A novel PCR strategy for high-efficiency, automated site-directed mutagenesis

We have developed a novel three-primer, one-step PCR-based method for site-directed mutagenesis. This method takes advantage of the fact that template plasmid DNA cannot be efficiently denatured at its reannealing temperature (T_ra), which is otherwise a troublesome problem in regular PCR. Two flanking primers and one mutagenic primer with different melting temperatures (T_m) are used together in a single PCR tube continuously without any intervention. A single-stranded mutagenic DNA (smDNA) is synthesized utilizing the high T_m mutagenic primer at a high annealing temperature, which prevents the priming of the low T_m primers (i.e. the two flanking primers). A megaprimer is then produced using this smDNA as the template at a denaturing temperature that prevents wild-type template DNA activity. The desired mutant DNA is then obtained by cycling again through these first two steps, resulting in a mutagenic efficiency of 100% in all tested cases. This highly automated method not only eliminates the necessity of any intermediate manipulation and accomplishes the mutagenesis process in a single round of PCR but, most notably, enables complete success of mutagenesis. This novel method is also both cost and time efficient and fully automated.     

Rapid high-efficiency site-directed mutagenesis by the phosphorothioate approach.

Several improvements to the existing phosphorothioate-based site-directed mutagenesis methodology are reported, and here it is demonstrated that the new procedure is able to produce large deletions, insertions and point mutations rapidly and with very high efficiency. The time required for the polymerization step has been reduced by using T7 DNA polymerase to extend the mutant oligonucleotide primer-template. The reaction produces good yields of double-stranded closed-circular DNA and some partially polymerized template. The reaction was treated with T5 D15 exonuclease to selectively destroy partially polymerized single-stranded phage DNA that may otherwise contribute to an increased background of wild-type transformants. The use of these enzymes greatly facilitates the implementation of the phosphorothioate-based site-directed mutagenesis method by requiring less template DNA and by allowing all the in vitro manipulations to be completed in a day. In its present form the method may easily be automated, enabling large systematic site-directed mutagenesis projects to be undertaken.     

Enzymatic inverse PCR: a restriction site independent, single-fragment method for high-efficiency, site-directed mutagenesis.

A new method is described for rapid site-directed mutagenesis of plasmid DNA. The new method, termed enzymatic inverse polymerase chain reaction (EIPCR), uses inverse PCR to amplify the entire plasmid. The key step to EIPCR is the incorporation of identical class 2s restriction sites in both primers. Class 2s restriction enzymes have a recognition site that is located 5' of the cut site (e.g., BsaI: GGTCTCN'NNNN,). Thus, after completing PCR, the ends of the full-length linearized plasmid are digested with the class 2s enzyme incorporated into the primers. The enzyme cuts off its entire recognition site and leaves the plasmid with compatible overhangs on both ends. Thus, in the ligation the only part that becomes part of the plasmid is the NNNN overhang, which can be made to be the native sequence. We have used the method for many plasmids and several class 2s enzymes. As an example, we report here the use of EIPCR for an insertion into pUC19 containing an inactive lacZ alpha-peptide, causing a frameshift that restores lacZ alpha-activity. Of 300 colonies evaluated, greater than 95% had the expected blue phenotype. The BsaI overhangs were correctly combined in all of the 35 blue colonies analyzed by restriction digestion and in all four clones that were sequenced. EIPCR is compared with four related PCR-based mutagenesis techniques. The major advantage of EIPCR over the other methods is the combination of greater than 95% correctly mutated clones with the need for only two PCR primers.     

Engineering the pH-optimum of activity of the GH12 family endoglucanase by site-directed mutagenesis

Endo-1,4-β-glucanase from Penicillium verruculosum (PvEGIII) belongs to family 12 of glycoside hydrolases (GH12). Analysis of the enzyme 3D model structure showed that the amino acid residue Asp98 may directly affect the pH-profile of enzyme activity since it is located at the distance of hydrogen bond formation from Glu203 that plays the role of a general acid in catalysis. The gene encoding the PvEGIII was cloned into Escherichia coli. After the deletion of two introns, a plasmid construction was obtained allowing the PvEGIII expression in E. coli. Using site-directed mutagenesis, the Asp98Asn mutant of the PvEGIII was obtained. Both the wild type and mutant PvEGIIIs were expressed in E. coli with a yield of up to 1 g/L and then isolated in a highly purified form. The enzyme specific activity against soluble carboxymethylcellulose was not changed after a single amino acid substitution. However, the pH-optimum of activity of the mutant PvEGIII was shifted from pH 4.0 to 5.1, compared to the wild type enzyme. The shift in the enzyme pH-optimum to more neutral pH was also observed on insoluble cellulose, in the process of enzymatic depigmentation of denim fabric. Similar situation featuring the effect of the Asp/Asn residue, located near the Glu catalytic residue, on the enzyme activity pH-profile has previously been described for xylanases of the GH11 family. Thus, the glycoside hydrolases belonging to the GH11 and GH12 families function by a rather similar mechanism of catalysis.     

Multiplex PCR for rapid differentiation of three species in the "Clostridium clostridioforme group"

Clostridium clostridioforme is a relatively antimicrobial resistant, phenotypically heterogeneous anaerobe that has been involved in a variety of infections. 16S rDNA sequencing analysis revealed three principal species in what has been called Clostridium clostridioforme - Clostridium bolteae, C. clostridioforme, and Clostridium hathewayi. Based on the 16S rDNA sequence information we obtained, we developed a cost-effective, timesaving one-step multiplex PCR assay for rapid and accurate differentiation of these three species. The established multiplex PCR identification scheme was applied to the identification of 88 clinical isolates that had previously been identified phenotypically as C. clostridioforme. The identification obtained from multiplex PCR assays showed 100% agreement with 16S rDNA sequencing identification. This scheme will permit more accurate assessment of the role of these three Clostridium species in infection and of the degree of antimicrobial resistance in each of the species.     

利用微卫星多重PCR技术鉴定剑尾鱼RR-B系

目的建立多重PCR技术鉴定选育剑尾鱼RR-B系的方法。方法根据可明显鉴定剑尾鱼RR-B系的6对特异性微卫星引物Msa012、Msa014、Msa033、Msb025、Msd003、Msd051,采用不同的组合方式对RR-B系剑尾鱼和红眼红体的非选育剑尾鱼进行多重PCR扩增。结果引物组合1(Msa014、Msb025、Msd003)和组合2(Msa012、Msa033、Msd051)两个三重PCR反应体系能清楚的扩增各个微卫星座位,且各目的片段之间无干扰,易于识别,引物组合1的排除概率为99.98%,引物组合2的排除概率为99.96%,能准确鉴定剑尾鱼RR-B系。结论本检测方法具有较高的稳定性,可快速准确鉴定剑尾鱼RR-B系。     

小型猪微卫星标记多重PCR体系的建立与应用

目的建立实验用小型猪微卫星标记的多重PCR体系和进行实验猪群的遗传监测。方法利用3种不同荧光标记的微卫星引物结合ABI3700遗传分析仪测序的方法,通过筛选和优化反应条件,建立可用于实验用小型猪遗传质量控制的稳定的多重PCR反应体系。在此基础上进一步检测实验用小型猪近交群体的遗传变异以验证建立体系的效率。结果筛选出了2组理想的组合：组合1包括SW742、S0228和S0218座位,复性温度58℃和56℃;组合2包括S0155、SW902和S0227三个座位,复性温度为60℃和58℃。组合内不同座位标记不同的荧光染料。还以此检测了实验用小型猪群体中的遗传变异。结论初步建立了中国三种实验用小型猪微卫星标记检测的多重PCR体系,为快速、大通量、准确的小型猪遗传监测提供了初步的技术基础。     

Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis

To develop a simple, speedy, economical and widely applicable method for multiple-site mutagenesis, we have substantially modified the Quik-Change™ Site-Directed Mutagenesis Kit protocol (Stratagene, La Jolla, CA). Our new protocol consists of (i) a PCR reaction using an in vitro technique, LDA (ligation-during-amplification), (ii) a DpnI treatment to digest parental DNA and to make megaprimers and (iii) a synthesis of double-stranded plasmid DNA for bacterial transformation. While the Quik Change™ Kit protocol introduces mutations at a single site, requiring two complementary mutagenic oligonucleotides, our new protocol requires only one mutagenic oligonucleotide for a mutation site, and can introduce mutations in a plasmid at multiple sites simultaneously. A targeting efficiency >70% was consistently achieved for multiple-site mutagenesis. Furthermore, the new protocol allows random mutagenesis with degenerative primers, because it does not use two complementary primers. Our mutagenesis strategy was successfully used to alter the fluorescence properties of green fluorescent protein (GFP), creating a new-color GFP mutant, cyan-green fluorescent protein (CGFP). An eminent feature of CGFP is its remarkable stability in a wide pH range (pH 4–12). The use of CGFP would allow us to monitor protein localization quantitatively in acidic organelles in secretory pathways.     

Multiplex PCR as a tool for validating plasmid content of Borrelia burgdorferi

Borrelia burgdorferi has an unusual genomic structure containing 21 plasmids. These plasmids carry genes that are essential for infectivity and survival of the spirochetes in vivo. Several plasmids are lost during cultivation in vitro, which might lead to a heterogeneous population after multiple passages and loss of infectivity in laboratory animals. Herein, we present a simple and inexpensive multiplex PCR method that detects the complete plasmid profile of B. burgdorferi B31 in just two PCR tubes.     

The paclitaxel site in tubulin probed by site-directed mutagenesis of Saccharomyces cerevisiae beta-tubulin

Previously, we created a paclitaxel-sensitive strain of Saccharomyces cerevisiae by mutating five amino acid residues in beta-tubulin in a strain that has a decreased level of the ABC multidrug transporters. We have used site-directed mutagenesis to examine the relative importance of the five residues in determining sensitivity of this strain to paclitaxel. We found that the change at position 19 from K (brain beta-tubulin) to A (yeast beta-tubulin) and at position 227 from H (brain beta-tubulin) to N (yeast beta-tubulin) had no effect on the activity of paclitaxel. On the other hand, the changes V23T, D26G and F270Y, drastically reduced sensitivity of AD1-8-tax to paclitaxel. Molecular modeling and computational studies were used to explain the results.     

Characterization of inhibitor binding to human kinesin spindle protein by site-directed mutagenesis

A number of inhibitors of kinesin spindle protein (KSP) have been described, which are known from X-ray crystallography studies to bind to an induced fit pocket defined by the L5 loop. We describe the characterization of eight mutant forms of KSP in which six residues that line this pocket have been altered. Mutants were analyzed by measuring rates of enzyme catalysis, in the presence and absence of six KSP inhibitors of four diverse structural classes and of varied ATP-competition status. Our analysis was in agreement with the model of binding established by the structural studies and suggests that binding energy is well distributed across functional groups in these molecules. The majority of the mutants retained significant enzymatic activity while diminishing inhibitor binding, indicating potential for the development of drug resistance. These data provide detailed information on interactions between inhibitor and binding pocket at the functional group level and enable the development of novel KSP inhibitors.     

Exposing a hidden functional site of C-reactive protein by site-directed mutagenesis

C-reactive protein (CRP) is a cyclic pentameric protein whose major binding specificity, at physiological pH, is for substances bearing exposed phosphocholine moieties. Another pentameric form of CRP, which exists at acidic pH, displays binding activity for oxidized LDL (ox-LDL). The ox-LDL-binding site in CRP, which is hidden at physiological pH, is exposed by acidic pH-induced structural changes in pentameric CRP. The aim of this study was to expose the hidden ox-LDL-binding site of CRP by site-directed mutagenesis and to generate a CRP mutant that can bind to ox-LDL without the requirement of acidic pH. Mutation of Glu(42), an amino acid that participates in intersubunit interactions in the CRP pentamer and is buried, to Gln resulted in a CRP mutant (E42Q) that showed significant binding activity for ox-LDL at physiological pH. For maximal binding to ox-LDL, E42Q CRP required a pH much less acidic than that required by wild-type CRP. At any given pH, E42Q CRP was more efficient than wild-type CRP in binding to ox-LDL. Like wild-type CRP, E42Q CRP remained pentameric at acidic pH. Also, E42Q CRP was more efficient than wild-type CRP in binding to several other deposited, conformationally altered proteins. The E42Q CRP mutant provides a tool to investigate the functions of CRP in defined animal models of inflammatory diseases including atherosclerosis because wild-type CRP requires acidic pH to bind to deposited, conformationally altered proteins, including ox-LDL, and available animal models may not have sufficient acidosis or other possible modifiers of the pentameric structure of CRP at the sites of inflammation.     

沙门菌、大肠杆菌和金黄色葡萄球菌的多重PCR检测

根据沙门菌invA基因、大肠杆菌phoA基因和金黄色葡萄球菌nuc基因序列,设计3对特异性引物进行多重PCR并对反应条件进行优化。结果表明3对引物能特异地扩增出284bp、622bp、484bp的目的条带;最佳反应条件为沙门菌、大肠杆菌、金黄色葡萄球菌的引物浓度分别为40nmol／L、40nmol／L、80nmol／L,Mg^2＋浓度2．4mmol／L,dNTP浓度2001μmol／L,Taq DNA聚合酶1.5u,退火温度55．0℃-57．4℃之间;在此条件下多重PCR同时检测DNA的敏感性分别是10．2pg、10．2pg、102．0pg,检测时间4h。建立的多重PCR是一种敏感、特异、准确、快速的方法,为同时检测食品中沙门菌、大肠杆菌和金黄色葡萄球菌奠定了基础。     

Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes

Site-directed mutagenesis followed by functional characterization is a widely used approach to obtain information on the structure-function relationship of proteins. Due to time and cost considerations, the number of amino acids studied is frequently reduced. To address the need for convenient parallel production of numerous point mutants of a protein, we developed an automated method to perform classical site-directed mutagenesis, protein purification, and characterization in a high-throughput manner. The process consists of a succession of six fully automated protocols that can be adapted to any automated liquid handling systems. Our procedure allows construction, validation, and characterization of hundreds of site-directed mutants of a given protein in just 4 days. The method is especially adapted to projects aiming at the study of unique or multiple mutants without the need to construct and screen large libraries of random mutants. The usefulness of the technique is illustrated by the construction and characterization of tens of single mutants of the penicillin-binding protein 2x (PBP2x) from Streptococcus pneumoniae. Moreover, seven mutations of PBP2x were obtained simultaneously in a single experiment with efficiency close to 90%.     

Multiplex genotyping of PCR products with MassTag-labeled primers.

A simple mass spectrometric based assay, the PinPoint assay, has previously been described for typing single nucleotide polymorphisms. The identity of a polymorphism is determined by mass differences of single base extended genotyping primers as determined by MALDI-TOF mass spectrometry. A simple method for multiplexing the assay is described, employing multiple primers with 5'oligo(dT) sequences (MassTags) which serve to mass discriminate the peaks of multiple extended and non-extended primers. The assay is extremely rapid and requires no labeling reagents.     

含非竞争性内标四重荧光RT-PCR方法检测手足口肠道病毒方法的研究

旨在了解手足口病的流行和感染情况,并进行快速准确的检测。建立了含非竞争性内标的同时检测肠道病毒通用型、肠道病毒EV71型及柯萨奇病毒CA16型的四重荧光RT-PCR方法,对该方法的特异性、灵敏度等进行评价,并对多份临床样本进行应用检测。结果表明,该检测方法特异性强,对肠道病毒及其他人类非肠道病毒进行检测,显示了良好的特异性;该检测方法对EV71型和CA16型的检测灵敏度分别达到31.25 TCID50和1.25×102TCID50;将浓度为1×104TCID50及5×102TCID50的EV71样本进行重复性试验,其变异系数均小于1.5%;将浓度为5×102-5×105TCID50的EV71和CA16样本进行线性试验,其相关系数R2值在0.982-0.998之间。采用本研究建立的方法检测40份疑似临床样本,最后检出31份肠道病毒阳性样本,其中8例EV71型阳性,13例CA16阳性。另外,试验数据表明,内标对监控PCR抑制物的存在具有重要作用。本方法能同时快速检测所有肠道病毒并进行EV71型及CA16型的分型,并且灵敏度高、特异性好、扩增效率高,由于加入了内标,能有效地监控假阴性的出现,适合于手足口病的临床检测。     

多重PCR检测无公害畜禽肉和水产品中4种致病菌

建立无公害畜禽肉和水产品中肠出血性大肠杆菌(EHEC)、沙门氏菌、副溶血性弧菌(VP)和单核细胞增生性李斯特氏菌(LM)的多重PCR检测方法,为这些致病菌的快速诊断提供实验依据。选择分别针对EHEC溶血素基因hlyAB、副溶血性弧菌属保守序列toxR基因、沙门氏菌侵袭基因invA和LM的iap基因特异的4对引物,先分别进行单重PCR扩增,再同时加入4对引物进行多重PCR扩增,扩增产物经测序验证。建立的多重PCR方法可简便、快速、灵敏地实现对EHEC、LM、沙门氏菌和VP的同时检测,在畜禽肉和水产品中的检测灵敏度达到10^3cfu/mL。     

High efficiency of site-directed mutagenesis mediated by a single PCR product.

We describe a highly efficient procedure for site-specific mutagenesis of double-stranded plasmids. The method relies on a single PCR primer which incorporates both the mutations at the selection site and the desired single base substitutions at the mutant site. This primer is annealed to the denatured plasmid and directs the synthesis of the mutant strand. After digestion with selection enzyme, the plasmid DNA is amplified into Escherichia coli strain BMH71-18 and subjected to a second digestion and amplification into the bacterial strain DH5alpha. A mutagenesis efficiency >80% was consistently achieved in the case of two unrelated plasmids.