DNA mutagenesis method


Ontology type: sgo:Patent     


Patent Info

DATE

1987-12-02T00:00

AUTHORS

BOTT, RICHARD RAY , FERRARI, EUGENIO , WELLS, JAMES ALLEN , ESTELL, DAVID AARON , HENNER, DENNIS JAMES

ABSTRACT

A method of DNA mutagenesis comprises (a) obtaining a DNA moiety encoding at least a portion of said precursor protein;(b) identifying a region within the moiety;(c) substituting nucleotides for those already existing within the region in order to create at least one restriction enzyme site unique to the moiety, whereby unique restriction sites 5' and 3' to the identified region are made available such that neither alters the amino acids coded for by the region as expressed;(d) synthesizing a plurality of oligonucleotides, the 5' and 3' ends of which each contain sequences capable of annealing to the restriction enzyme sites introduced in step (c) and which, when ligated to the moiety, are expressed as substitutions, deletions and/or insertions of at least one amino acid in or into said precursor protein;(e) digesting the moiety of step (c) with restriction enzymes capable of cleaving the unique sites;(f) ligating each of the oligonucleotides of step (d) into the digested moiety of step (e) whereby a plurality of mutant DNA moieties are obtained; and optionally the further steps of(g) expressing each of said moieties as a mutant protein in a suitable host;(h) recovering the mutant proteins of step (g); and(i) screening the step (h) mutant proteins for the desirable characteristic. More... »

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A method of DNA mutagenesis comprises\n

  • (a) obtaining a DNA moiety encoding at least a portion of said precursor protein;
  • (b) identifying a region within the moiety;
  • (c) substituting nucleotides for those already existing within the region in order to create at least one restriction enzyme site unique to the moiety, whereby unique restriction sites 5' and 3' to the identified region are made available such that neither alters the amino acids coded for by the region as expressed;
  • (d) synthesizing a plurality of oligonucleotides, the 5' and 3' ends of which each contain sequences capable of annealing to the restriction enzyme sites introduced in step (c) and which, when ligated to the moiety, are expressed as substitutions, deletions and/or insertions of at least one amino acid in or into said precursor protein;
  • (e) digesting the moiety of step (c) with restriction enzymes capable of cleaving the unique sites;
  • (f) ligating each of the oligonucleotides of step (d) into the digested moiety of step (e) whereby a plurality of mutant DNA moieties are obtained; and optionally the further steps of
  • (g) expressing each of said moieties as a mutant protein in a suitable host;
  • (h) recovering the mutant proteins of step (g); and
  • (i) screening the step (h) mutant proteins for the desirable characteristic.

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