A Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics

With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.     

Cloning single-stranded DNA

DNA sequence and expression analyses have greatly benefited from using M13 and pUC derived cloning vectors and their polycloning sites. A chronology of the original concepts and experiments is reviewed.     

通路克隆系统:DNA重组技术的新进展

近几年新发展了一种载体间DNA片段相互灵活转化的多功能系统 ,即通路克隆系统(gatewaycloningsystem)。它是一种位点特异的DNA重组技术 ,包括PCR产物的定向克隆 ,DNA片断高效、广泛的亚克隆 ,氨基或羧基末端的融合蛋白表达等。重点阐述了该系统的作用原理、特点及其应用。     

DNA片段的高效克隆

用Taq酶进行PCR扩增时,其PCR产物的3末端有一个附加的A碱基。因此,目前在克隆PCR产物时一般使用T－VCctor。但T—Vector的价格比较昂贵,而使用本试剂盒也可在短时间内使PCR产物与平滑末端载体进行高效连接。PCR产物与平滑末端的载体连接时,有必要除去3廉端的附加碱基,并使其5末端磷酸化。使用TaKaRaBKLKit（BllllltillgKillstiollLigstiollKit）可使这一连串的反应在短时间内完成。PCR产物的末端平滑化与磷酸化反应在一个反应体系内同时进行,一次反应后便可得到能够用于连接的DNA片段。用于连接反应的PCR产物无需进…     

Cloning of single-stranded synthetic DNA

The method for cloning a single-stranded synthetic DNA with the short complementary oligonucleotides, that form corresponding restriction sites, is proposed. The potency of the method is demonstrated by cloning a single-stranded polynucleotide A (93 nucleotide residues (n. r.] in plasmid vector pBR327. The polynucleotide A includes a leader structure of the human fibroblast interferon gene. Oligonucleotides (IV) (20 n. r.) and (VI) (16 n. r.) were taken as strengthening complements and to create the sticky ends for the restrictases HindIII and EcoRI. 72% of the obtained clones appeared to be hybrid. Four hybrid clones were analyzed, and three of them carried the desirable insertion. The primary structures of these insertions are confirmed by sequencing.     

Cloning of epoxide hydratase complementary DNA

Tightly membrane-bound polysomes were isolated from livers of rats administered trans-stilbene oxide. Epoxide hydratase mRNA was enriched from these polysomes using immunochemical techniques and oligo(dT)-cellulose chromatography. This resulted in an increase in message concentration over that found in noninduced membrane-bound cDNA, synthesized from enriched mRNA, was inserted into the ampicillin resistance gene of pBR322 using oligo(dG)-oligo(dC) tailing. Clones containing sequences complementary to epoxide hydratase mRNA were selected by differential colony hybridization using [32P]cDNA synthesized from immunoenriched mRNA and [32P]cDNA synthesized from nonenriched mRNA. Plasmids from four clones, which only annealed with the enriched probe, were isolated and shown to specifically hybridize with epoxide hydratase mRNA by hybrid selection-translation. A composite restriction endonuclease map of the plasmid inserts was constructed which spanned 1310 base pairs and represented approximately 80% of the message sequence. The 3'-5' orientation of this map relative to the epoxide hydratase mRNA was also determined.     

Cloning of a mammalian DNA methyltransferase

Cloning and sequencing of cDNA clones has shown that mammalian DNA (cytosine-5)-methyltransferase comprises a 1000-amino acid (aa) N-terminal region of unknown function and a 570-aa C-terminal region that is clearly related to bacterial type-II cytosine restriction methyltransferases. These findings indicate that the mammalian enzyme contains at least two structural domains and suggest a common evolutionary origin for mammalian and prokaryotic DNA (cytosine-5)-methyltransferases.     

Cloning and characterization ofDaphnia mitochondrial DNA

Summary The mitochondrial genome ofDaphnia pulex (Crustacea, Cladocera) was cloned as a single fragment into the plasmid vector pUC12. The genome size, estimated from restriction endonuclease fragment lengths, is 15,400±200 base pairs. The GC content, estimated from thermal denaturation studies, is 42%. The positions of 39 cleavage sites were mapped for 14 restriction enzymes. The distribution of these sites within the genome is random (P=0.44). Heterologous hybridizations withDrosophila sylvestris mitochondrial DNA (mtDNA) probes indicate that gene orders withinDaphnia andDrosophila mtDNAs are similar.     

Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair.

Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, a umu-like function of S. typhimurium has been found; the phenotype of this function is weaker than that of its E. coli counterpart, which is consistent with the weak mutagenic response of S. typhimurium to UV compared with the response in E. coli.     

Cloning multiple copies of a DNA segment

A method for self-ligation of DNA segments, which is based on the rotational non-equivalence of ends produced by AvaI cleavage, has been developed. Using this method and an initiator molecule to encourage the formation of long polymers, we have obtained a plasmid containing 34 repeats of a 123-bp rat DNA segment. All of the repeats are in the same orientation, and the plasmid is quite stable. It should be possible to polymerize any DNA segment by this method. Potential uses of the procedure include production of large amounts of small, homogeneous DNAs for physical studies such as X-ray crystallography, and increasing the expression of cloned genes in bacteria.     

快速克隆DNA大片段的方法

文章采用菌落PCR的技术,快速扩增发根农杆菌Ri质粒rol（包括rolA、rolB、rolC）基因的DNA片段并用于测序分析。克服了由于Ri质粒过大,提取困难等问题。     

一种新的DNA分子克隆方法

DNA分子克隆是基本的分子生物学实验技术,传统的分子克隆方法大多需经过酶切链接过程,但在某些情况下,没有合适的酶切位点往往会成为阻碍克隆进行的障碍.本文描述了一种新的分子克隆方法,称为不依赖酶切和链接的分子克隆（RLIC）.利用RLIC,将3种不同大小的DNA片段克隆到3种不同载体,证明了这种方法的有效性和可靠性.由于该方法不受限制性酶切序列限制,省去了酶切连接步骤,因此具有很大的灵活性和简便性,在分子生物学研究方面有广泛应用前景.     

Nuclease-Assisted Suppression of Human DNA Background in Sepsis

Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C₀t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C₀t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.     

DNA克隆载体的发展和应用

ＤＮＡ克隆载体的发展和应用刘建喜林爱星丁翔陈永福（中国农业大学农业生物技术国家重点实验室,北京１０００９４）ＴｈｅＤｅｖｅｌｏｐｍｅｎｔａｎｄＡｐｐｌｉｃａｔｉｏｎｏｆＤＮＡＣｌｏｎｉｎｇＶｅｃｔｏｒｓＬｉｕＪｉａｎｘｉＬｉｎＡｉｘｉｎＤｉｎｇＸｉａ...