Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.     

One step cloning of defined DNA fragments from large genomic clones

Recently, the nucleotide sequences of entire genomes became available. This information combined with older sequencing data discloses the exact chromosomal location of millions of nucleotide markers stored in the databases at NCBI, EMBO or DDBJ. Despite having resolved the intron/exon structures of all described genes within these genomes with a stroke of a pen, the sequencing data opens up other interesting possibilities. For example, the genomic mapping of the end sequences of the human, murine and rat BAC libraries generated at The Institute for Genomic Research (TIGR), reveals now the entire encompassed sequence of the inserts for more than a million of these clones. Since these clones are individually stored, they are now an invaluable source for experiments which depend on genomic DNA. Isolation of smaller fragments from such clones with standard methods is a time consuming process. We describe here a reliable one-step cloning technique to obtain a DNA fragment with a defined size and sequence from larger genomic clones in less than 48 hours using a standard vector with a multiple cloning site, and common restriction enzymes and equipment. The only prerequisites are the sequences of ends of the insert and of the underlying genome.     

一种用于构建红色红曲霉基因组Cosmid文库的大片段DNA制备方法

摘要：【目的】制备用于构建红色红曲霉cosmid文库的大片段基因组DNA。【方法】采用优化的酚氯仿抽提法制备DNA,并利用Sau3AI切割至平均大小为40 kb,然后使用Stratagene包装蛋白构建cosmid文库。基于PCR法使用同源探针从该文库中进行了目的基因的筛选。【结果】制备了浓度为5 μg/μL,平均片段大小大于48 kb的红色红曲霉大片段基因组DNA。利用该DNA构建的cosmid文库基因组覆盖倍数为10,并筛选到了含有目的片段的cosmid。【结论】通过该方法制备红色红曲霉大片段基因组D     

PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.     

放线菌基因组大片段克隆载体及异源表达宿主改造的研究进展

大规模基因组测序发现放线菌基因组内包含有极丰富的天然产物合成基因, 是非常有价值的资源。放线菌基因组中负责天然产物合成的基因通常成簇存在。要想完整地克隆这些较大的基因簇并且进行功能研究, 或者通过异源表达激活原本沉默的天然产物合成基因簇, 需要大容量的载体系统和合适的异源宿主。本文重点介绍了放线菌中常用于基因组大片段克隆的载体及异源表达宿主改造的研究进展。     

Two-dimensional electrophoretic display of restriction fragments from genomic DNA

We have developed a procedure for the resolution of restriction enzyme digests of mammalian genomic DNA in two dimensions. Fragments from a first digestion are separated on a column of purified agarose containing a second restriction enzyme in the absence of the divalent cation required for enzyme activity. After enzyme activation and digestion, the fragments are resolved on an agarose slab gel. We have digested rat genomic DNA and found in the ethidium-stained pattern a variety of features which have not been described previously.     

福尔马林固定铜鱼基因组DNA的提取与扩增

铜鱼（Coreius heterodon）作为长江中、上游的重要经济鱼类是研究三峡大坝阻隔效应的重要材料之一。然而,过去的铜鱼标本都保存在福尔马林溶液中,有必要探讨从福尔马林固定的铜鱼标本中有效提取基因组DNA的方法以及这些DNA用于微卫星和线粒体分析的可行性。本实验通过改进的酒精梯度浸泡法去除标本中的甲醛,然后用酚-氯仿抽提法成功地提取到了铜鱼标本的基因组DNA;设计引物后进行了线粒体和微卫星的PCR扩增,扩增产物经银染检测。微卫星扩增结果显示只有部分个体可以扩出目的带,而线粒体控制区部分区段在所有个体中均能稳定重复的扩出;mtDNA SSCP分析显示带型一致。结果表明,福尔马林固定的铜鱼标本可以被用来开展短片段的扩增和遗传变异分析等方面的相关研究。     

Kindred DNA amplification from two distinct populations of cDNA fragments

Kindred DNA amplification is a novel and cost-effective method developed to isolate common cDNA fragments between two distinct cDNA populations. Unlike subtractive hybridization, which discards common sequences, kindred DNA amplification isolates and amplifies these sequences within a single hybridization procedure. The utility of this method is demonstrated by cloning the genes in common between two different but metabolically homologous muscles, murine ventricular myocardium and soleus. The reliability of kindred DNA amplification was confirmed by Southern hybridization.     

A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50-to 100-kb genomic fragments for direct DNA sequencing

A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employed the yeast Flp/FRTelements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392–2398]. To extend the applicability of such a system to many species, we describe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep (TrfA) protein from the promiscuous RK2/RP4 plasmid.We have constructed insertion plasmids which carry the FRT and oriV sites. To introduce such plasmids into the appropriate position in the host genome, a short genomic sequence homologous to this position was cloned into the multiple cloning site (MCS) of the FRT/oriVinsertion plasmid and then recombined into this position in the genome by RecA-mediated recombination. In such a manner, many strains with single FRT/oriV insertions at various positions could be generated. Subsequent genetic crosses or phage transduction allow two neighboring FRT/oriVsites (less than 150 kb apart) to be brought into a single genome. In the present report, the lacZ and phoB sites, which are 51 kb apart in the E. coli genome, were used for the introduction of the FRT/oriV sites.To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the P_tet promoter/operator which is tightly regulated by the TetR repressor. The addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) excision of the 51-kb genomic segment between the two FRTsites (in lacZ and in phoB), and (ii) its amplification.     

Chemical regulated production of cDNAs from genomic DNA fragments in plants

We have developed a new chemical inducible genetic system that allows for the isolation of any cDNA molecule from in vitro generated genomic transgenes in transgenic plants. This system, termed regulated in vivo cDNA generation (RIDE), permits both targeted isolation of individual full-length cDNA molecules and random isolation of any partial or full-length cDNA from in planta genomic libraries. The RIDE system makes use of the 17-beta estradiol-inducible promoter system linked to intron donor and acceptor sites in a new binary vector configuration. In transgenic Arabidopsis and tobacco plants, we show that the RIDE system can isolate low-abundance full-length cDNAs previously unattainable by conventional means at high efficiencies (75-85%). The ability to randomly isolate individual exons and exons spliced together from genomic libraries in planta suggest that this system can be used for the isolation of any cDNA molecules. The RIDE system thus appears to be an efficient and versatile system for the generation of potentially any cDNA molecule. Moreover, the ORF structural data generated will be of value in both verifying and correcting computational ORF predications in the databases available to the scientific community.     

Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1

Especial conditions were developed for the amplification of five DNA segments from US region of BHV-1 by polymerase chain reaction. In order to eliminate most nonspecific products it was found that addition of three cosolvents DMSO, glycerol and NP 40 was a simple method for increasing the specificity of amplification.     

Extraction, amplification and characterization of wood DNA from dipterocarpaceae

A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations of wood tissue. Genotypes, the origin of sampled material, and species can be identified based on an investigation of wood if suitable information on genetic variation patterns within and among species is available. Potential applications are in forensics and in the control of the timber and wood trade. We extracted DNA from wood of Dipterocarpaceae, a family that dominates rainforests and comprises many important timber species in Southeast Asia. Several different DNA isolation techniques were compared and optimized for wood samples from natural populations and from wood processing enterprises. The quality of the DNA was tested by spectrophotometry, PCR amplification, and PCR inhibitor tests. An average DNA yield of 2.2 μg was obtained per 50–100 mg of dried wood sample. Chloroplast DNA (cpDNA) regions of different length were amenable to PCR amplification from the extracted DNA. Modification of DNA isolation techniques by the addition of polyvinylpyrrolidone (PVP) addition up to 3.1% into lysis buffer reduced PCR inhibition effectively. In order to evaluate the extraction method, we analyzed leaves and wood from the same tree by PCR amplification, genotyping and sequencing of chloroplast microsatellites.     

Direct cloning and transplanting of large DNA fragments from Escherichia coli chromosome

We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts: cma (which has a homologous sequence with cmb) and cmb, each of which has no resistance separately unless the two parts are fused together. The cmb sequence was integrated into one flank of a target cloning region in the chromosome, and a linear vector containing the cma sequence was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned an approximately 48 kb DNA fragment from the E. coli DH1-Z chromosome with a positive frequency of approximately 80%. Combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to successfully replace the corresponding region of the E. coli DH36 chromosome and knock out four non-essential genomic regions in one step. This strategy could provide a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways for genome assembly and for the functional study of DNA sequences dozens of kilobases in length.