Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.     

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.     

Characterizing seamless ligation cloning extract for synthetic biological applications

Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular and synthetic biology projects.     

Construction of DNA Marker Plasmids Based on Taq Tailing Activity and Selective Recovery of Ligation Products

DNA fragments with standard molecular weights (DNA markers, which are usually commercial products) are routinely electrophoresed in conjunction with DNA samples in molecular biology labs to serve as references for DNA molecular weight; this is done by referencing their relative molecular weights. In this study, we present a new technical strategy for constructing super-plasmids for homemade DNA marker production with single restriction enzyme digestion. In this study, two super-plasmids for DNA marker production have been developed, based on tailing activity of Taq polymerase and selective recovery of ligation products following agarose gel electrophoresis.     

DNA克隆和组装技术研究进展

DNA克隆和组装技术是重要的分子生物学工具。近年来,随着合成生物学的飞速发展,对大片段DNA元件的快速有效组装就显得尤为关键。同时,各种DNA克隆和组装技术也竞相发展起来。通过对基于非典型酶切连接、PCR、同源重组、单链退火拼接等原理发展起来的各种DNA克隆和组装技术进行综述,为合成生物学的进一步发展提供有效的操作工具。     

Available methods for assembling expression cassettes for synthetic biology

Studies in the structural biology of the multicomponent protein complex, metabolic engineering, and synthetic biology frequently rely on the efficient over-expression of these subunits or enzymes in the same cell. As a first step, constructing the multiple expression cassettes will be a complicated and time-consuming job if the classic and conventional digestion and ligation based cloning method is used. Some more efficient methods have been developed, including (1) the employment of a multiple compatible plasmid expression system, (2) the rare-cutter-based design of vectors, (3) in vitro recombination (sequence and ligation independent cloning, the isothermally enzymatic assembly of DNA molecules in a single reaction), and (4) in vivo recombination using recombination-efficient yeast (in vivo assembly of overlapping fragments, reiterative recombination for the chromosome integration of foreign expression cassettes). In this review, we systematically introduce these available methods.     

Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries

High-throughput genomics, proteomics and synthetic biology studies require ever more efficient and economical strategies to clone complex DNA libraries or variants of biological modules. In this paper, we provide a protocol for a sequence-independent approach for cloning complex individual or combinatorial DNA libraries, and routine or high-throughput cloning of single or multiple DNA fragments. The strategy, called circular polymerase extension cloning (CPEC), is based on polymerase overlap extension and is therefore free of restriction digestion, ligation or single-stranded homologous recombination. CPEC is highly efficient, accurate and user friendly. Once the inserts and the linear vector have been prepared, the CPEC reaction can be completed in 10 min to 3 h, depending on the complexity of the gene libraries.     

一步法快速构建多片段连接的同源臂载体

目的：建立一种简便、高效,可一步完成多个片段连接,从而构建含同源臂的载体的方法。方法：按照酶切后可产生前后片段相匹配的粘性末端接头的原则设计PCR引物,在目的片段两端均引入BsaⅠ酶切位点。以G160基因为例,PCR扩增打靶用左右同源臂片段、示踪基因CMV-EGFP片段、载体骨架pMD19－T等4个片段,纯化后一起加入一个反应管中,并加入BsaⅠ限制性内切酶和T7DNA连接酶及相应缓冲液,进行酶切、酶连接共10～50个循环反应,一步构建含同源臂载体的质粒;产物经高温处理后,直接转化感受态细胞,并进行重组子PCR鉴定;对pMD19－T载体进行优化,突变载体上的BsaⅠ酶切位点,把示踪基因CMV-EGFP片段引入pHSG298－T载体,再选择不同的G160基因同源臂片段组合对构建系统进行验证。结果：重组质粒酶切和PCR结果表明,应用一步法可成功连接多个片段来构建含同源臂及示踪基因的克隆载体;用优化后的pMD19－T－O载体体系,在2d内即完成了6种各含4个片段的载体的构建。结论：多个基因片段一步无缝连接的方法简便、易行、可靠,不仅可快速构建某类载体系统,还可对基因进行精确的点突变,该系统可用于快速构建基因打靶载体。     

Tandem PCR: a novel and efficient unit amplification model for the preparation of small DNA fragments

The present DNA marker preparation with PCR amplification, one primer pair for one target DNA fragment, was very tedious and labor intensive. To develop a simple and efficient system for the preparation of small DNA fragments, a novel PCR amplification pattern was designed and tested, of which targeted small DNA fragments were amplified in groups as a unit with a specific synthetic vector as template DNA. The amplified units can be different dependent on the identities of the employed primers and give out variable combinations of small DNA fragments through complete or partial restrictive digestion with EcoRI. The novel pattern made the PCR amplification of small DNA fragments not only more efficient but also more economic than ever before. The tandem PCR pattern, as the most efficient and high throughput method for small DNA fragment preparation, has wide application for the production of various DNA markers and a good complementation to the larger DNA fragment preparation by complex synthetic vector fermentation.     

A primer-based approach to genome walking

A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.     

Rapid and efficient subcloning of DNA without dephosphorylation or gel electrophoresis

Conventional subcloning into plasmid vectors often involves dephosphorylation, gel electrophoresis, DNA extraction, and purification to isolate the target insert and the cleaved plasmid. This is not only time-consuming but very often problematic. We have developed strategies that can circumvent these steps by mixing digested donor and recipient plasmids together for ligation. These strategies capitalizes on: (1) the ability to enhance the ligation efficiency of desired DNA fragments into the target vector by the generation and removal of small (<50 bp) fragments from nontarget DNA using peripheral restriction sites and spin column technology and (2) the elimination of undesired ligation products after ligation by using the Lac Z gene, differences in antibiotic resistance among plasmid vectors, and unique restriction sites situated in nontarget DNA fragments.     

一个RNAi载体上反向重复片段的测序策略

相邻的反向重复DNA片段有形成单链内二级结构的倾向,属于一种测序困难的DNA模板。解决RNAi载体插入的反向重复片段的测序问题,为该类载体正确性的测序验证奠定基础。采用常规分子克隆方法构建表达小麦TaATG2串联反向重复片段的RNAi载体,设计2种策略对经菌落PCR初步鉴定的载体进行测序验证:一种是以完整的载体质粒为模板进行测序;另一种是先对载体进行酶切处理,切除反向重复片段中的一个后对保留另一个片段的线性载体进行测序。结果表明,第一种测序策略受到串联反向重复片段形成的单链内部二级结构的影响,测序信号在反向重复片段处出现衰减或乱峰,无法读取序列。第二种测序策略排除了2个反向重复片段之间的干扰,保留在载体上的片段测序信号清晰,序列准确。采用酶切切除一个片段后进行测序的方法,经过2次酶切和2次测序可以有效地对载体上的2个反向重复片段分别进行序列测定,进而确认构建载体的正确性。     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

耐热DNA连接酶介导的TLCR技术在简化DNA重组实验中的应用

传统的DNA重组方法Type Ⅱ型限制酶技术受到片段纯化的限制,无法做到复杂混合体系中DNA片段的特异性连接。为解决这个问题,本研究将耐热连接酶链式反应(Thermostable ligase chain reaction, TLCR)引入DNA片段的连接与捕获。该技术利用耐热型DNA连接酶的特性,在热循环反应中配合针对目的片段末端序列设计的单链寡核苷酸连接模板--Helper,实现目的片段的特异性连接和产物数量的指数性增长。两个质粒构建实验被用于验证TLCR技术的可行性和应用效果。首先利用TLCR技术从一个未经纯化的含有7种不同大小片段的混杂体系中特异性地将一段1.5 kb的片段捕获进载体,随机抽取的克隆样品经检验准确率达到80%,验证TLCR技术在复杂混合体系中特异性连接DNA片段的可行性和准确性。在另一个质粒构建实验中,TLCR技术从λ噬菌体基因组Hind消化物中将两段总长达27 kb的片段按顺序捕获进载体,随机抽取的克隆样品经检验同样达到了80%的准确率。结果表明,TLCR技术能够简化DNA重组实验的操作,并且适用于多片段和大片段的连接,可以为生物学研究提供便利。     

A rapid method for analyzing the ligation products of synthetic oligodeoxyribonucleotides

A method is described for the rapid analysis of DNA ligation products in the assembly of synthetic genes and gene fragments. The method is based on the simultaneous analysis of multiple ligation reactions where a single but different DNA oligomer is radiolabelled per ligation reaction. After each ligation the reaction mixture is electrophoresed on a denaturing, as well as a non-denaturing, polyacrylamide gel allowing one to monitor the ligation reaction products. In addition, a unique method for generating single stranded DNA sizing standards up to approximately 300 nucleotides in length is described.     

利用Red重组系统快速构建基因打靶载体

基因敲除小鼠模型是在哺乳动物体内研究基因功能最可靠的方法之一。利用常规的分子克隆的方法构建基因打靶载体往往工作周期长,对于难度特别大的基因有时甚至无法完成打靶载体的构建。通过合理应用Red重组系统和低拷贝中间载体,利用50bp的同源重组序列直接从BAC载体中克隆了长片段的小鼠基因组序列;将得到的基因组序列再次通过重组和改造,构建了Gpr56等基因的完全敲除并带有报告基因的打靶载体,实现了打靶载体的快速构建。     

Positive selection improves the efficiency of DNA assembly

With the advent of synthetic biology and cell engineering, the demand for large synthetic DNA fragments has been steadily increasing. Consequently, a number of multi-fragment cloning technologies optimized for the assembly of sizable DNA constructs have been developed. Still, screening for the right clone can be tedious because the high incidence of illegitimate assembly results in a relatively large proportion of missing or shuffled DNA elements. To mitigate this risk, we have developed a strategy that reduces the rate of fragment mis-assembly and is compatible with a variety of cloning methodologies. The approach is based on the positive selection of truncated plasmid markers, which are rendered active by providing their missing sequences during the assembly process. The method has been successfully validated in the context of complex in vivo and in vitro homologous recombination workflows, but it could be readily adapted to other cloning strategies, including those based on restriction endonucleases.     

Characterization of proteolytic fragments of bacteriophage T7 DNA ligase.

Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion. These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively. The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion. The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of [alpha-32P]ATP, confirming that it contains the active site lysine residue. In common with the intact enzyme, the C-terminal portion of the protein retains the ability to band shift DNA fragments of various lengths, implicating it in DNA binding. It can also inhibit ligation by the intact protein, apparently by competing for target sites on DNA. We conclude that the N-terminal region, which contains the putative active site lysine, plays a role in the transfer of AMP from the enzyme-adenylate complex to the 5'phosphate at the nick site, while the C-terminal 26 kDa fragment appears to position the enzyme at the target site on DNA.     

End-labeling of long DNA fragments with biotin and detection of DNA immobilized on magnetic beads

To immobilize DNA fragments onto magnetic beads coated with streptavidin for isolation purpose, it is important to label one biotin molecule at one terminus of DNA fragment. After failure to label long DNA with biotin by PCR and filling-in reaction, a 9.2 kb DNA was labeled with biotin by a modified ligation strategy. A simple method is also reported to detect the quantity and integrity of DNA immobilized on the magnetic beads.     

A normalization strategy applied to HiCEP (an AFLP-based expression profiling) analysis: Toward the strict alignment of valid fragments across electrophoretic patterns

Background  

Gene expression analysis based on comparison of electrophoretic patterns is strongly dependent on the accuracy of DNA fragment sizing. The current normalization strategy based on molecular weight markers has limited accuracy because marker peaks are often masked by intense peaks nearby. Cumulative errors in fragment lengths cause problems in the alignment of same-length fragments across different electropherograms, especially for small fragments (< 100 bp). For accurate comparison of electrophoretic patterns, further inspection and normalization of electrophoretic data after fragment sizing by conventional strategies is needed.