Site-Directed Mutagenesis

  • John R. Adair
  • T. Paul Wallace
Part of the Springer Protocols Handbooks book series (SPH)


The ability to create defined mutations and to generate precise fusions of protein domams by modifications at the DNA level is now an accepted and routine technique in molecular biology This ability has developed over the past 10–l5 years and rests on a foundation of experiments on DNA.DNA hybridization that stretches back to the 1960s The 1993 Nobel lectures (1,2) provide a timely, historical survey of the development of key stages in the development of current methods.


  1. 1.
    Smith, M (1993) Synthetic DNA and Biology The Nobel Foundation 1994, Stockholm,SwedenGoogle Scholar
  2. 2.
    Mulhs, K B (1993) The Polymerase Chain Reaction The Nobel Foundation 1994,Stockholm, SwedenGoogle Scholar
  3. 3.
    Carter, P (1986) Site-directed mutagenesis Biochem J 237, 1–7PubMedGoogle Scholar
  4. 4.
    Lehman, I R (1981) DNA polymerase I of Escherrchla co in The Enzymes vol 14A (Boyer, P D, ed ), Academic, New York, pp 16–38Google Scholar
  5. 5.
    Klenow, H and Henmngsen, I (1970) Selected ehmmation of the exonuclease activity of the deoxyrlbonuclelc acid polymerase from Escherzchia coli B by limited proteolysis Proc Natl Acad SCI USA 65, 168–175PubMedCrossRefGoogle Scholar
  6. 6.
    Kienow, H, Overgaard-Hansen, K, and Patkar, S A (1971) Proteolytlc cleavage of native DNA polymerase into two different catalytic fragments Influence of assay condotions on the change of exonuclease activity and polymerase activity Eur J Biochem 22, 371–381CrossRefGoogle Scholar
  7. 7.
    Nossal, N G (1974) DNA synthesis on a double stranded DNA template by the T4 bacteriophage DNA polymerase and the T4 gene 32 DNA unwmding protein J Biol Chem 249,5668–5676PubMedGoogle Scholar
  8. 8.
    Tabor, S, Huber, H E, and Richardson, C C (1987) Escherlchia coli thloredoxm confers processlvlty on the DNA polymerase activity of the gene 5 protein of bacteriophage T7 J Biol Chem 262, 16,212–16,223PubMedGoogle Scholar
  9. 9.
    Nossal, N G (1984) Prokaryotlc DNA replication systems Annu Rev Biochem 8, pp 581–615Google Scholar
  10. 10.
    Sayers, J R, Krekel, C, and Eckstem, F (1992) Rapid high-efficiency site-directed mutagenesis by the phosphorothloate approach BioTechnzques 13,592–596Google Scholar
  11. 11.
    Kunkel, T A (1985) Rapid and efficient site specific mutagenesis without phenotyplc selection Proc Natl Acad Sci USA 82,488–492PubMedCrossRefGoogle Scholar
  12. 12.
    Kramer, W, Schugart, K, and Fritz, H-J (1982) Directed mutagenesis of DNA cloned in filamentous phage influence of hemlmethylated GATC sites on marker recovery from restrIction fragments Nuclerc Acids Res 10,6475–6485CrossRefGoogle Scholar
  13. 13.
    Fritz, H-J (1885) The ohgonucleotlde-directed construction of mutations in recomblnant filamentous phage, in DNA Cloning A Practzcal Approach, vol I (Glover, D M, ed ), IRL Press Ltd, Oxford, UK, pp 15 1–163Google Scholar
  14. 14.
    Kramer, B, Kramer, W, and Fritz, H-J (1984) Different base/base mismatches are corrected with differing effclencles by the methyl-directed DNA mismatch-repair system of E co11 Cell 38,879–887PubMedCrossRefGoogle Scholar
  15. 15.
    Carter, P, Bedouelle, H, and Winter, G (1985) Improved ohgonucleotlde site-dn-ected mutagenesis using in 13 vectors Nucleic Acids Res 13,4431–4443PubMedCrossRefGoogle Scholar
  16. 16.
    Laengle-Rouault, F, Maenhaut-Mlchel, G, and Radman, M (1986) GATC sequence and mismatch repair in Escherzchza colt EMBO J 5,2009–2013Google Scholar
  17. 17.
    Deng, W P and Nlckloff, J A (1992) Site directed mutagenesis of virtually any plasmld by ehmmating a unique site Anal Biochem 200, 81–88PubMedCrossRefGoogle Scholar
  18. 18.
    Kramer, W, Drutsa, V, Jansen, H-W, Kramer, B, Pflugfelder, M, and Fritz, H-J (1984) The gapped duplex DNA approach to ohgonucleotlde-directed mutation construction Nucleic Acids Res 12,9441–9456PubMedCrossRefGoogle Scholar
  19. 19.
    Waye, M in Y (1993) Use of in 13 Ping-Pong vectors and T4 DNA polymerase in ohgonucleotlde-dlrected mutagenesis Meth Enzymol 217,259–270Google Scholar
  20. 20.
    Salki, R. K, Scharf, S, Faloona, F, Mulhs, K B, Horn, G T, Erhch, H A, and Arnhelm, N (1985) Eniymattc amphfication of P-globm genomlc sequences and restrlction site analysis for diagnosis of sickle anemia Science 230, 1350–1354CrossRefGoogle Scholar
  21. 21.
    Newton, C R and Graham, A (1994) PCR BIOS, Oxford, UKGoogle Scholar
  22. 22.
    McPherson, M J, Qmrke, P, and Taylor, G R (eds ) (1992) Polymerase Charn ReacttonA Practzcal Approach Oxford University Press, Oxford, UKGoogle Scholar
  23. 23.
    Abramson, R D and Myers, T W (1992) Nucleic acid amphfication technologies Curr Open Biotech 4,41–47CrossRefGoogle Scholar
  24. 24.
    Ausubel, F in, Brent, R, Kingston, R E, Moore, D D, Seldman, J G, Smith, J A, and Struhl, K (eds ) (1991) Current Protocols in Molecular Biology Wiley, New YorkGoogle Scholar
  25. 25.
    Sarkar, G and Sommer, S S (1990) The “Megapnmer” method of site-directed mutagenesis Biotechniques 8,404–407.PubMedGoogle Scholar
  26. 26.
    Hlguchr, R, Krummel, B, and Salki, R K (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments study of protein and DNA mteractions Nucleic Acids Res 15, 7351–7367Google Scholar
  27. 27.
    Ho, S N, Hunt, H D, Horton, R in, Pullen, J K, and Pease, L R (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction Gene 77, 51–59PubMedCrossRefGoogle Scholar
  28. 28.
    Horton, R in, Cal, Z, Ho, S N, and Pease, L R (1990) Gene sphcing by overlap extension tailor made genes using the polymerase chain reaction Biotechniques 8,528–535PubMedGoogle Scholar
  29. 29.
    Jayaraman, K, Fingar, S A, Shah, J, and Fyles, J (1991) Polymerase chain reactionmediated gene synthesis synthesis of a gene coding for lsoiyme c of horseradish peroxldase Proc Nati Acad Sci USA 88,4084–4088CrossRefGoogle Scholar
  30. 30.
    Lewis, A P and Crowe, J S (1991) Immunoglobulin complementanty determining region grafting by recombinant polymerase cham reaction to generate humamsed monoclonal antibodies Gene 101,297–302PubMedCrossRefGoogle Scholar
  31. 31.
    Sate, K, Tsuchlya, M, Saldanha, J, Kolshlhara, Y, Ohsugl, Y, Klshlmoto, T, and Ben&g, M in (1994) Humamiation of a mouse anti-human mterleukm-6 receptor antlbody comparing two methods for selecting human framework regions Mol Immunol 31, 371–381CrossRefGoogle Scholar
  32. 32.
    Dillon, P J and Rosen, C A (1990) A rapid method for the construction of synthetic genes using the polymerase chain reaction. Biotechniques 9, 298–300PubMedGoogle Scholar
  33. 33.
    Daugherty, B L, DeMartmo, J A, Law, M-F, Kawka, D W, Singer, I I, and Mark, G E (1991) Polymerase chain reaction facilitates the cloning, CDR-grafting and rapid expression of a murme monoclonal antibody directed agamst the CD 18 component of leukocyte mtegrms Nucleic Acids Res 19,2471–2476PubMedCrossRefGoogle Scholar
  34. 34.
    Prodromou, C and Pearl, L H (1992) Recursive PCR a novel technique for total gene synthesis Prot Eng 5,827–829CrossRefGoogle Scholar
  35. 35.
    Deng, S-J, MacKenzie, C R, and Narang, S A (1993) Simultaneous randonuiation of antlbody CDRs by a synthetic hgase chain reaction strategy Nucleic Acids Res 21,4418–4419PubMedCrossRefGoogle Scholar
  36. 36.
    Eckert, K A and Kunkel, T A (1991) DNA polymerase fidehty and the polymerase chain reaction PCR Methods Appl 1, 17–24Google Scholar
  37. 37.
    Ling, L L, Keohavong, P, Dlas, C, and Thilly, W G (1991) Optimliation of the polymerase chain reaction with regard to fidelity modified T7, Taq, and Vent DNA Polymerases PCR Methods Appl 1,63–69Google Scholar
  38. 38.
    Clark, J in (1988) Novel non-ternplated nucleotlde addition reactions catalyzed by procaryotlc and eucaryotlc DNA polymerases Nucleic Acids Res 20,9677–9686CrossRefGoogle Scholar
  39. 39.
    Verhoeyen, M, Mlistem, C, and Winter, G (1988) Reshaping human antibodies grafting an antllysoiyme activity Science 239, 1534–1536PubMedCrossRefGoogle Scholar
  40. 40.
    Rlechmann, L, Clark, M, Waldmann, H, and Winter, G (1988) Reshaping human antlbodies for therapy Nature 332,323–327CrossRefGoogle Scholar
  41. 41.
    Tempest, P R, Barbanti, E, Bremner, P, Carr, F J, Ghisheri, M, Rifaldl, B, and Marcuccl, M (1994) A humamzed anti-tumour necrosis factor-a monoclonal antibody that acts as a partial, competltlve antagonist of the template antibody Hybirdoma 13, 183–190CrossRefGoogle Scholar
  42. 42.
    Tempest, P R, White, P, Wllhamson, E D., Tltball, R W, Kelly, D C, Kemp, G J L, Gray, P in D, Forster, S J, Carr, F J, and Harris, W H (1994) Efficient generation of a reshaped human mAb specific for the a toxm of Clostndlum perfrlngens Prot Eng 7, 1501–1507CrossRefGoogle Scholar
  43. 43.
    Hsiao, K, Bajorath, J, and Hams, L J (1994) Humanization of 60 3, an anti-CD18 antibody, importance of the L2 loop Prot Eng 7,815–822CrossRefGoogle Scholar
  44. 44.
    Huston, J S, McCartney, J, Tat, M S, Mottola-Carlshorn, C, Jm, D, Wartren, F, Keck, P, and Oppermann, H (1993) Medical applications of single-cham antibodies Intern Rev Immunol 10,195–217CrossRefGoogle Scholar
  45. 45.
    Hayashl, N, Welschof, M, Zewe, M, Braunagel, M, Dubel, S, Breltling, F, and Little, M (1994) Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR Biotechniques 17,310–315Google Scholar
  46. 46.
    Clackson, T, Gussow, D, and Jones, P (1991) General applications of PCR to gene cloning and manipulation, in Polymerase Chain Reaction A Practical Approach (McPherson, M J, Qmrke, P, and Taylor, G R, eds ), Oxford University Press, Oxford, UK, pp 187–214Google Scholar
  47. 47.
    McCafferty, J, Griffiths, A D, Winter, G, and Chiswell, D J (1990) Phage antlbodles filamentous phage displaying antlbody variable domains Nature 348,552–554PubMedCrossRefGoogle Scholar
  48. 48.
    Clackson, T, Hoogenboom, H R, Griffiths, A D, and Winter, G (1991) Making antlbody fragments using phage display hbrarles Nature 352,624–628PubMedCrossRefGoogle Scholar
  49. 49.
    Adalr, J, Bodmer, M W, Mountain, A, and Owens, R J (1992) CDR Graffited An&CEA Antlbodles and Their Production WO92/01059Google Scholar

Copyright information

© Humana Press Inc , Totowa, NJ. 1998

Authors and Affiliations

  • John R. Adair
    • 1
  • T. Paul Wallace
    • 1
  1. 1.Axis Genetics PLCUK

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