Overlap Extension PCR Method Research Articles

Unraveling the peculiarities and development of novel inhibitors of leishmanial arginyl-tRNA synthetase.

Aminoacylation by tRNA synthetase is a crucial part of protein synthesis and is widely recognized as a therapeutic target for drug development. Unlike the arginyl-tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of Leishmania donovani (LdArgRS) that can follow the canonical two-step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE-PCR) method to create a deletion mutant (ΔIns-LdArgRS) devoid of this region to investigate its function. Notably, the purified LdArgRS and ΔIns-LdArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full-length protein showed better catalytic efficiency than ΔIns-LdArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo-coumarin derivative (Comp-7j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of LdArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for ΔIns-LdArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid-specific insertion and Rossmann fold motif, LdArgRS revealed typical structural characteristics of ArgRSs, and Comp-7j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNAArg near the insertion region enhanced the stability and compactness of LdArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti-leishmanial agents.

DNA Data Storage in Perl

Here we report a simple and flexible method for DNA data storage based on Perl script. For this approach, the text data of the preamble of the “Universal Declaration of Human Rights” consisting of 2,046 words was encoded into the corresponding 8,148 base pairs of DNA using Perl-based encoding with a hash table. The encoded DNA sequences were then artificially synthesized for storage. The information DNA consisted of a total of 22 chemically synthesized DNA fragments with 400 nucleotides each, which were inserted into a cloning vector to multiply the plasmid DNA. The nucleotide integrity of the data-carrying DNA sequences were ensured under the accelerated aging conditions. Also, an erroneous nucleotide in the information DNA sequences was successfully corrected using the overlap extension PCR method. The stored DNA was read by sequencing, and the resulting DNA sequence information was successfully decoded to convert the DNA records back to the original document. Our results indicate that textual data can be stored in DNA using a simple, easy, and flexible Perl by running a script from the command line.

Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis.

The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.

Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination

To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector.

Construction and evaluation of a novel humanized HER2-specific chimeric receptor.

IntroductionThe human epidermal growth factor receptor 2 (HER2) represents one of the most studied tumor-associated antigens (TAAs) for cancer immunotherapy. The monoclonal antibody (mAb) trastuzumab has improved the outcomes of patients with HER2+ breast cancer. However, a large number of HER2+ tumors are not responsive to, or become resistant to, trastuzumab-based therapy, and thus more effective therapies targeting HER2 are needed.MethodsHER2-specific T cells were generated by the transfer of genes that encode chimeric antigen receptor (CAR). Using a multistep overlap extension PCR method, we constructed a novel, humanized HER2 CAR-containing, chA21 single-chain variable fragment (scFv) region of antigen-specific mAb and T-cell intracellular signaling chains made up of CD28 and CD3ζ. An interferon γ and interleukin 2 enzyme-linked immunosorbent assay and a chromium-51 release assay were used to evaluate the antitumor immune response of CAR T cells in coculture with tumor cells. Furthermore, SKBR3 tumor–bearing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were treated with HER2 CAR T cells to evaluate antitumor activity. Human CD3+ T cell accumulation in tumor xenograft was detected by immunohistochemistry.ResultschA21-28z CAR was successfully constructed, and both CD4+ and CD8+ T cells were transduced. The expanded HER2 CAR T cells expressed a central memory phenotype and specifically reacted against HER2+ tumor cell lines. Furthermore, the SKBR3 tumor xenograft model revealed that HER2 CAR T cells significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed robust accumulation of human CD3+ T cells in regressing SKBR3 lesions.ConclusionsThe results of this study show that novel chA21 scFv-based, HER2-specific CAR T cells not only recognized and killed HER2+ breast and ovarian cancer cells ex vivo but also induced regression of experimental breast cancer in vivo. Our data support further exploration of the HER2 CAR T-cell therapy for HER2-expressing cancers.

Construction and expression of nm23-H1 gene with different enzyme activities and resistant specific shRNA in eukaryotic expression vector

背景与目的已有的研究证明nm23-H1基因是一个重要的肿瘤转移抑制基因，但其抑制肿瘤转移的生化机理尚不完全清楚。Nm23-H1基因结构和功能异常与肿瘤的侵袭转移有密切关系。我们前期已构建了nm23-H1的短发夹RNA（short hairpin RNA, shRNA）载体以及可抵抗此shRNA降解的nm23-H1的cDNA的表达载体，在此基础上我们欲应用基因定点突变技术构建具有不同酶活性并能抵抗此shRNA降解的nm23-H1cDNA真核表达载体，并通过恢复实验验证其表达，为进一步研究肿瘤抑制基因nm23-H1的分子机制提供理论基础和实验依据。方法以pcDNA3.1(+)-shRNA-resistant-nm23-H1质粒为突变模板，应用重叠延伸PCR方法引入nm23-H1基因四个单点突变和一个联合位点突变，并将突变基因片段克隆到真核表达载体pcDNA3.1Hygro(+)。将突变质粒转染人肺腺癌细胞株A549/nm23-H1-shRNA（稳定沉默nm23-H1基因），利用Western blot技术验证不同突变体nm23-H1蛋白的表达。结果成功构建了shRNA抵抗的nm23-H1S44A、nm23-H1P96S、nm23-H1H118F、nm23-H1S120G、nm23-H1P96S-S120G五个突变型真核表达载体，经DNA序列分析突变的碱基序列与实验设计完全一致，经Western blot验证nm23-H1蛋白表达正常。结论成功构建了五个具有不同突变位点的shRNA抵抗的nm23-H1基因真核表达载体，并且突变蛋白质nm23-H1表达正常，同时也表明重叠延伸PCR技术是一种高效、便捷、经济的DNA定点突变方法。

Generation of Human Immunodeficiency Virus–1 Long Terminal Repeat Reporter Genes by Rapid Polymerase Chain Reaction–Mediated Mutagenesis

Objective: The development of a human immunodeficiency virus (HIV)–1 promoter reporter system is critical for investigating transcription activity of the virus. We designed a modified overlap extension PCR method for single round site-directed mutagenesis of the long terminal repeat (LTR) segment of HIV-1. Methods: The procedure consists of overlap extension PCR, DpnI digestion, and product transformation. Wild-type and serial sequence deletion clones of HIV-1 LTR were constructed. Basal transcription activity was also measured. Results: The overall efficiency for obtaining the desired products was 53.2%. The reporter assay indicated the importance of the second NF-ĸB cis-element and surrounding regions in mediating the regulation of HIV-1 transcription. Conclusion: The technique we investigated is suitable for highthroughput functional studies of virus-host interaction.

Effects of Thioredoxin: SUMO and Intein on Soluble Fusion Expression of an Antimicrobial Peptide OG2 in Escherichia coli

OG2 is a modified antimicrobial peptide of Palustrin-OG1 (OG1), which is derived from Odorrana grahami frog. OG2 has shown much higher selective antimicrobial activity and lower hemolytic activity than OG1, indicating OG2 may be a promising antimicrobial agent. In this study, we investigated three fusion partners, including thioredoxin, Mxe GyrA intein, and small ubiquitin-like modifier (SUMO), each fused with OG2, and examined their effects on the expression level and solubility of OG2 in Escherichia coli. The codon-optimized OG2 gene was cloned into pET32a (+) and pTWIN1 for fusion with thioredoxin and Mxe GyrA intein, respectively. In addition, the SUMO-OG2 gene was amplified by splice overlap extension PCR method and was cloned into pET30a (+). All recombinant plasmids were then transformed into E. coli BL21(DE3)pLysS, and the expressed fusion proteins were verified. Upon isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, OG2 fused with thioredoxin (Trx-OG2) showed the highest yield as a soluble fusion protein (50 mg/L), followed by Mxe GyrA intein (44 mg/L) and SUMO (11 mg/L). The thioredoxin-fused protein (Trx-OG2) was then purified by nickel-nitrilotriacetic acid chromatography and desalted by Sephadex G25. The OG2 released by both tobacco etch virus protease and enterokinase from Trx-OG2 showed strong antimicrobial activity against Staphylococcus aureus ATCC25923.

Effects of Thioredoxin: SUMO and Intein on Soluble Fusion Expression of an Antimicrobial Peptide OG2 in Escherichia coli

OG2 is a modified antimicrobial peptide of Palustrin-OG1 (OG1), which is derived from Odorrana grahami frog. OG2 has shown much higher selective antimicrobial activity and lower hemolytic activity than OG1, indicating OG2 may be a promising antimicrobial agent. In this study, we investigated three fusion partners, including thioredoxin, Mxe GyrA intein, and small ubiquitin-like modifier (SUMO), each fused with OG2, and examined their effects on the expression level and solubility of OG2 in Escherichia coli. The codon-optimized OG2 gene was cloned into pET32a (+) and pTWIN1 for fusion with thioredoxin and Mxe GyrA intein, respectively. In addition, the SUMO-OG2 gene was amplified by splice overlap extension PCR method and was cloned into pET30a (+). All recombinant plasmids were then transformed into E. coli BL21(DE3)pLysS, and the expressed fusion proteins were verified. Upon isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, OG2 fused with thioredoxin (Trx-OG2) showed the highest yield as a soluble fusion protein (50 mg/L), followed by Mxe GyrA intein (44 mg/L) and SUMO (11 mg/L). The thioredoxin-fused protein (Trx-OG2) was then purified by nickel-nitrilotriacetic acid chromatography and desalted by Sephadex G25. The OG2 released by both tobacco etch virus protease and enterokinase from Trx-OG2 showed strong antimicrobial activity against Staphylococcus aureus ATCC25923.

Soluble Monomeric IgG1 Fc

Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.

Experimental Study on Construction of Human Hiwi Adenovirus Vector and Apoptosis of K562 Cells Induced by Transfected Vector

Abstract 4721 Background:Leukemia stem cells are responsible for the genesis, progression, drug resistance and relapse of leukemia. Hiwi gene is an important divisive regulatory factor, which plays a role in maintaining the resting stage and down-regulating the cell cycle of stem/progenitor cells. We acquired human overall Hiwi coding region genes and constructed an adenovirus vector carrying human Hiwi with fluorescin. This study will not only establish the foundation of the further study in function and mechanism of Hiwi induce the differentiation and apoptosis of leukemia stem cells, but also provide the theoretical basis for searching new therapeutic target and method for leukemia. Methods:Using the overlap extension PCR method to amplify overall Hiwi coding region genes and insert overall Hiwi coding region genes into Flag-IRES –hrGFP vector that carrying green fluorescin with Gateway clone technology to construct pDown-Hiwi-3×flag-IRES-hrGFP. After transform the vector to Stb13, we screen the positive clone with PCR method, extract the plasmid and process recombination reaction between pDown-Hiwi-3×flag-IRES-hrGFP clone vector and pAV.Des1d expression vector to abtain the pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP adenovirus vector recombinant. Screening the positive clone with PCR and extracting the plasmid which were digested by enzyme and then examined by sequencing consequences. At last packaged Ad-Hiwi-3×flag-IRES-hrGFP adenovirus vector recombinant is obtained. Transfect the recombinant adenovirus vector to K562 cells, and then identify the function of the cells. Results:Successfully clone human overall Hiwi coding region genes with the technology of overlap extension PCR and construct the adenovirus vector recombinant with enzyme cutting identification and gene sequencing examination. Conclusions:Successfully construct Ad-Hiwi-3×flag-IRES-hrGFP adenovirus vector recombinant and establish the foundation for the further study of Hiwi gene in leukemia stem cells. The transfected recombinant adenovirus vector can induce K562 cells to apoptosis. [U1]ɾ3ý£z Disclosures:No relevant conflicts of interest to declare.

Heparin Impairs Angiogenesis through Inhibition of MicroRNA-10b

Heparin, which has been used as an anticoagulant drug for decades, inhibits angiogenesis, whereas thrombin promotes tumor-associated angiogenesis. However, the mechanisms underlying the regulation of angiogenesis by heparin and thrombin are not well understood. Here, we show that microRNA-10b (miR-10b) is down-regulated by heparin and up-regulated by thrombin in human microvascular endothelial cells (HMEC-1). Overexpression of miR-10b induces HMEC-1 cell migration, tube formation, and angiogenesis, and down-regulates homeobox D10 (HoxD10) expression via direct binding of miR-10b to the putative 3' UTR of HoxD10. In addition, HMEC-1 cell migration and tube formation are induced by HoxD10 knockdown, whereas angiogenesis is arrested when HoxD10 expression is increased after anti-miR-10b or heparin treatments. Furthermore, expression of miR-10b and its transcription factor Twist are up-regulated by thrombin, whereas HoxD10 expression is impaired by thrombin. Using quartz crystal microbalance analysis, we show that heparin binds to thrombin, thereby inhibiting thrombin-induced expression of Twist and miR-10b. However, the expression of miR-10b is not attenuated by heparin any more after thrombin expression is silenced by its siRNA. Interestingly, we find that heparin attenuates miR-10b expression and induces HoxD10 expression in vivo to inhibit angiogenesis and impair the growth of MDA-MB-231 tumor xenografts. These results provide insight into the molecular mechanism by which heparin and thrombin regulate angiogenesis.

Funnel-like Hexameric Assembly of the Periplasmic Adapter Protein in the Tripartite Multidrug Efflux Pump in Gram-negative Bacteria

Gram-negative bacteria expel diverse toxic chemicals through the tripartite efflux pumps spanning both the inner and outer membranes. The Escherichia coli AcrAB-TolC pump is the principal multidrug exporter that confers intrinsic drug tolerance to the bacteria. The inner membrane transporter AcrB requires the outer membrane factor TolC and the periplasmic adapter protein AcrA. However, it remains ambiguous how the three proteins are assembled. In this study, a hexameric model of the adapter protein was generated based on the propensity for trimerization of a dimeric unit, and this model was further validated by presenting its channel-forming property that determines the substrate specificity. Genetic, in vitro complementation, and electron microscopic studies provided evidence for the binding of the hexameric adapter protein to the outer membrane factor in an intermeshing cogwheel manner. Structural analyses suggested that the adapter covers the periplasmic region of the inner membrane transporter. Taken together, we propose an adapter bridging model for the assembly of the tripartite pump, where the adapter protein provides a bridging channel and induces the channel opening of the outer membrane factor in the intermeshing tip-to-tip manner.

Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6×His tag in Pichia pastoris

Human mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) catalyzes the oxidation of acetaldehyde to acetic acid. Therefore, ALDH2 has therapeutic potential in detoxification of acetaldehyde. Furthermore, ALDH2 catalyzes nitroglycerin to nitrate and 1, 2-glyceryldinitrate during therapy for angina pectoris, myocardial infarction, and heart failure. Large quantities of ALDH2 will be needed for potential clinical practice. In this study, Pichia pastoris was used as a platform for expression of human ALDH2. Based on the ALDH2*1 cDNA sequence, we designed ALDH2 cDNA by choosing the P. pastoris preferred codons and by decreasing the G + C content level. The sequence was synthesized using the overlap extension PCR method. The cDNA and 6xHis tags were subcloned into the plasmid pPIC9K. The recombinant protein was expressed in P. pastoris GS115 and purified using Ni(2+)-Sepharose affinity chromatography. The amount of secreted protein in the culture was 80 mg/L in shake-flask cultivation and 260 mg/L in high-density bioreactor fermentation. Secreted ALDH2 was easily purified from the culture supernatant by using Ni(2+)-Sepharose affinity chromatography. After purification of the fermentation supernatant, the enzyme had a specific activity of 1.2 U/mg protein. The yield was about 16 mg/L in a shake flask culture of P. pastoris GS115 which contained the original human ALDH2*1 cDNA.

Detection of antibodies to Epstein-Barr virus with ELISA based on fusion capsid protein

Objective To develop enzyme-linked immunosorbent assay (ELISA) for detecting antibody to viral capsid antigen (VCA) of Epstein-Barr virus (EBV) by using fusion capsid protein of p23 and p18 as antigen and evaluate its diagnostic implication. Methods Full-length p23 (amino acids 1-162) and carboxy half of p18 (105-176) gene fragment were connected by a (Gly4Ser)3 linker using overlap extension PCR method. The prokaryotic expressed fusion protein of p23-p18 was purified by nickel-chelating sepharose resin affinity chromatography and used as the antigens for ELISA detection of IgG and IgM antibodies to VCA. Western blot was used to detect its antigenicity and specificity. The serum samples from 60 patients with infectious mononucleosis (IM), fifty-six chronic renal dysfunction patients on hemodialysis and 326 serum samples from healthy blood donors were examined for the presence of VCA-specific IgG and IgM by fusion protein ELISA and indirect immunoflurorescene assay (IFA) simultaneously. The specificity, sensitivity and accuracy of ELISA were detected. Results (1) Identification of the fusion protein : p23-p18 fusion gene was constructed correctly and expressed successfully in the prokaryotic expression vector. The fusion protein showed good reactivity with 10 cases of VCA IgM and 10 cases of IgG positive sera in Western blot, while no reactive bands were found with 8 cases of VCA IgM and IgG negative sera. (2)Clinical tests: IFA and ELISA with fusion proteins were used to detect VCA IgG and IgM in 442 serum samples listed above. As compared with IFA, the sensitivity, specificity and accuracy of the recombinant protein IgG ELISA were 97.3% (392/403), 97.4% (38/39) and 97.3% (430/442), respectively,and those of IgM ELISA were 94.2% (65/69), 99.5% (371/373 ), and 98.6% (436/442), respectively. Conclusion The ELISA based on fusion viral capsid proteins is sensitive, specific and accurate method for determining antibodies to VCA of EBV for both clinical diagnosis and epidemiology studies. Key words: Herpesvirus 4; human; Antigens; viral; Capsid proteins; Recombinant fusion proteins; Enzyme-linked immunosorbont assay

Role of the ϵ Subunit of Thermophilic F1-ATPase as a Sensor for ATP

The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the alpha(3)beta(3)gamma complex of TF(1). The ATPase activity of the resulting alpha(3)beta(3)gammaepsilon complexes was measured, and the extent of inhibition by the mutant epsilon subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the epsilon subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF(1) by fixing the epsilon subunit into the folded conformation.

Aeromonas Exoenzyme T of Aeromonas salmonicida Is a Bifunctional Protein That Targets the Host Cytoskeleton

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.

Asymmetric overlap extension PCR method bypassing intermediate purification and the amplification of wild-type template in site-directed mutagenesis

By combining asymmetric PCR and overlap extension, we developed a novel asymmetric overlap extension PCR (AOE-PCR) method for site-directed mutagenesis which bypassed the need for intermediate purification and excluded the amplification of a wild-type template. This method was used to introduce single base mutations into a small GTPase gene from cotton and to simultaneously introduce two mutations just by repeating this method using the first round AOE-PCR products as template. Our results suggested that the AOE-PCR method represents a valuable improvement of the original overlap extension PCR for site-directed mutagenesis.

Assembling and cloning genes for fusion proteins using reverse transcription one-step overlap extension PCR method

The need to construct fusion genes for diVerent purposes arises in various Welds of molecular biology [1,2]. PCR is one of the most popular methods to generate fusions of diVerent DNA sequences. Examples include constructs where (i) diVerent lengths of a particular promoter sequence are fused to the coding region of another gene to determine which sequences are involved in the regulation of gene expression or which promoter expresses a particular gene more eYciently, (ii) fusion genes are created to code for hybrid proteins, and (iii) DNA sequences representing precise deletion mutants are constructed. The construction of such fused or deleted genes usually involves ligation of diVerent restriction fragments, a tedious and time-consuming process that frequently is restricted by the availability of proper cleavage sites [3,4]. The overlap extension (OE) technique is widely used in genetic engineering to overcome such limitations. Specially designed oligonucleotides and PCR methods are applied to generate two DNA fragments with overlapping ends. These fragments are combined in a fusion reaction to anneal the overlapping ends of the appropriate strands. Both 3 ends serve as primers extending the complementary strands. The resulting fusion product is ampliWed further by subsequent PCR. SpeciWc alterations can be introduced into the PCR product through incorporation of changes into the overlapping primers [5]. A variation of the OE techniques, the onestep overlap extension PCR (OOE–PCR), was described by Urban and coworkers [6]. The OOE–PCR method runs in

Site-Directed Mutagenesis Using Pfu DNA Polymerase and T4 DNA Ligase

Site-Directed Mutagenesis Using Pfu DNA Polymerase and T4 DNA Ligase