Nucleic Acids Research, 1984, Vol. 12, No. 24 9441-9456
© 1984
MOLECULAR BIOLOGY |
The gapped duplex DNA approach to oligonucleotide-directed mutation construction
Institut für Genetik, Universität zu Köln Weyertal 121, D-5000 Köln 41 3Max-Planck-Institut für Molekulare Genetik Ihnestrasse 63, D-1000 Berlin 33, FRG
*To whom correspondence should be addressed
Received November 12, 1984. Accepted November 22, 1984.
A simple and efficient method is described to introduce structurally pre-determined mutations into recombinant genomes of filamentous phage M13. The method rests on gapped duplex DNA (gdDNA) molecules of the phage M13 genome as the key intermediate. In this gdDNA, the (+) and the (shorter) (–)strand carry different genetic markers in such a way, that a rigorous selection can be applied for phage carrying the markers of the (–)strand. For introduction of the mutation, a synthetic oligonucleotide with partial homology to a target site within the single stranded DNA region is annealed to the gdDNA. The oligonucleotide subsequently becomes part of the (–)strand by enzymatic DNA gap filling and sealing. This physical linkage is preserved at the genetic level after transfection of a recipient E.coli strain deficient in DNA mismatch correction, so that the synthetic marker can be selected from the phage progeny independent from its potential phenotype. It is demonstrated that by this method mutants can be constructed with marker yields in excess of 70%.
1Present address: Max-Planck-Institut für Biochemie, Abteilung Zellbiologie, Am Klopferspitz 2, D-8033 Martinsried, FRG
2Present address: A.N.Belozersky Laboratory of Moscow State University, Moscow, W-234, 119899, USSR
4Present address: Biozentrun der Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Chen, R. Jin, H.-Y. Dong, and Y.-M. Feng In Vitro Refolding/Unfolding Pathways of Amphioxus Insulin-like Peptide: IMPLICATIONS FOR FOLDING BEHAVIOR OF INSULIN FAMILY PROTEINS J. Biol. Chem., December 31, 2004; 279(53): 55224 - 55233. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Vinje, S. J. G. Oudejans, J. R. Stewart, M. D. Sobsey, and S. C. Long Molecular Detection and Genotyping of Male-Specific Coliphages by Reverse Transcription-PCR and Reverse Line Blot Hybridization Appl. Envir. Microbiol., October 1, 2004; 70(10): 5996 - 6004. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Gorke, L. Fraysse, and A. Galinier Drastic Differences in Crh and HPr Synthesis Levels Reflect Their Different Impacts on Catabolite Repression in Bacillus subtilis J. Bacteriol., May 15, 2004; 186(10): 2992 - 2995. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Gorke Regulation of the Escherichia coli Antiterminator Protein BglG by Phosphorylation at Multiple Sites and Evidence for Transfer of Phosphoryl Groups between Monomers J. Biol. Chem., November 21, 2003; 278(47): 46219 - 46229. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-Y. Jia, Z.-Y. Guo, Y. Wang, Y. Xu, S.-S. Duan, and Y.-M. Feng Peptide models of four possible insulin folding intermediates with two disulfides Protein Sci., November 1, 2003; 12(11): 2412 - 2419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. MARMIER-GOURRIER, A. CLERY, V. SENTY-SEGAULT, B. CHARPENTIER, F. SCHLOTTER, F. LECLERC, R. FOURNIER, and C. BRANLANT A structural, phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA RNA, July 1, 2003; 9(7): 821 - 838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-h. Fang, B.-J. Wang, C.-H. Wang, S.-J. Lee, Y.-T. Chang, Y.-K. Chuang, and C.-N. Lee DNA Loop Repair by Escherichia coli Cell Extracts J. Biol. Chem., June 13, 2003; 278(25): 22446 - 22452. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yan, Z.-Y. Guo, X.-W. Gong, D. Xi, and Y.-M. Feng A peptide model of insulin folding intermediate with one disulfide Protein Sci., April 1, 2003; 12(4): 768 - 775. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Toyama, M. Yamashita, M. Yoneda, A. Zaborowski, M. Nagato, H. Ono, N. Hirayama, and Y. Murooka Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis Protein Eng. Des. Sel., June 1, 2002; 15(6): 477 - 483. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Barreiro, E. González-Lavado, and J. F. Martín Organization and Transcriptional Analysis of a Six-Gene Cluster around the rplK-rplA Operon of Corynebacterium glutamicum Encoding the Ribosomal Proteins L11 and L1 Appl. Envir. Microbiol., May 1, 2001; 67(5): 2183 - 2190. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. Jacquenet, D. Ropers, P. S. Bilodeau, L. Damier, A. Mougin, C. M. Stoltzfus, and C. Branlant Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing Nucleic Acids Res., January 15, 2001; 29(2): 464 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Krispin and R. Allmansberger The Bacillus subtilis galE Gene Is Essential in the Presence of Glucose and Galactose J. Bacteriol., April 15, 1998; 180(8): 2265 - 2270. [Abstract] [Full Text]
|
|
|
|
|
|
W.-h. Fang, J.-Y. Wu, and M.-J. Su Methyl-directed Repair of Mismatched Small Heterologous Sequences in Cell Extracts from Escherichia coli J. Biol. Chem., September 5, 1997; 272(36): 22714 - 22720. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Dhoest, Z. Zhao, B. De Geest, E. Deridder, A. Sillen, Y. Engelborghs, D. Collen, and P. Holvoet Role of the Arg123-Tyr166 Paired Helix of Apolipoprotein A-I in Lecithin:Cholesterol Acyltransferase Activation J. Biol. Chem., June 20, 1997; 272(25): 15967 - 15972. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Kilic, M. B. Dalton, S. K. Burrell, J. P. Mayer, S. D. Patterson, and M. Sinensky In Vitro Assay and Characterization of the Farnesylation-dependent Prelamin A Endoprotease J. Biol. Chem., February 21, 1997; 272(8): 5298 - 5304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Unno, H. Shimada, Y. Toba, R. Makino, and Y. Ishimura Role of Arg112 of Cytochrome P450cam in the Electron Transfer from Reduced Putidaredoxin. ANALYSES WITH SITE-DIRECTED MUTANTS J. Biol. Chem., July 26, 1996; 271(30): 17869 - 17874. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. H.F. a. J. W. Engels and S. H. Fass Influence of Specific Signal Peptide Mutations on the Expression and Secretion of the alpha -Amylase Inhibitor Tendamistat in Streptomyces lividans J. Biol. Chem., June 21, 1996; 271(25): 15244 - 15252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-P. Nicolas, Gér. Lambeau, and M. Lazdunski Identification of the Binding Domain for Secretory Phospholipases A(2) on Their M-type 180-kDa Membrane Receptor J. Biol. Chem., December 1, 1995; 270(48): 28869 - 28873. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. G. Horrevoets, A. E. Smilde, J. C. Fredenburgh, H. Pannekoek, and M. E. Nesheim The Activation-resistant Conformation of Recombinant Human Plasminogen Is Stabilized by Basic Residues in the Amino-terminal Hinge Region J. Biol. Chem., June 30, 1995; 270(26): 15770 - 15776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Pilon, H. Wienk, W. Sips, M. de Swaaf, I. Talboom, R. van 't Hof, G. de Korte-Kool, R. Demel, P. Weisbeek, and B. de Kruijff Functional Domains of the Ferredoxin Transit Sequence Involved in Chloroplast Import J. Biol. Chem., February 24, 1995; 270(8): 3882 - 3893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kim, S.-Y. Namgoong, M. Jayaram, and R. M. Harshey Step-arrest Mutants of Phage Mu Transposase J. Biol. Chem., January 20, 1995; 270(3): 1472 - 1479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Creutz, N. G. Kambouris, S. L. Snyder, H. C. Hamman, M. R. Nelson, W. Liu, and P. Rock Effects of the expression of mammalian annexins in yeast secretory mutants J. Cell Sci., December 1, 1992; 103(4): 1177 - 1192. [Abstract] [PDF]
|
|
|
|
 |
|
 O Halevy, D Michalovitz, and M Oren Different tumor-derived p53 mutants exhibit distinct biological activities Science, October 5, 1990; 250(4977): 113 - 116. [Abstract] [PDF]
|
|
|
|
|
|
E Blachly-Dyson, S Peng, M Colombini, and M Forte Selectivity changes in site-directed mutants of the VDAC ion channel: structural implications Science, March 9, 1990; 247(4947): 1233 - 1236. [Abstract] [PDF]
|
|
|
|
|
|
C Guerrier-Takada, N Lumelsky, and S Altman Specific interactions in RNA enzyme-substrate complexes Science, December 22, 1989; 246(4937): 1578 - 1584. [Abstract] [PDF]
|
|
|
|
|
|
O. Kuipers, M. Thunnissen, P de Geus, B. Dijkstra, J Drenth, H. Verheij, and G. de Haas Enhanced activity and altered specificity of phospholipase A2 by deletion of a surface loop Science, April 7, 1989; 244(4900): 82 - 85. [Abstract] [PDF]
|
|
|
|
|
|
K Luger, U Hommel, M Herold, J Hofsteenge, and K Kirschner Correct folding of circularly permuted variants of a beta alpha barrel enzyme in vivo Science, January 13, 1989; 243(4888): 206 - 210. [Abstract] [PDF]
|
|
|
|
|
|
M Better, C. Chang, R. Robinson, and A. Horwitz Escherichia coli secretion of an active chimeric antibody fragment Science, May 20, 1988; 240(4855): 1041 - 1043. [Abstract] [PDF]
|
|
|
|
|
|
A Bachmair, D Finley, and A Varshavsky In vivo half-life of a protein is a function of its amino-terminal residue Science, October 10, 1986; 234(4773): 179 - 186. [Abstract] [PDF]
|
|
|
|
|
|
D Botstein and D Shortle Strategies and applications of in vitro mutagenesis Science, September 20, 1985; 229(4719): 1193 - 1201. [PDF]
|
|
|
|
|
|
S. Jacquenet, A. Mereau, P. S. Bilodeau, L. Damier, C. M. Stoltzfus, and C. Branlant A Second Exon Splicing Silencer within Human Immunodeficiency Virus Type 1 tat Exon 2 Represses Splicing of Tat mRNA and Binds Protein hnRNP H J. Biol. Chem., October 26, 2001; 276(44): 40464 - 40475. [Abstract] [Full Text] [PDF]
|
|