Site-directed mutagenesis using Pfu DNA polymerase and T4 DNA ligase
2005; Future Science Ltd; Volume: 38; Issue: 6 Linguagem: Inglês
10.2144/05386bm03
ISSN1940-9818
AutoresYair Adereth, Kristen J. Champion, Tien Hsu, Vincent Dammai,
Tópico(s)Molecular Biology Techniques and Applications
ResumoBioTechniquesVol. 38, No. 6 BenchmarksOpen AccessSite-directed mutagenesis using Pfu DNA polymerase and T4 DNA ligaseYair Adereth, Kristen J. Champion, Tien Hsu & Vincent DammaiYair AderethMedical University of South Carolina, Charleston, SC, USA, Kristen J. ChampionMedical University of South Carolina, Charleston, SC, USA, Tien HsuMedical University of South Carolina, Charleston, SC, USA & Vincent Dammai*Address correspondence to: Vincent Dammai, Medical University of South Carolina, Department of Pathology and Laboratory Medicine, 321 Hollings Cancer Center, 86 Jonathan Lucas Street, Charleston, SC 29425, USA. e-mail: E-mail Address: dammaiv@musc.eduMedical University of South Carolina, Charleston, SC, USAPublished Online:30 May 2018https://doi.org/10.2144/05386BM03AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit IntroductionHere we describe a simple and cost-effective PCR-based method to introduce deletions, point mutations, frame-shift, or truncations with very high efficiency. The scope and additional applications of the method are discussed.Site-directed mutagenesis involves the introduction of mutations at the DNA level to alter the primary amino acid sequence of proteins (1,2). To approach the desired level of reliability in generating mutants, several methods have been described, some of which are technically demanding and/or require special reagents (1–6). Megaprimer-based methods for site-directed mutagenesis utilize three oligonucleotide primers (4) or only one mutagenic primer (5) in two rounds of PCR. However, when longer fragments are mutagenized, the efficiency of the methods decreases dramatically and the number of side products increases. Inverse PCR can generate mutations with two primers but solely utilizes plasmid DNA templates (6). We describe a highly efficient, cost-effective, and reliable method to generate mutations along the entire length of the cDNA. The strategy involves the following steps. (i) Four primers (e.g., Figure 1, primers #1–#4) amplify the cDNA in the first-round PCR. Primer pairs (#1 and #2) and (#3 and #4) generate the N- and C-terminal PCR products, respectively. Primers #2 and #3 are purchased as 5′ phosphorylated primers. Primers #1 and #4 are unphosphorylated. (ii) T4 DNA ligasemediated joining of the PCR products immediately following the first-round PCR. (iii) Second-round amplification of the ligated product using primers #1 and #4. (iv) Restriction digestion of the PCR product (from step 3) and cloning into any vector of choice. In our case, primer #2 contained the desired mutation (S65L, S65W, or R167Q). The PCRs contained the following components: 5.0 ng template DNA, 2.5 µL forward primers (0.05 µg/µL, 10× stock), and 2.5 µL reverse primers (0.05 µg/µL, 10× stock), 2.5 µL 2 mM dNTP, 0.5 µL 100 mM MgSO4, 2.5 µL 10× Pfu buffer (Stratagene, La Jolla, CA, USA), 0.5 µL Pfu DNA polymerase (5 U/µL; Stratagene), and sterile water to 25 µL. Standard PCR cycling conditions were used (1 cycle of 94°C for 4 min, 54°C for 1 min, 72°C for 1 min; 28 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 7 min). One microliter (approximately 100 ng) of each PCR product was directly used for blunt-end ligation [1 h at room temperature, 1 µL 10× buffer (New England Biolabs, Beverly, MA, USA), 1 µL each of N- and C-terminal PCR products, 0.5 µL T4 DNA ligase (100,000 U/mL), and 6.5 µL sterile water]. One microliter (approximately 10 ng) of the ligation reaction was used for the second-round PCR under the conditions described above. Final PCR products were digested with restriction enzymes and gel-purified using the Zymoclean Gel RNA Recovery Kit™ (Zymo Research, Orange, CA, USA). Fifty nanograms of insert were ligated to compatible sites in 50 ng mammalian expression vector, pEGFP-C1, and transformed into chemically competent bacteria.Figure 1. Strategy for site-directed mutagenesis.Step 1. Introduction of a deletion shown as an example. Note that gene-specific primers or alternate primers (T7 or T3 promoter primers) are used to achieve a minimum size of a 114-bp product. Step 2. Ligation of the PCR products. Step 3. Amplification of the ligated product to generate cDNA containing the desired mutation. Step 4. Restriction digestion of the final PCR product and ligation into the expression vector.Pfu DNA polymerase is used to ensure blunt ends and avoid second-site mutations. Initially, gel-purified PCR products from step 1 were used for ligation (step 2). Subsequently, we found that PCR products could be directly used without removing PCR primers (Figure 2). Moreover, Pfu buffer was found to be compatible with ligase buffer; thus, PCR products can be directly ligated without removing the Pfu buffer. The minimum size of the PCR product amplified was 114 bp (Figure 2, lane 15). Thus, if mutations close to translation initiation or termination sites are needed, alternate primers complementary to the vector (Figure 1, T7 or T3 promoter primers) or the untranslated regions (UTRs) of the cDNA can be used to amplify at least a 114-bp PCR product. In the second round of PCR, primers are designed to only amplify the open reading frame (ORF).Figure 2. Applications of the strategy.(A) Generation of point mutants and a deletion of the VHL protein. Lanes 1 and 13, 1 kb DNA ladder (Promega, Madison, WI, USA); lanes 12 and 14, 100-bp DNA ladder (New England Biolabs); lanes 2, 4, and 6, N-terminal PCR products of VHL mutants S65L, S65W, or R167Q, respectively; lanes 3, 5, and 7, C-terminal PCR products of VHL mutants S65L, S65W, or R167Q, respectively; lanes 8–10, full-length S65L, S65W, and R167Q point mutants after second-round PCR; lane 11, simultaneous introduction of a point mutation and deletion of amino acids 66–166 by joining S65L N-terminal PCR product (lane 2) with 167–213 C-terminal PCR product (lane 7). (B) Expected PCR products for VHL. (C) Fusion of membrane localization signal (mem) of integrin alpha-3 (114 bp, lane 15) to EGFP-integrin UTR (2.2 kb, lane 16) to create mem-EGFP-integrin 3′ UTR (lane 17). VHL, Von Hippel-Lindau; EGFP, enhanced green fluorescent protein; UTR, untranslated region.Using our method, we cloned a number of mutants (examples shown in Figure 2) of the Von Hippel-Lindau (VHL) protein in a single day. The procedure can also be adapted to generate novel fusion proteins containing distinct sequence elements. For example, we generated fusions containing the membrane localization signal of integrin alpha-3 and a reporter [enhanced green fluorescent protein (EGFP)] containing the 3′ UTR of integrin alpha-3 (Figure 2C) to study messenger RNA (mRNA) transport and protein localization of the reporter fusion. All the constructs were confirmed by sequencing.The strategy has several advantages. (i) The method is cost-effective and can be completed in a single day. (ii) The amplified PCR product is significantly smaller, allowing direct manipulation of larger cDNAs. (iii) Any vector, DNA fragment, or PCR product containing the cDNA can be the starting template. (iv) Small epitope tags can be fused in-frame to the protein of interest (see use of alternate primers). (v) Sufficient quantities of the final PCR product are available for cloning into multiple vectors, thus reducing the time and effort spent on subcloning. (vi) As opposed to the ligase-independent overlap-extension PCR method (7), "mixing and matching" various N-terminal and C-terminal PCR products (Figure 2A, lane 11) generates a higher number of deletion mutants with fewer step 1 PCRs. This is advantageous where a large number of random deletions are required; for example, in mapping protein-protein interaction domains. (vii) The strategy is technically very simple and involves no intermediate purification steps.AcknowledgementsThis work is supported by a MUSC-DOD Phase VII (Geocenters) grant to V.D.Competing Interests StatementThe authors declare no competing interests.References1. Braman, J. (Ed.). 2002. In Vitro Mutagenesis Protocols, 2nd ed. Humana Press, Totowa, NJ.Crossref, Google Scholar2. Ishii, T.M., P. Zerr, X.M. Xia, C.T. Bond, J. Maylie, and J.P. Adelman. 1998. Site-directed mutagenesis. Methods Enzymol. 293:53–71.Crossref, Medline, CAS, Google Scholar3. Chiu, J., P.E. March, R. Lee, and D. Tillett. 2004. Site-directed igase-independent mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h. Nucleic Acids Res. 32:e174.Crossref, Medline, Google Scholar4. Sarkar, G. and S.S. Sommer. 1990. The "megaprimer" method of site-directed mutagenesis. BioTechniques 8:404–407.Medline, CAS, Google Scholar5. Nagy, Z.B., F. Felfoldi, L. Tamas, and L.G. Puskas. 2004. A one-tube two-step polymerase chain reaction-based site-directed mutagenesis method with simple identification of the mutated product. Anal. Biochem. 324:301–303.Crossref, Medline, CAS, Google Scholar6. Stemmer, W.P. and S.K. Morris. 1992. Enzymatic inverse PCR: a restriction site independent, single fragment method for high-efficiency site-directed mutagenesis. BioTechniques 13:214–220.Medline, CAS, Google Scholar7. Lee, J., H.J. Lee, M.K. Shin, and W.S. Ryu. 2004. Versatile PCR-mediated insertion or deletion mutagenesis. BioTechniques 36:398–400.Link, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByCapsids and Portals Influence Each Other's Conformation During Assembly and MaturationJournal of Molecular Biology, Vol. 432, No. 7Molecular and structural analysis of a mechanical transition of helices in the L. donovani coronin coiled-coil domainInternational Journal of Biological Macromolecules, Vol. 143An Efficient Approach for Two Distal Point Site-Directed Mutagenesis from Randomly Ligated PCR Products1 July 2019 | Applied Biochemistry and Biotechnology, Vol. 189, No. 4Flexible Connectors between Capsomer Subunits that Regulate Capsid AssemblyJournal of Molecular Biology, Vol. 429, No. 16On the catalytic mechanism of bacteriophage HK97 capsid crosslinkingVirology, Vol. 506GCM2 -Activating Mutations in Familial Isolated HyperparathyroidismThe American Journal of Human Genetics, Vol. 99, No. 5Correct Assembly of the Bacteriophage T5 Procapsid Requires Both the Maturation Protease and the Portal ComplexJournal of Molecular Biology, Vol. 428, No. 1Why Johnny can't clone: Common pitfalls and not so common solutionsIchiro Matsumura3 April 2018 | BioTechniques, Vol. 59, No. 3Transient Contacts on the Exterior of the HK97 Procapsid That Are Essential for Capsid AssemblyJournal of Molecular Biology, Vol. 426, No. 10Calcium/Calmodulin-Dependent Protein Kinase Is Negatively and Positively Regulated by Calcium, Providing a Mechanism for Decoding Calcium Responses during Symbiosis Signaling 24 December 2013 | The Plant Cell, Vol. 25, No. 12Pseudomonas putida F1 has multiple chemoreceptors with overlapping specificity for organic acidsMicrobiology, Vol. 159, No. Pt_6Taxis of Pseudomonas putida F1 toward Phenylacetic Acid Is Mediated by the Energy Taxis Receptor Aer2Applied and Environmental Microbiology, Vol. 79, No. 7A rapid and efficient one-step site-directed deletion, insertion, and substitution mutagenesis protocolAnalytical Biochemistry, Vol. 434, No. 2A Discussion of Molecular Biology Methods for Protein Engineering30 September 2011 | Molecular Biotechnology, Vol. 51, No. 1A Rapid and Versatile PCR-Based Site-Directed Mutagenesis Protocol for Generation of Mutations Along the Entire Length of a Cloned cDNA19 March 2010Enhancing the Apoptotic Potential of Insulin-Like Growth Factor-Binding Protein-3 in Prostate Cancer by Modulation of CK2 PhosphorylationMolecular Endocrinology, Vol. 23, No. 10Specific Expression of BMP2/4 Ortholog in Biomineralizing Tissues of Corals and Action on Mouse BMP Receptor16 September 2008 | Marine Biotechnology, Vol. 11, No. 2Soybean Nodule Autoregulation Receptor Kinase Phosphorylates Two Kinase-associated Protein Phosphatases in VitroJournal of Biological Chemistry, Vol. 283, No. 37Poly(adp-ribose) Polymerase-1 Regulates Tracp Gene Promoter Activity During RANKL-Induced Osteoclastogenesis19 November 2007 | Journal of Bone and Mineral Research, Vol. 23, No. 4An Evolutionarily Conserved Nuclear Export Signal Facilitates Cytoplasmic Localization of the Tbx5 Transcription Factor27 March 2023 | Molecular and Cellular Biology, Vol. 28, No. 5Differential Binding of Poly(ADP-Ribose) Polymerase-1 and JunD/Fra2 Accounts for RANKL-Induced Tcirg1 Gene Expression During Osteoclastogenesis9 April 2007 | Journal of Bone and Mineral Research, Vol. 22, No. 7RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis21 August 2006 | Journal of Bone and Mineral Research, Vol. 21, No. 11Endocytic Function of von Hippel-Lindau Tumor Suppressor Protein Regulates Surface Localization of Fibroblast Growth Factor Receptor 1 and Cell MotilityJournal of Biological Chemistry, Vol. 281, No. 17 Vol. 38, No. 6 Follow us on social media for the latest updates Metrics History Received 9 March 2005 Accepted 25 March 2005 Published online 30 May 2018 Published in print June 2005 Information© 2005 Author(s)AcknowledgementsThis work is supported by a MUSC-DOD Phase VII (Geocenters) grant to V.D.Competing Interests StatementThe authors declare no competing interests.PDF download
Referência(s)