Nucleic Acids Research, 1994, Vol. 22, No. 13 2587-2591
© 1994
RNA |
A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase
Temple University School of Medicine, Department of Microbiology and Immunology and The Sol Sherry Thrombosis Research Center 3400 N. Broad Street, Philadelphia, PA 19140, USA
*To whom correspondence should be addressed at: Department of Microbiology/Immunology, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, PA 19140, USA
Received March 22, 1994. Revised May 24, 1994. Accepted May 24, 1994.
A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Stahl, J. Beck, and M. Nassal Chaperones Activate Hepadnavirus Reverse Transcriptase by Transiently Exposing a C-Proximal Region in the Terminal Protein Domain That Contributes to {varepsilon} RNA Binding J. Virol., December 15, 2007; 81(24): 13354 - 13364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ohkuri and A. Yamagishi The Effects of Mutations at Position 253 on the Thermostability of the Bacillus subtilis 3-Isopropylmalate Dehydrogenase Subunit Interface J. Biochem., June 1, 2007; 141(6): 791 - 797. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Gardsvoll and M. Ploug Mapping of the Vitronectin-binding Site on the Urokinase Receptor: INVOLVEMENT OF A COHERENT RECEPTOR INTERFACE CONSISTING OF RESIDUES FROM BOTH DOMAIN I AND THE FLANKING INTERDOMAIN LINKER REGION J. Biol. Chem., May 4, 2007; 282(18): 13561 - 13572. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-W. Chen, C.-M. Chiang, T.-H. Tseng, and S.-M. Yu Interaction between Rice MYBGA and the Gibberellin Response Element Controls Tissue-Specific Sugar Sensitivity of {alpha}-Amylase Genes PLANT CELL, September 1, 2006; 18(9): 2326 - 2340. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-H. Kang, W.-S. Lim, S.-H. Hwang, J.-W. Park, H.-S. Choi, and K.-H. Kim Importance of the C-terminal domain of soybean mosaic virus coat protein for subunit interactions J. Gen. Virol., January 1, 2006; 87(1): 225 - 229. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. TSAGKALIA, F. LEONTIADOU, M. A. XAPLANTERI, G. PAPADOPOULOS, D. L. KALPAXIS, and T. CHOLI-PAPADOPOULOU Ribosomes containing mutants of L4 ribosomal protein from Thermus thermophilus display multiple defects in ribosomal functions and sensitivity against erythromycin RNA, November 1, 2005; 11(11): 1633 - 1639. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tatematsu, S. Ward, O. Leyser, Y. Kamiya, and E. Nambara Identification of cis-Elements That Regulate Gene Expression during Initiation of Axillary Bud Outgrowth in Arabidopsis Plant Physiology, June 1, 2005; 138(2): 757 - 766. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. van der Sloot, M. M. Mullally, G. Fernandez-Ballester, L. Serrano, and W. J. Quax Stabilization of TRAIL, an all-{beta}-sheet multimeric protein, using computational redesign Protein Eng. Des. Sel., September 1, 2004; 17(9): 673 - 680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. K. Ganser-Pornillos, U. K. von Schwedler, K. M. Stray, C. Aiken, and W. I. Sundquist Assembly Properties of the Human Immunodeficiency Virus Type 1 CA Protein J. Virol., March 1, 2004; 78(5): 2545 - 2552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Ortiz-Salmeron, M. Nuccetelli, A. J. Oakley, M. W. Parker, M. L. Bello, and L. Garcia-Fuentes Thermodynamic Description of the Effect of the Mutation Y49F on Human Glutathione Transferase P1-1 in Binding with Glutathione and the Inhibitor S-Hexylglutathione J. Biol. Chem., November 21, 2003; 278(47): 46938 - 46948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ohkuri and A. Yamagishi Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface Protein Eng. Des. Sel., August 1, 2003; 16(8): 615 - 621. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yunoue, H. Tokuo, K. Fukunaga, L. Feng, T. Ozawa, T. Nishi, A. Kikuchi, S. Hattori, J. Kuratsu, H. Saya, et al. Neurofibromatosis Type I Tumor Suppressor Neurofibromin Regulates Neuronal Differentiation via Its GTPase-activating Protein Function toward Ras J. Biol. Chem., July 11, 2003; 278(29): 26958 - 26969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Kuhn, A. Nemeth, S. Meyer, and E. Wahle The RNA Binding Domains of the Nuclear poly(A)-binding Protein J. Biol. Chem., May 2, 2003; 278(19): 16916 - 16925. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Yung, F. Dela Cruz, S. Hamren, J. Zhu, M. Tsutsumi, J. W. Bloom, M. Caudle, S. Roczniak, T. Todd, L. Lemoine, et al. Generation of Highly Selective VPAC2 Receptor Agonists by High Throughput Mutagenesis of Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-activating Peptide J. Biol. Chem., March 14, 2003; 278(12): 10273 - 10281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Song, A. Patel, K. Thamatrakoln, C. Liu, D. Feng, C. Clayberger, and A. M. Krensky Functional Domains and DNA-binding Sequences of RFLAT-1/KLF13, a Kruppel-like Transcription Factor of Activated T Lymphocytes J. Biol. Chem., August 9, 2002; 277(33): 30055 - 30065. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Handa, N. Acharya, and U. Varshney Effects of mutations at tyrosine 66 and asparagine 123 in the active site pocket of Escherichia coli uracil DNA glycosylase on uracil excision from synthetic DNA oligomers: evidence for the occurrence of long-range interactions between the enzyme and substrate Nucleic Acids Res., July 15, 2002; 30(14): 3086 - 3095. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Fletcher and R. J. Jackson Pestivirus Internal Ribosome Entry Site (IRES) Structure and Function: Elements in the 5' Untranslated Region Important for IRES Function J. Virol., April 16, 2002; 76(10): 5024 - 5033. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Linnemann, C. Kiel, P. Herter, and C. Herrmann The Activation of RalGDS Can Be Achieved Independently of Its Ras Binding Domain. IMPLICATIONS OF AN ACTIVATION MECHANISM IN Ras EFFECTOR SPECIFICITY AND SIGNAL DISTRIBUTION J. Biol. Chem., March 1, 2002; 277(10): 7831 - 7837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Sommer, F. Drepper, and M. Hippler The Luminal Helix l of PsaB Is Essential for Recognition of Plastocyanin or Cytochrome c6 and Fast Electron Transfer to Photosystem I in Chlamydomonas reinhardtii J. Biol. Chem., February 15, 2002; 277(8): 6573 - 6581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Furger, J. Monks, and N. J. Proudfoot The Retroviruses Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Adopt Radically Different Strategies To Regulate Promoter-Proximal Polyadenylation J. Virol., December 1, 2001; 75(23): 11735 - 11746. [Abstract] [Full Text]
|
|
|
|
|
|
M. Wagner, C. Fingerhut, H. J. Gross, and A. Schon The first phytoplasma RNase P RNA provides new insights into the sequence requirements of this ribozyme Nucleic Acids Res., June 15, 2001; 29(12): 2661 - 2665. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Cicero, S. T. Hubl, C. J. Harrison, O. Littlefield, J. A. Hardy, and H. C. M. Nelson The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity Nucleic Acids Res., April 15, 2001; 29(8): 1715 - 1723. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. L. Keplinger, X. Guo, J. Quine, Y. Feng, and D. R. Cavener Complex Organization of Promoter and Enhancer Elements Regulate the Tissue- and Developmental Stage-Specific Expression of the Drosophila melanogaster Gld Gene Genetics, February 1, 2001; 157(2): 699 - 716. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. G. Baginski, D. C. Pevear, M. Seipel, S. C. C. Sun, C. A. Benetatos, S. K. Chunduru, C. M. Rice, and M. S. Collett Mechanism of action of a pestivirus antiviral compound PNAS, June 23, 2000; (2000) 140220397. [Abstract] [Full Text]
|
|
|
|
|
|
D. Solecki, G. Bernhardt, M. Lipp, and E. Wimmer Identification of a Nuclear Respiratory Factor-1 Binding Site within the Core Promoter of the human polio virus receptor/CD155 Gene J. Biol. Chem., April 21, 2000; 275(17): 12453 - 12462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. T. Klein Gunnewiek, R. I. Hussein, Y. van Aarssen, D. Palacios, R. de Jong, W. J. van Venrooij, and S. I. Gunderson Fourteen Residues of the U1 snRNP-Specific U1A Protein Are Required for Homodimerization, Cooperative RNA Binding, and Inhibition of Polyadenylation Mol. Cell. Biol., March 15, 2000; 20(6): 2209 - 2217. [Abstract] [Full Text]
|
|
|
|
|
|
U. Bertsch, C. Deschermeier, W. Fanick, I. Girkontaite, K. Hillemeier, H. Johnen, W. Weglohner, F. Emmrich, and G. W. Mayr The Second Messenger Binding Site of Inositol 1,4,5-Trisphosphate 3-Kinase Is Centered in the Catalytic Domain and Related to the Inositol Trisphosphate Receptor Site J. Biol. Chem., January 21, 2000; 275(3): 1557 - 1564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Wierzbicka-Patynowski, S. Niewiarowski, C. Marcinkiewicz, J. J. Calvete, M. M. Marcinkiewicz, and M. A. McLane Structural Requirements of Echistatin for the Recognition of alpha vbeta 3 and alpha 5beta 1 Integrins J. Biol. Chem., December 31, 1999; 274(53): 37809 - 37814. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Gardsvoll, K. Dano, and M. Ploug Mapping Part of the Functional Epitope for Ligand Binding on the Receptor for Urokinase-type Plasminogen Activator by Site-directed Mutagenesis J. Biol. Chem., December 31, 1999; 274(53): 37995 - 38003. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Suzuki, B. Chen, and D. L. Nuss Mapping of a Hypovirus p29 Protease Symptom Determinant Domain with Sequence Similarity to Potyvirus HC-Pro Protease J. Virol., November 1, 1999; 73(11): 9478 - 9484. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Verbeek, J. Lekstrom-Himes, D. J. Park, P. M.-C. Dang, P. T. Vuong, S. Kawano, B. M. Babior, K. Xanthopoulos, and H. P. Koeffler Myeloid Transcription Factor C/EBPvarepsilon Is Involved in the Positive Regulation of Lactoferrin Gene Expression in Neutrophils Blood, November 1, 1999; 94(9): 3141 - 3150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-L. Zhu, C.-I Pao, E. Hunter Jr., K.-w. M. Lin, G.-j. Wu, and L. S. Phillips Identification of Core Sequences Involved in Metabolism-Dependent Nuclear Protein Binding to the Rat Insulin-Like Growth Factor I Gene Endocrinology, October 1, 1999; 140(10): 4761 - 4771. [Abstract] [Full Text]
|
|
|
|
|
|
N. Fischer, P. Setif, and J.-D. Rochaix Site-directed Mutagenesis of the PsaC Subunit of Photosystem I. FB IS THE CLUSTER INTERACTING WITH SOLUBLE FERREDOXIN J. Biol. Chem., August 13, 1999; 274(33): 23333 - 23340. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Bos, D. Hogan, and R. J. Belland Homologue Scanning Mutagenesis Reveals CD66 Receptor Residues Required for Neisserial Opa Protein Binding J. Exp. Med., August 2, 1999; 190(3): 331 - 340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Raychaudhuri, L. Niu, J. Conrad, B. G. Lane, and J. Ofengand Functional Effect of Deletion and Mutation of the Escherichia coli Ribosomal RNA and tRNA Pseudouridine Synthase RluA J. Biol. Chem., July 2, 1999; 274(27): 18880 - 18886. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Bauer, G. Mirey, I. R. Vetter, J. A. Garcia-Ranea, A. Valencia, A. Wittinghofer, J. H. Camonis, and R. H. Cool Effector Recognition by the Small GTP-binding Proteins Ras and Ral J. Biol. Chem., June 18, 1999; 274(25): 17763 - 17770. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Linnemann, M. Geyer, B. K. Jaitner, C. Block, H. R. Kalbitzer, A. Wittinghofer, and C. Herrmann Thermodynamic and Kinetic Characterization of the Interaction between the Ras Binding Domain of AF6 and Members of the Ras Subfamily J. Biol. Chem., May 7, 1999; 274(19): 13556 - 13562. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. van den Berghe, R. H. Cool, and A. Wittinghofer Discriminatory Residues in Ras and Rap for Guanine Nucleotide Exchange Factor Recognition J. Biol. Chem., April 16, 1999; 274(16): 11078 - 11085. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Linari, M. Ueffing, F. Manson, A. Wright, T. Meitinger, and J. Becker The retinitis pigmentosa GTPase regulator, RPGR, interacts with the delta subunit of rod cyclic GMP phosphodiesterase PNAS, February 16, 1999; 96(4): 1315 - 1320. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Solecki, E. Wimmer, M. Lipp, and G. Bernhardt Identification and Characterization of the cis-Acting Elements of the Human CD155 Gene Core Promoter J. Biol. Chem., January 15, 1999; 274(3): 1791 - 1800. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Landis and L. A. Loeb Random Sequence Mutagenesis and Resistance to 5-Fluorouridine in Human Thymidylate Synthases J. Biol. Chem., October 2, 1998; 273(40): 25809 - 25817. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Ho, K. Badellino, F. A. Baglia, and P. N. Walsh A Binding Site for Heparin in the Apple 3 Domain of Factor XI J. Biol. Chem., June 26, 1998; 273(26): 16382 - 16390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hippler, F. Drepper, W. Haehnel, and J.-D. Rochaix The N-terminal domain of PsaF: Precise recognition site for binding and fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii PNAS, June 23, 1998; 95(13): 7339 - 7344. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Martin-Bermudo, O. M. Dunin-Borkowski, and N. H. Brown Modulation of Integrin Activity is Vital for Morphogenesis J. Cell Biol., May 18, 1998; 141(4): 1073 - 1081. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-A. Lu, E.-K. Lim, and S.-M. Yu Sugar Response Sequence in the Promoter of a Rice alpha -Amylase Gene Serves as a Transcriptional Enhancer J. Biol. Chem., April 24, 1998; 273(17): 10120 - 10131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Tubulekas and P. Liljestrom Suppressors of Cleavage-Site Mutations in the p62 Envelope Protein of Semliki Forest Virus Reveal Dynamics in Spike Structure and Function J. Virol., April 1, 1998; 72(4): 2825 - 2831. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Mo and T. C. Holland Determination of the Transmembrane Topology of Herpes Simplex Virus Type 1 Glycoprotein K (gK) J. Biol. Chem., December 26, 1997; 272(52): 33305 - 33311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Fritz and B. Kaina rhoB Encoding a UV-inducible Ras-related Small GTP-binding Protein Is Regulated by GTPases of the Rho Family and Independent of JNK, ERK, and p38 MAP Kinase J. Biol. Chem., December 5, 1997; 272(49): 30637 - 30644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Arai, F. S. Monteclaro, C.-L. Tsou, C. Franci, and I. F. Charo Dissociation of Chemotaxis from Agonist-induced Receptor Internalization in a Lymphocyte Cell Line Transfected with CCR2B. EVIDENCE THAT DIRECTED MIGRATION DOES NOT REQUIRE RAPID MODULATION OF SIGNALING AT THE RECEPTOR LEVEL J. Biol. Chem., October 3, 1997; 272(40): 25037 - 25042. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. S. Monteclaro and I. F. Charo The Amino-terminal Domain of CCR2 Is Both Necessary and Sufficient for High Affinity Binding of Monocyte Chemoattractant Protein 1. RECEPTOR ACTIVATION BY A PSEUDO-TETHERED LIGAND J. Biol. Chem., September 12, 1997; 272(37): 23186 - 23190. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Yeh, S. Wu, J. T. Murphy, and J. C. Lagarias A Cyanobacterial Phytochrome Two-Component Light Sensory System Science, September 5, 1997; 277(5331): 1505 - 1508. [Abstract] [Full Text]
|
|
|
|
|
|
E. Ersdal-Badju, A. Lu, Y. Zuo, V. Picard, and S. C. Bock Identification of the Antithrombin III Heparin Binding Site J. Biol. Chem., August 1, 1997; 272(31): 19393 - 19400. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Gosling, F. S. Monteclaro, R. E. Atchison, H. Arai, C.-L. Tsou, M. A. Goldsmith, and I. F. Charo Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity PNAS, May 13, 1997; 94(10): 5061 - 5066. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Basile, A. Sica, E. d'Aniello, F. Breviario, G. Garrido, M. Castellano, A. Mantovani, and M. Introna Characterization of the Promoter for the Human Long Pentraxin PTX3. ROLE OF NF-kappa B IN TUMOR NECROSIS FACTOR-alpha AND INTERLEUKIN-1beta REGULATION J. Biol. Chem., March 28, 1997; 272(13): 8172 - 8178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M P Ashe, P Griffin, W James, and N J Proudfoot Poly(A) site selection in the HIV-1 provirus: inhibition of promoter-proximal polyadenylation by the downstream major splice donor site. Genes & Dev., December 1, 1995; 9(23): 3008 - 3025. [Abstract] [PDF]
|
|
|
|
 |
|
 M P Weiner and G L Costa Rapid PCR site-directed mutagenesis. Genome Res., December 1, 1994; 4(3): S131 - S136. [PDF]
|
|
|
|
|
|
R. Dorfman and P. N. Walsh Noncovalent Interactions of the Apple 4 Domain That Mediate Coagulation Factor XI Homodimerization J. Biol. Chem., February 23, 2001; 276(9): 6429 - 6438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Boudreaux, F. MacMillan, C. Teutloff, R. Agalarov, F. Gu, S. Grimaldi, R. Bittl, K. Brettel, and K. Redding Mutations in Both Sides of the Photosystem I Reaction Center Identify the Phylloquinone Observed by Electron Paramagnetic Resonance Spectroscopy J. Biol. Chem., September 28, 2001; 276(40): 37299 - 37306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Inagaki, R. Maitra, K. Satoh, and H. B. Pakrasi Amino Acid Residues That Are Critical for in Vivo Catalytic Activity of CtpA, the Carboxyl-terminal Processing Protease for the D1 Protein of Photosystem II J. Biol. Chem., August 3, 2001; 276(32): 30099 - 30105. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. H. Wilkinson, F. S. London, and P. N. Walsh Residues 88-109 of Factor IXa Are Important for Assembly of the Factor X Activating Complex J. Biol. Chem., February 15, 2002; 277(8): 5725 - 5733. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. G. Baginski, D. C. Pevear, M. Seipel, S. C |