Europe PMC

This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy.

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

Author information

ORCIDs linked to this article

Proceedings of the National Academy of Sciences of the United States of America, 01 Nov 1982, 79(21):6409-6413
https://doi.org/10.1073/pnas.79.21.6409 PMID: 6983070 PMCID: PMC347135

Free full text in Europe PMC

Abstract 

We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.

Free full text 

Logo of pnasLink to Publisher's site
Proc Natl Acad Sci U S A. 1982 Nov; 79(21): 6409–6413.
PMCID: PMC347135
PMID: 6983070

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

G Dalbadie-McFarland, L W Cohen, A D Riggs, C Morin, K Itakura, and J H Richards
Copyright and License information Disclaimer

Abstract

We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.7M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.
 
icon of scanned page 6409
6409
icon of scanned page 6410
6410
icon of scanned page 6411
6411
icon of scanned page 6412
6412
icon of scanned page 6413
6413
 

Images in this article

Image
Image
on p.6411
Image
Image
on p.6411
Image
Image
on p.6412
Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Shortle D, Nathans D. Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome. Proc Natl Acad Sci U S A. 1978 May;75(5):2170–2174. [Europe PMC free article] [Abstract] [Google Scholar]
  • Müller W, Weber H, Meyer F, Weissmann C. Site-directed mutagenesis in DNA: generation of point mutations in cloned beta globin complementary dna at the positions corresponding to amino acids 121 to 123. J Mol Biol. 1978 Sep 15;124(2):343–358. [Abstract] [Google Scholar]
  • Razin A, Hirose T, Itakura K, Riggs AD. Efficient correction of a mutation by use of chemically synthesized DNA. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4268–4270. [Europe PMC free article] [Abstract] [Google Scholar]
  • Hutchison CA, 3rd, Phillips S, Edgell MH, Gillam S, Jahnke P, Smith M. Mutagenesis at a specific position in a DNA sequence. J Biol Chem. 1978 Sep 25;253(18):6551–6560. [Abstract] [Google Scholar]
  • Gillam S, Astell CR, Smith M. Site-specific mutagenesis using oligodeoxyribonucleotides: isolation of a phenotypically silent phi X174 mutant, with a specific nucleotide deletion, at very high efficiency. Gene. 1980 Dec;12(1-2):129–137. [Abstract] [Google Scholar]
  • Gillam S, Jahnke P, Astell C, Phillips S, Hutchison CA, 3rd, Smith M. Defined transversion mutations at a specific position in DNA using synthetic oligodeoxyribonucleotides as mutagens. Nucleic Acids Res. 1979 Jul 11;6(9):2973–2985. [Europe PMC free article] [Abstract] [Google Scholar]
  • Gillam S, Smith M. Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: II. In vitro selection of mutant DNA. Gene. 1979 Dec;8(1):99–106. [Abstract] [Google Scholar]
  • Montell C, Fisher EF, Caruthers MH, Berk AJ. Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site. Nature. 1982 Feb 4;295(5848):380–384. [Abstract] [Google Scholar]
  • Kudo I, Leineweber M, RajBhandary UL. Site-specific mutagenesis on cloned DNAs: generation of a mutant of Escherichia coli tyrosine suppressor tRNA in which the sequence G-T-T-C corresponding to the universal G-T-pseudouracil-C sequence of tRNAs is changed to G-A-T-C. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4753–4757. [Europe PMC free article] [Abstract] [Google Scholar]
  • Simons GF, Veeneman GH, Konings RN, van Boom JH, Schoemakers JG. Oligonucleotide-directed mutagenesis of gene IX of bacteriophage M13. Nucleic Acids Res. 1982 Feb 11;10(3):821–832. [Europe PMC free article] [Abstract] [Google Scholar]
  • Wallace RB, Johnson PF, Tanaka S, Schöld M, Itakura K, Abelson J. Directed deletion of a yeast transfer RNA intervening sequence. Science. 1980 Sep 19;209(4463):1396–1400. [Abstract] [Google Scholar]
  • Hall A, Knowles JR. Directed selective pressure on a beta-lactamase to analyse molecular changes involved in development of enzyme function. Nature. 1976 Dec 23;264(5588):803–804. [Abstract] [Google Scholar]
  • Shortle D, Koshland D, Weinstock GM, Botstein D. Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5375–5379. [Europe PMC free article] [Abstract] [Google Scholar]
  • Koshland D, Botstein D. Secretion of beta-lactamase requires the carboxy end of the protein. Cell. 1980 Jul;20(3):749–760. [Abstract] [Google Scholar]
  • Shortle D, Grisafi P, Benkovic SJ, Botstein D. Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1588–1592. [Europe PMC free article] [Abstract] [Google Scholar]
  • Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [Abstract] [Google Scholar]
  • Sutcliffe JG. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3737–3741. [Europe PMC free article] [Abstract] [Google Scholar]
  • Fisher J, Belasco JG, Khosla S, Knowles JR. beta-Lactamase proceeds via an acyl-enzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin. Biochemistry. 1980 Jun 24;19(13):2895–2901. [Abstract] [Google Scholar]
  • Pratt RF, Loosemore MJ. 6-beta-bromopenicillanic acid, a potent beta-lactamase inhibitor. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4145–4149. [Europe PMC free article] [Abstract] [Google Scholar]
  • Knott-Hunziker V, Waley SG, Orlek BS, Sammes PG. Penicillinase active sites: labelling of serine-44 in beta-lactamase I by 6beta-bromopenicillanic acid. FEBS Lett. 1979 Mar 1;99(1):59–61. [Abstract] [Google Scholar]
  • Knott-Hunziker V, Orlek BS, Sammes PG, Waley SG. 6 beta-Bromopenicillanic acid inactivates beta-lactamase I. Biochem J. 1979 Jan 1;177(1):365–367. [Europe PMC free article] [Abstract] [Google Scholar]
  • Loosemore MJ, Cohen SA, Pratt RF. Inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid: kinetics. Biochemistry. 1980 Aug 19;19(17):3990–3995. [Abstract] [Google Scholar]
  • Cohen SA, Pratt RF. Inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopencillanic acid: mechanism. Biochemistry. 1980 Aug 19;19(17):3996–4003. [Abstract] [Google Scholar]
  • Ambler RP. The structure of beta-lactamases. Philos Trans R Soc Lond B Biol Sci. 1980 May 16;289(1036):321–331. [Abstract] [Google Scholar]
  • Boyer HW, Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. [Abstract] [Google Scholar]
  • Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. [Europe PMC free article] [Abstract] [Google Scholar]
  • Vincent WS, 3rd, Goldstein ES. Rapid preparation of covalently closed circular DNA by acridine yellow affinity chromatography. Anal Biochem. 1981 Jan 1;110(1):123–127. [Abstract] [Google Scholar]
  • Norgard MV. Rapid and simple removal of contaminating RNA from plasmid DNA without the use of RNase. Anal Biochem. 1981 May 1;113(1):34–42. [Abstract] [Google Scholar]
  • Ish-Horowicz D, Burke JF. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. [Europe PMC free article] [Abstract] [Google Scholar]
  • Ito H, Ike Y, Ikuta S, Itakura K. Solid phase synthesis of polynucleotides. VI. Further studies on polystyrene copolymers for the solid support. Nucleic Acids Res. 1982 Mar 11;10(5):1755–1769. [Europe PMC free article] [Abstract] [Google Scholar]
  • Gergen JP, Stern RH, Wensink PC. Filter replicas and permanent collections of recombinant DNA plasmids. Nucleic Acids Res. 1979 Dec 20;7(8):2115–2136. [Europe PMC free article] [Abstract] [Google Scholar]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [Abstract] [Google Scholar]
  • Maxam AM, Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. [Abstract] [Google Scholar]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [Europe PMC free article] [Abstract] [Google Scholar]
Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Full text links 

Read article at publisher's site: https://doi.org/10.1073/pnas.79.21.6409

Read article for free, from open access legal sources, via Unpaywall: https://europepmc.org/articles/pmc347135?pdf=renderUnpaywall

Citations & impact 

Impact metrics

103
Citations
Jump to Citations

Citations of article over time

Alternative metrics

Altmetric item for https://www.altmetric.com/details/41389628
Altmetric
Discover the attention surrounding your research
https://www.altmetric.com/details/41389628

Smart citations by scite.ai
Smart citations by scite.ai include citation statements extracted from the full text of the citing article. The number of the statements may be higher than the number of citations provided by EuropePMC if one paper cites another multiple times or lower if scite has not yet processed some of the citing articles.
Explore citation contexts and check if this article has been supported or disputed.
https://scite.ai/reports/10.1073/pnas.79.21.6409

Supporting
Mentioning
Contrasting
0
79
1

Article citations

Go to all (103) article citations

Similar Articles 

To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.

Funding 

Funders who supported this work.

NIGMS NIH HHS (3)