Nucleic Acids Research, 1989, Vol. 17, No. 16 6545-6551
© 1989
ENZYMOLOGY |
A simple method for site-directed mutagenesis using the polymerase chain reaction
Department of Molecular Biology, University of Southern California Los Angeles, CA 90089-1340, USA 1Department of Biochemistry, University of California Berkeley, CA 94720, USA
*To whom correspondence should be addressed
Received June 5, 1989. Revised July 12, 1989. Accepted July 12, 1989.
We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification by the polymerase chain reaction (PCR). The method is based on the amplification of the entire plasmid using primers that include the desired changes. The method is rapid, simple in its execution, and requires only minute amounts of plasmid template DNA. It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to be manipulated. In our system the yield of transformants was high and the fraction of them harboring plasmids with only the desired change was consistently about 80%. The generality of the method should make it useful for the direct alteration of most cloned genes. The only limitation may be the total length of the plasmid to be manipulated. During the study we found that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the newly synthesized chains. These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene sequence.
+ Present address: Department of Biological Sciences, University of California, Santa Barbara, CA 93106, USA
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. M. Cartwright, E.-K. Lim, C. Kleanthous, and D. J. Bowles A Kinetic Analysis of Regiospecific Glucosylation by Two Glycosyltransferases of Arabidopsis thaliana: DOMAIN SWAPPING TO INTRODUCE NEW ACTIVITIES J. Biol. Chem., June 6, 2008; 283(23): 15724 - 15731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sanchez-Hidalgo, M. Martinez-Bueno, A. M Fernandez-Escamilla, E. Valdivia, L. Serrano, and M. Maqueda Effect of replacing glutamic residues upon the biological activity and stability of the circular enterocin AS-48 J. Antimicrob. Chemother., June 1, 2008; 61(6): 1256 - 1265. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. van Ooijen, G. Mayr, M. Albrecht, B. J. C. Cornelissen, and F. L.W. Takken Transcomplementation, but not Physical Association of the CC-NB-ARC and LRR Domains of Tomato R Protein Mi-1.2 is Altered by Mutations in the ARC2 Subdomain Mol Plant, May 1, 2008; 1(3): 401 - 410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. van Ooijen, G. Mayr, M. M. A. Kasiem, M. Albrecht, B. J. C. Cornelissen, and F. L. W. Takken Structure-function analysis of the NB-ARC domain of plant disease resistance proteins J. Exp. Bot., April 4, 2008; (2008) ern045v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Elliott, M. T. McIntosh, H. D. Hosgood III, S. Chen, G. Zhang, P. Baevova, and K. A. Joiner Four distinct pathways of hemoglobin uptake in the malaria parasite Plasmodium falciparum PNAS, February 19, 2008; 105(7): 2463 - 2468. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Xie, D. Liu, Y. Feng, L. Shan, and J. Wang Folding Stability and Cooperativity of the Three Forms of 1-110 Residues Fragment of Staphylococcal Nuclease Biophys. J., March 15, 2007; 92(6): 2090 - 2107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Chen, D. M. Choudhury, and S. W. Craig Coincidence of Actin Filaments and Talin Is Required to Activate Vinculin J. Biol. Chem., December 29, 2006; 281(52): 40389 - 40398. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. MAURISSE, J. CHEUNG, J. WIDDICOMBE, and D. C GRUENERT Modification of the Pig CFTR Gene Mediated by Small Fragment Homologous Replacement Ann. N.Y. Acad. Sci., October 1, 2006; 1082(1): 120 - 123. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Trujillo, M. Sakagashira, and N. L. Eberhardt The Human Growth Hormone Gene Contains a Silencer Embedded within an Alu Repeat in the 3'-Flanking Region Mol. Endocrinol., October 1, 2006; 20(10): 2559 - 2575. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Petschacher and B. Nidetzky Engineering Candida tenuis Xylose Reductase for Improved Utilization of NADH: Antagonistic Effects of Multiple Side Chain Replacements and Performance of Site-Directed Mutants under Simulated In Vivo Conditions Appl. Envir. Microbiol., October 1, 2005; 71(10): 6390 - 6393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Parker and R. Meyer Mechanisms of Strand Replacement Synthesis for Plasmid DNA Transferred by Conjugation J. Bacteriol., May 15, 2005; 187(10): 3400 - 3406. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Lassila, J. R. Keeffe, P. Oelschlaeger, and S. L. Mayo Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity Protein Eng. Des. Sel., April 1, 2005; 18(4): 161 - 163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Chiu, P. E. March, R. Lee, and D. Tillett Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h Nucleic Acids Res., December 7, 2004; 32(21): e174 - e174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. K. Lamb, J. Ren, A. Park, C. Johnson, K. Leslie, S. Cocklin, P. Thompson, C. Mee, A. Cooper, D. K. Stammers, et al. Modulation of the ligand binding properties of the transcription repressor NmrA by GATA-containing DNA and site-directed mutagenesis Protein Sci., December 1, 2004; 13(12): 3127 - 3138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Park, H. K. Lamb, C. Nichols, J. D. Moore, K. A. Brown, A. Cooper, I. G. Charles, D. K. Stammers, and A. R. Hawkins Biophysical and kinetic analysis of wild-type and site-directed mutants of the isolated and native dehydroquinate synthase domain of the AROM protein Protein Sci., August 1, 2004; 13(8): 2108 - 2119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-M. Lee, J.-R. Chen, H.-L. Lee, W.-M. Leu, L.-Y. Chen, and N.-T. Hu Functional Dissection of the XpsN (GspC) Protein of the Xanthomonas campestris pv. campestris Type II Secretion Machinery J. Bacteriol., May 15, 2004; 186(10): 2946 - 2955. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Saha, J. Hill, M. A. Utton, A. A. Asuni, S. Ackerley, A. J. Grierson, C. C. Miller, A. M. Davies, V. L. Buchman, B. H. Anderton, et al. Parkinson's disease {alpha}-synuclein mutations exhibit defective axonal transport in cultured neurons J. Cell Sci., March 1, 2004; 117(7): 1017 - 1024. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. O. Duan and P. J. Simpson-Haidaris Functional Analysis of Interleukin 6 Response Elements (IL-6REs) on the Human {gamma}-Fibrinogen Promoter: BINDING OF HEPATIC Stat3 CORRELATES NEGATIVELY WITH TRANSACTIVATION POTENTIAL OF TYPE II IL-6REs J. Biol. Chem., October 17, 2003; 278(42): 41270 - 41281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Jin, E. Turcott, S. Englehardt, G. J. Mize, and D. R. Morris The Two Upstream Open Reading Frames of Oncogene mdm2 Have Different Translational Regulatory Properties J. Biol. Chem., July 3, 2003; 278(28): 25716 - 25721. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-C. Wu, F. J. Castellino, and S.-L. Wong A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy J. Biol. Chem., May 9, 2003; 278(20): 18199 - 18206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Spiers, H. K. Lamb, S. Cocklin, K. A. Wheeler, J. Budworth, A. L. Dodds, M. J. Pallen, D. J. Maskell, I. G. Charles, and A. R. Hawkins PDZ Domains Facilitate Binding of High Temperature Requirement Protease A (HtrA) and Tail-specific Protease (Tsp) to Heterologous Substrates through Recognition of the Small Stable RNA A (ssrA)-encoded Peptide J. Biol. Chem., October 11, 2002; 277(42): 39443 - 39449. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. M. Panin, L. Shao, L. Lei, D. J. Moloney, K. D. Irvine, and R. S. Haltiwanger Notch Ligands Are Substrates for Protein O-Fucosyltransferase-1 and Fringe J. Biol. Chem., August 9, 2002; 277(33): 29945 - 29952. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Chauhan and R. Bhatnagar Identification of Amino Acid Residues of Anthrax Protective Antigen Involved in Binding with Lethal Factor Infect. Immun., August 1, 2002; 70(8): 4477 - 4484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ma and S. Taylor A 15-Residue Bifunctional Element in D-AKAP1 Is Required for Both Endoplasmic Reticulum and Mitochondrial Targeting J. Biol. Chem., July 19, 2002; 277(30): 27328 - 27336. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. Feister, B. J. Auerbach, L. A. Cole, B. R. Krause, and S. K. Karathanasis Identification of an IL-6 response element in the human LCAT promoter J. Lipid Res., June 1, 2002; 43(6): 960 - 970. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Cleary, F.-C. Tan, K.-A. Nakrieko, S. J. Thompson, P. M. Mullineaux, G. P. Creissen, E. von Stedingk, E. Glaser, A. G. Smith, and C. Robinson Isolated Plant Mitochondria Import Chloroplast Precursor Proteins in Vitro with the Same Efficiency as Chloroplasts J. Biol. Chem., February 8, 2002; 277(7): 5562 - 5569. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Guth, T. O. Tange, E. Kellenberger, and J. Valcarcel Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing Mol. Cell. Biol., November 15, 2001; 21(22): 7673 - 7681. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Azad, D. R. Stanford, S. Sarkar, and A. K. Hopper Role of Nuclear Pools of Aminoacyl-tRNA Synthetases in tRNA Nuclear Export Mol. Biol. Cell, May 1, 2001; 12(5): 1381 - 1392. [Abstract] [Full Text]
|
|
|
|
 |
|
 B. Gajewska, J. Kaminska, A. Jesionowska, N. C. Martin, A. K. Hopper, and T. Zoladek WW Domains of Rsp5p Define Different Functions: Determination of Roles in Fluid Phase and Uracil Permease Endocytosis in Saccharomyces cerevisiae Genetics, January 1, 2001; 157(1): 91 - 101. [Abstract] [Full Text]
|
|
|
|
|
|
N. S. Chernyaeva and E. J. Murgola Covariance of Complementary rRNA Loop Nucleotides Does Not Necessarily Represent Functional Pseudoknot Formation In Vivo J. Bacteriol., October 15, 2000; 182(20): 5671 - 5675. [Abstract] [Full Text]
|
|
|
|
|
|
J. Carmichael, J. Chatellier, A. Woolfson, C. Milstein, A. R. Fersht, and D. C. Rubinsztein Bacterial and yeast chaperones reduce both aggregate formation and cell death in mammalian cell models of Huntington's disease PNAS, July 30, 2000; (2000) 170280697. [Abstract] [Full Text]
|
|
|
|
 |
|
 S Sotillos and S Campuzano DRacGAP, a novel Drosophila gene, inhibits EGFR/Ras signalling in the developing imaginal wing disc Development, January 12, 2000; 127(24): 5427 - 5438. [Abstract] [PDF]
|
|
|
|
|
|
S. Guth, C. Martinez, R. K. Gaur, and J. Valcarcel Evidence for Substrate-Specific Requirement of the Splicing Factor U2AF35 and for Its Function after Polypyrimidine Tract Recognition by U2AF65 Mol. Cell. Biol., December 1, 1999; 19(12): 8263 - 8271. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Martin, C. Steinkuhler, M. Brunetti, A. Pessi, R. Cortese, R. De Francesco, and M. Sollazzo A loop-mimetic inhibitor of the HCV-NS3 protease derived from a minibody Protein Eng. Des. Sel., November 1, 1999; 12(11): 1005 - 1011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Luo, Y. Zhou, and B. S. Cooperman Antichymotrypsin Interaction with Chymotrypsin. INTERMEDIATES ON THE WAY TO INHIBITED COMPLEX FORMATION J. Biol. Chem., June 18, 1999; 274(25): 17733 - 17741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ruiz-Lozano, M. L. Hixon, M. W. Wagner, A. I. Flores, S. Ikawa, A. S. Baldwin Jr., K. R. Chien, and A. Gualberto p53 Is a Transcriptional Activator of the Muscle-specific Phosphoglycerate Mutase Gene and Contributesin Vivo to the Control of Its Cardiac Expression Cell Growth Differ., May 1, 1999; 10(5): 295 - 306. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. Kanzler, A. W. Lohse, A. Keil, J. Henninger, H. P. Dienes, P. Schirmacher, S. Rose-John, K. H. Meyer Zum Buschenfelde, and M. Blessing TGF-beta 1 in liver fibrosis: an inducible transgenic mouse model to study liver fibrogenesis Am J Physiol Gastrointest Liver Physiol, April 1, 1999; 276(4): G1059 - G1068. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Hiddinga and N. L. Eberhardt Intracellular Amyloidogenesis by Human Islet Amyloid Polypeptide Induces Apoptosis in COS-1 Cells Am. J. Pathol., April 1, 1999; 154(4): 1077 - 1088. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Thompson, C. Robinson, and A. Mant Dual Signal Peptides Mediate the Signal Recognition Particle/Sec-independent Insertion of a Thylakoid Membrane Polyprotein, PsbY J. Biol. Chem., February 12, 1999; 274(7): 4059 - 4066. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kong Au and P. Chow Leung Identification of the Binding and Inhibition Sites in the Calmodulin Molecule for Ophiobolin A by Site-Directed Mutagenesis Plant Physiology, November 1, 1998; 118(3): 965 - 973. [Abstract] [Full Text]
|
|
|
|
|
|
X. Gu, B. Trigatti, S. Xu, S. Acton, J. Babitt, and M. Krieger The Efficient Cellular Uptake of High Density Lipoprotein Lipids via Scavenger Receptor Class B Type I Requires Not Only Receptor-mediated Surface Binding but Also Receptor-specific Lipid Transfer Mediated by Its Extracellular Domain J. Biol. Chem., October 9, 1998; 273(41): 26338 - 26348. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. F. Vanzo, Y. S. Li, B. Py, E. Blum, C. F. Higgins, L. C. Raynal, H. M. Krisch, and A. J. Carpousis Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome Genes & Dev., September 1, 1998; 12(17): 2770 - 2781. [Abstract] [Full Text]
|
|
|
|
|
|
J. Chatellier, F. Hill, P. A. Lund, and A. R. Fersht In vivo activities of GroEL minichaperones PNAS, August 18, 1998; 95(17): 9861 - 9866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. L. Law, H. Itoh, D. J. Law, G. J. Mize, J. L. Merchant, and D. R. Morris Transcription Factor ZBP-89 Regulates the Activity of the Ornithine Decarboxylase Promoter J. Biol. Chem., August 7, 1998; 273(32): 19955 - 19964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Culí and J. Modolell Proneural gene self-stimulation in neural precursors: an essential mechanism for sense organ development that is regulated by Notch signaling Genes & Dev., July 1, 1998; 12(13): 2036 - 2047. [Abstract] [Full Text]
|
|
|
|
|
|
Y. Lee, T. Shioi, H. Kasahara, S. M. Jobe, R. J. Wiese, B. E. Markham, and S. Izumo The Cardiac Tissue-Restricted Homeobox Protein Csx/Nkx2.5 Physically Associates with the Zinc Finger Protein GATA4 and Cooperatively Activates Atrial Natriuretic Factor Gene Expression Mol. Cell. Biol., June 1, 1998; 18(6): 3120 - 3129. [Abstract] [Full Text]
|
|
|
|
|
|
X.-C. Wu, R. Ye, Y. Duan, and S.-L. Wong Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis: Streptokinase with Longer Functional Half-Life Appl. Envir. Microbiol., March 1, 1998; 64(3): 824 - 829. [Abstract] [Full Text]
|
|
|
|
 |
|
 K. P. Giese, N. B. Fedorov, R. K. Filipkowski, and A. J. Silva Autophosphorylation at Thr286 of the  Calcium-Calmodulin Kinase II in LTP and LearningScience, February 6, 1998; 279(5352): 870 - 873. [Abstract] [Full Text]
|
|
|
|
 |
|
 G. Vaduva, N. C. Martin, and A. K. Hopper Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton J. Cell Biol., December 29, 1997; 139(7): 1821 - 1833. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Braun, B. Persson, H. R. Kaback, and G. von Heijne Alanine Insertion Scanning Mutagenesis of Lactose Permease Transmembrane Helices J. Biol. Chem., November 21, 1997; 272(47): 29566 - 29571. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Di Marco, I. Gloaguen, A. Demartis, I. Saggio, R. Graziani, G. Paonessa, and R. Laufer Agonistic and Antagonistic Variants of Ciliary Neurotrophic Factor (CNTF) Reveal Functional Differences between Membrane-bound and Soluble CNTF alpha -Receptor J. Biol. Chem., September 12, 1997; 272(37): 23069 - 23075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. Herzig, S. M. Jobe, H. Aoki, J. D. Molkentin, A. W. Cowley Jr., S. Izumo, and B. E. Markham Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload PNAS, July 8, 1997; 94(14): 7543 - 7548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Schneider and G. Scheiner-Bobis Involvement of the M7/M8 Extracellular Loop of the Sodium Pump alpha Subunit in Ion Transport. STRUCTURAL AND FUNCTIONAL HOMOLOGY TO P-LOOPS OF ION CHANNELS J. Biol. Chem., June 27, 1997; 272(26): 16158 - 16165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. O'Malley, S. A. Nair, H. Rubin, and B. S. Cooperman The Kinetic Mechanism of Serpin-Proteinase Complex Formation. AN INTERMEDIATE BETWEEN THE MICHAELIS COMPLEX AND THE INHIBITED COMPLEX J. Biol. Chem., February 21, 1997; 272(8): 5354 - 5359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Shiota and H. Nakayama UV endonuclease of Micrococcus luteus, a cyclobutane pyrimidine dimer-DNA glycosylase/abasic lyase: Cloning and characterization of the gene PNAS, January 21, 1997; 94(2): 593 - 598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Fiedler and G. Scheiner-Bobis Transmembrane Topology of alpha - and beta -Subunits of Na+,K+-ATPase Derived from beta -Galactosidase Fusion Proteins Expressed in Yeast J. Biol. Chem., November 15, 1996; 271(46): 29312 - 29320. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Feller, O. l. Bussy, C. Houssier, and C. Gerday Structural and Functional Aspects of Chloride Binding to Alteromonas haloplanctis alpha -Amylase J. Biol. Chem., September 27, 1996; 271(39): 23836 - 23841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Guo, L.-H. Wang, K.-H. Ruan, and R. J. Kulmacz Role of Val509 in Time-dependent Inhibition of Human Prostaglandin H Synthase-2 Cyclooxygenase Activity by Isoform-selective Agents J. Biol. Chem., August 9, 1996; 271(32): 19134 - 19139. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Veiga-da-Cunha, S. Courtois, A. Michel, E. Gosselain, and E. Van Schaftingen Amino Acid Conservation in Animal Glucokinases J. Biol. Chem., March 15, 1996; 271(11): 6292 - 6297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Patstone and P. Maher Copper and Calcium Binding Motifs in the Extracellular Domains of Fibroblast Growth Factor Receptors J. Biol. Chem., February 16, 1996; 271(7): 3343 - 3346. [Abstract] [Full Text] [PDF]
|