be inactivated, at high temperatures employed in an industrial process, or a therapeutic protein may be short lived due to protease sensitivity necessitating administration of high, somewhat toxic, doses. Random or directed mutagenesis can be employed to alter the nucleotide sequence encoding a protein to improve its stability, activity, specificity, cofactor requirements, or protease resistance. Straightforward protocols have been developed to introduce nucleotide substitutions into a gene on an oligonucleotide primer by PCR. When the specific amino acids that contribute to a property are known in advance, the defined nucleotide changes can be introduced on an oligonucleotide by overlap extension or inverse PCR. When the amino acid changes that will result in the desired property of a protein are unknown, libraries of randomly mutated sequences can be generated by performing a PCR under conditions that increase the error rate or by employing degenerate oligonucleotide primers. Most of the mutations will decrease the function of the encoded protein and therefore the libraries must be screened to identify proteins with desired characteristics. Shuffling of DNA segments from two or more genes creates a large number of hybrid proteins that can also be screened for unique biological activity.
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