved to be effective in SNP discovery, producing 7000–11 000 and 14 751 SNPs within and among three pine species, respectively, from a PstI library. This investigation provides guidance for the design and analysis of GBS experiments, particularly for organisms for which genomic information is lacking.     

An Arrayed Bacteriophage P1 Genomic Library of Pneumocystis carinii

We have constructed an arrayed, large insert, multiple coverage genomic library of Pneumocystis carinii DNA using the bacteriophage P1 cloning system. The library consists of ∽4800 independent clones with an average insert size of ∽55 kbp individually arrayed in 50 microtiter plates, and is readily screened on ten or fewer microtiter plate-sized filters using a high density colony replicating device. Screening of the library for unique P. carinii sequences detected an average of 4–5 positive clones for each, consistent with a several-fold coverage of the ∽10-mbp P. carinii genome. Restriction and hybridization analyses demonstrated that the P1 clones in this library are quite stable and contain few, if any, chimeric inserts. Thus, this arrayed, large insert library off. carinii genomic DNA will be a valuable tool in the future genetic dissection of this important pathogen.     

Target-specific PCR primers can detect and differentiate ophiostomatoid fungi from microbial communities associated with the mountain pine beetle Dendroctonus ponderosae

The aim of this study was to develop DNA probes that could identify the major fungal species associated with mountain pine beetles (MPB). The beetles are closely associated with fungal species that include ophiostomatoid fungi that can be difficult to differentiate morphologically. The most frequently isolated associates are the pine pathogens Grosmannia clavigera and Leptographium longiclavatum, the less pathogenic Ophiostoma montium, and an undescribed Ceratocystiopsis species (Cop. sp.). Because growing, isolating and extracting DNA from fungi vectored by MPB can be time and labour intensive, we designed three rDNA primer sets that specifically amplify short rDNA amplicons from O. montium, Cop. sp. and the pine Leptographium clade. We also designed two primer sets on a gene of unknown function that can differentiate G. clavigera and L. longiclavatum. We tested the primers on 76 fungal isolates that included MPB associates. The primers reliably identified their targets from DNA obtained from pure fungal cultures, pulverized beetles, beetle galleries, and tree phloem inoculated with G. clavigera. The primers will facilitate large-scale work on the ecology of the MPB-fungal-lodgepole pine ecosystem, as well as phytosanitary/quarantine sample screening.     

Characterization of a pine multigene family containing elicitor- responsive stilbene synthase genes

Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial -glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.     

Construction and extensive characterization of a goat Bacterial Artificial Chromosome library with threefold genome coverage

A goat Bacterial Artificial Chromosome (BAC) library of 61,440 independent clones was constructed and characterized. The average size of the inserts was estimated at 153 kilobases by analyzing almost 500 clones using Not1 digestion followed by FIGE (Field Inverted Gel Electrophoresis) analysis. The library represents about three genome equivalents, which yields a theoretical probability of 0.95 of isolating a particular DNA sequence. After individual growth, the clones were arrayed in 40 superpools, which were organized in three dimension pools. A rapid technique for pool DNA preparation by microwave treatment was set up. This technique was compatible with PCR analysis. Primer pairs from 166 sequences (microsatellites, coding sequences from goat, and conserved Expressed Sequence Tags (ESTs) from humans) enabled the library to be successfully searched in 165 cases, with an average of 3.52 positive superpools. Only one sequence could not be found. The degree of chimerism was evaluated by FISH analysis with DNA from over 110 clones and was estimated at 4%. This BAC library will constitute an invaluable tool for positional cloning in ruminants, as well as for more general comparative mapping studies in mammals. Received: 2 July 1997 / Accepted: 13 September 1997     

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)。     

一种新的线粒体基因组DNA捕获探针的制备及初步应用

目的:建立新的线粒体基因组DNA杂交捕获探针制备方法并用进行初步应用。方法:通过PCR技术扩增特异线粒体DNA片段,并与生物素偶联,最后与标记磁珠的亲和素混合获得捕获探针。并自行制备的线粒体基因组DNA文库捕获探针与肝癌全基因组测序文库进行液相杂交。分离捕获产物后PCR扩增并进行测序分析。结果:成功建立了线粒体基因组杂交捕获探针制备方法并成功分离线粒体基因组DNA;对测序数据的分析显示:90%以上测序数据来自线粒体基因组DNA,且覆盖率达到100%,且均一性良好。检测到的同质性变异位点数量和异质性变异位点数量与全基因组测序数据产生的结果接近(P=0.9152,P=0.8409)。结论:新方法制备的线粒体基因组DNA杂交捕获探针可以从全基因组文库中高效捕获线粒体基因组DNA测序文库。     

Generation of a P1 artificial chromosome library of the Southern pufferfish

We describe the generation of a P1 artificial chromosome genomic library from the Southern pufferfish, Spheroides nephelus. The arrayed library consists of approximately 30,000 clones and has an average insert size of 125-150 kb. The coverage is estimated to encompass seven to eight genome equivalents. The library has been used for isolating numerous genomic clones and for establishing contigs of several multigene families. Analysis of several of the clones from this library suggests a preponderance of CA repeat tracts relative to their abundance in humans. The library and high-density filters have been made available to the scientific public through genomics distribution companies.