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

Area of Science:

• Molecular Biology
• Enzyme Engineering
• Plasmid Genetics

Background:

• The beta-lactamase gene on plasmid pBR322 confers ampicillin resistance.
• The enzyme's active site is hypothesized to involve a serine-threonine dyad at residues 70 and 71.
• Site-directed mutagenesis is a key tool for studying enzyme function.

Purpose of the Study:

• To investigate the role of the serine-threonine dyad in beta-lactamase activity.
• To generate a mutant enzyme with altered ampicillin resistance.
• To demonstrate a generalizable method for creating specific DNA mutations.

Main Methods:

• Oligonucleotide-directed mutagenesis was employed to alter the beta-lactamase gene.
• A chemically synthesized 16-base oligodeoxyribonucleotide was used to prime in vitro DNA synthesis.
• The Ser-Thr dyad at residues 70-71 was inverted to Thr-Ser.
• Mutant detection utilized colony hybridization at the DNA level.

Main Results:

• A specific mutation in the beta-lactamase gene was successfully introduced.
• The resulting mutant exhibited an ampicillin-sensitive phenotype.
• The double-mismatch mutagenesis method proved effective for generating the desired mutation.
• The method demonstrated generality, independent of enzyme phenotype.

Conclusions:

• The study successfully inverted the serine-threonine dyad in the beta-lactamase active site.
• This inversion resulted in a loss of ampicillin resistance, confirming the dyad's importance.
• The described double-mismatch mutagenesis technique offers a simple, versatile approach for genetic manipulation.