A PCR-after-ligation method for cloning of multiple DNA inserts

Here we present a novel and simple PCR-after-ligation method for efficient assembly of multiple DNA inserts. After initial ligation of multiple inserts and vector, the ligation mixture is used as template for a PCR using a pair of primers flanking the cloning sites on the vector. The fragment with correct size is gel purified and inserted into the vector by conventional two-way ligation. With this method, a recombinant plasmid containing four DNA inserts was correctly constructed. As a control, all of the constructs obtained directly from DNA ligation were found to be self-ligation of the vector.     

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。     

Ligase-free subcloning: a versatile method to subclone polymerase chain reaction (PCR) products in a single day.

Often, it is convenient to subclone polymerase chain reaction (PCR) products into a plasmid vector for subsequent replication in bacteria, but conventional subcloning methods often fail. We report a rapid and versatile method to subclone PCR products directionally into a specific site of virtually any plasmid vector. The procedure requires only four primers, does not require DNA ligase, and may be accomplished in a single day. Ligase-free subcloning is performed by incorporating into the PCR primers sequences at the 5' ends that result in PCR products whose 3' ends are complementary to the 3' ends of the recipient linearized plasmid. The PCR product and the linearized plasmid are spliced together in a second PCR reaction in which Taq polymerase extends the complementary overlapping 3' ends (ligation by overlap extension). Denaturation followed by heterologous reannealing and cyclization results in a cyclic recombinant plasmid with two nicks that may be used directly to transform competent Escherichia coli. In our hands, ligase-free subcloning is rapid, and offers many advantages over existing strategies.     

Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning.

A simple and rapid method for cloning of amplification products directly from the polymerase chain reaction (PCR) has been developed. The method is based on the addition of a 12-base dUMP-containing sequence (CUACUACUACUA) to the 5' end of PCR primers. Incorporation of these primers during PCR results in the selective placement of dUMP residues into the 5' end of amplification products. Selective degradation of the dUMP residues in the PCR products with uracil DNA glycosylase (UDG) disrupts base pairing at the termini and generates 3' overhangs. Annealing of 3' protruding termini to vector DNA containing complementary 3' ends results in chimeric molecules which can be transformed, with high efficiency, without in vitro ligation. Directional cloning of PCR products has also been accomplished by incorporating different dU-containing sequences at the end of each PCR primer. Substitution of all dT residues in PCR primers with dU eliminates cloning of aberrant "primer dimer" products and enriches cloning of genuine PCR products. The method has been applied to cloning of inter-Alu DNA sequences from human placental DNA. Using a single primer, DNA sequences between appropriately oriented Alu sequences were amplified and cloned. Cloning of cDNA for the glyceraldehyde-3'-phosphate dehydrogenase gene from rat brain RNA was also demonstrated. The 3' end region of this gene was amplified by the 3' RACE method and the amplified DNA was cloned after UDG digestion. Characterization of cloned DNAs by sequence analysis showed accurate repair of the cloning junctions. The ligase-free cloning method with UDG should prove to be a widely applicable procedure for rapid cloning of PCR-amplified DNA.     

T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends

Dozens of PCR-based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation-mediated PCR and randomly primed PCR. However, none of them has been generally applied for this purpose, because they are either difficult or inefficient. Here we describe a simple and efficient PCR strategy—T-linker-specific ligation PCR (T-linker PCR) for gene or chromosome walking. The strategy amplifies the template molecules in three steps. First, genomic DNA is digested with 3′ overhang enzymes. Secondly, primed by a specific primer, a strand of the target molecule is replicated by Taq DNA polymerase and a single A tail is generated on the 3′ unknown end of the target molecule, and then a 3′ overhang-T linker (named T-linker) is specifically ligated onto the target. Thirdly, the target is amplified by two rounds of nested PCR with specific primers and T-linker primers. T-linker PCR significantly improves the existing PCR methods for walking because it uses specific T/A ligation instead of arbitrary ligation or random annealing. To show the feasibility and efficiency of T-linker PCR, we have exploited this method to identify vector DNA or T-DNA insertions in transgenic plants.     

Ligation-independent cloning of PCR products (LIC-PCR).

A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.     

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。     

应用RD-PCR技术制备基因芯片靶基因片段

应用RD－PCR技术分离SH－SY5Y细胞的基因片段。从正常培养的SH－SY5Y细胞中提取总RNA,经oligo(dT）纤维素柱纯化分离出mRNA,然后以oligo(dT18)为锚定引物反转录生成单链cDNA,再以此为模板合成DNA的第二条链;将双链DNA经Sau3AI酶切之后,接上接头,经通用引物和选择性引物进行扩增;然后与载体pMD18-T相连,克隆鉴定、筛选、测序。所分离的cDNA片段经过扩增后用于制备基因芯片的靶基因,杂交检测的结果表明,此种方法所分离的基因片段可以用于基因芯片的靶基因片段,所制备的芯片将为进一步研究神经细胞基因表达提供了条件。     

RF cloning: a restriction-free method for inserting target genes into plasmids

Restriction-free (RF) cloning provides a simple, universal method to precisely insert a DNA fragment into any desired location within a circular plasmid, independent of restriction sites, ligation, or alterations in either the vector or the gene of interest. The technique uses a PCR fragment encoding a gene of interest as a pair of primers in a linear amplification reaction around a circular plasmid. In contrast to QuickChange site-directed mutagenesis, which introduces single mutations or small insertions/deletions, RF cloning inserts complete genes without the introduction of unwanted extra residues. The absence of any alterations to the protein as well as the simplicity of both the primer design and the procedure itself makes it suitable for high-throughput expression and ideal for structural genomics.     

基于重叠延伸PCR法的定点突变技术

目的:建立一种高效而经济的定点突变方法。方法:采用重叠延伸PCR定点突变技术,引物设计时引入目的突变,以前两次PCR产物为模板,进行第三次PCR,即可获得突变后的目的DNA片段。将此片段连入pMDTM18-T载体后测序验证突变结果。结果:DNA测序表明,待突变位点已由ATTGG突变为ATTTT。结论:成功实现了目的位点的定点突变,重叠延伸PCR法是一种高效且经济的定点突变方法。     

Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR)

A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5′ ends of PCR product strands contain built-in amplification primers, abasic sites within the primers result in the formation of 5′ single-stranded overhangs at the ends of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This “autosticky PCR” (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5′ overhang.     

PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae.

We developed a novel method for synthesizing marker-disrupted alleles of yeast genes. The first step is PCR amplification of two sequences located upstream and downstream of the reading frame to be disrupted. Due to the addition of non-specific single A overhangs by Taq DNA polymerase, each PCR product can be ligated with a marker DNA which has T residues at its 3' ends. After amplification of individual ligation products through the second PCR, both products are mixed and annealed, and the single strand is converted to a double strand by an extension reaction. The final step is PCR amplification of the fragment composed of a selectable marker and two flanking sequences with the outermost primers. This method is rapid and needs only short oligonucleotides as primers.     

家蚕核型多角体病毒囊膜蛋白GP64的原核表达

通过Primer Premier 5.0设计1对特异性引物,用于扩增家蚕核型多角体病毒囊膜蛋白GP64的部分DNA片段,对PCR扩增得到的DNA片段进行纯化,并对纯化后的双酶切DNA片段与经同样双酶切后的原核表达载体pET28a进行连接;对捕获有重组质粒pET28a-GP64的大肠埃希菌BL21（DE3）细胞进行IPTG诱导,通过SDS-PAGE对诱导产物进行电泳分析,结果表明扩增的目的片段获得了表达;通过His单抗对诱导产物进行Western Blot印迹分析,其结果表明诱导蛋白带为融合有组氨酸的目的蛋白。对原核表达的GP64截短蛋白和免疫佐剂进行充分研磨,将充分研磨后的匀浆液对昆明小鼠进行皮下多点注射,以制备的GP64多抗对家蚕核型多角体病毒粒子感染的BmN细胞总蛋白进行Western Blot印迹分析,结果在杂交膜上出现一条特异杂交带、其分子量大小约为64 ku,表明制备的GP64多抗可用于其功能的进一步研究。     

Restriction site-free insertion of PCR products directionally into vectors

A restriction site-free cloning method has been developed for inserting a PCR product into a vector flexibly and precisely at any desired location with high efficiency. The method uses a pair of DNA integration primers with two portions. The 3' portion isolates the inserts by PCR, and the 5' portion integrates the PCR products into the homologous region of the vector. For mutagenesis, a third portion of mutation-generating sequences can be placed in between the 3' and 5' portions. This method has been used to clone the E. coli gene that codes for peptidyl-tRNA hydrolase, expressing it as a native protein and as a glutathione S-transferase fusion protein. It was also applied to convert a construct of the E. coli fatty acid biosynthesis protein with an N-terminal hexa-histidine tag into a construct with a C-terminal hexa-histidine tag.     

Use of PCR primers containing a 3'-terminal ribose residue to prevent cross-contamination of amplified sequences.

Cross-contamination with previously amplified products poses a serious limitation in the use of PCR for clinical testing and in certain research applications as well. In the present study we report the use of novel primers containing a 3'-terminal ribose residue to circumvent this problem. Extension of the primer by Taq DNA polymerase generates a cleavable ribonucleotide linkage within the amplified product. Cleavage of the primer by base or with a ribonuclease interferes with further replication of the product should carry over to another sample occur. Primers terminating in any of the 4 ribose residues function equally well as all DNA primers. Taq DNA polymerase is thus able to both efficiently extend and copy the single ribose residue. In translating from all DNA primers to ones containing a 3'-ribose residue no modification of the PCR protocol is required. The products formed can be used in all applications of the PCR. Since neither the original sample DNA, the primers or the extension products are modified by base or ribonuclease treatment both pre- and post-amplification sterilization can be carried out. Pre-amplification treatment with RNase A can yield as high as 10(4)-fold sterilization. Under these conditions the addition of beta-mercaptoethanol or other sulfhydryl reducing agent is necessary to inactivate the enzyme during thermocycling. Post-amplification treatment with NaOH readily yields at least 10(6)-fold sterilization. This alone is sufficient for most, if not all, applications of PCR. It is especially useful for quantitative RT-PCR, since the original target RNA sequence, which may be present in high copy numbers, is also destroyed.     

Universal TA cloning

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs. The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs. The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species. This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency. The universal TA cloning method is thus both convenient and labor-saving.     

PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors

We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5′- and 3′-regions of a target gene. Of the four primers used in amplification of the 5′- and 3′-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5′- or 3′-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5′ and 3′ PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.     

A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.

A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification of intermediate products. This method which we have named one-step overlap extension PCR (OOE-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector.     

Novel Strategies to Construct Complex Synthetic Vectors to Produce DNA Molecular Weight Standards

DNA molecular weight standards (DNA markers, nucleic acid ladders) are commonly used in molecular biology laboratories as references to estimate the size of various DNA samples in electrophoresis process. One method of DNA marker production is digestion of synthetic vectors harboring multiple DNA fragments of known sizes by restriction enzymes. In this article, we described three novel strategies—sequential DNA fragment ligation, screening of ligation products by polymerase chain reaction (PCR) with end primers, and “small fragment accumulation”—for constructing complex synthetic vectors and minimizing the mass differences between DNA fragments produced from restrictive digestion of synthetic vectors. The strategy could be applied to construct various complex synthetic vectors to produce any type of low-range DNA markers, usually available commercially. In addition, the strategy is useful for single-step ligation of multiple DNA fragments for construction of complex synthetic vectors and other applications in molecular biology field. Zhe Chen and Jianbing Wu contributed to this work equally.