Random-Scanning Mutagenesis

Oligonucleotide-mediated mutagenesis is a useful tool for engineering nucleotide changes at defined positions in a DNA sequence. Oligonucleotide-based approaches are commonly used to introduce missense mutations at individual codons in a gene or gene segment, thereby revealing the functional importance of specific amino acid residues in a protein. For mutagenesis studies involving tracts of polypeptide sequence, investigators typically change each successive residue to alanine or to a limited number of alternative amino acids. Although these strategies can provide useful information, it is sometimes desirable to test a broader spectrum of amino acid changes at the targeted positions. This article describes a facile, oligonucleotide-based method for generating all 19 possible replacements at individual amino acid sites within a protein. This technique is known as “random-scanning mutagenesis” and is illustrated herein using examples from our studies of a conserved polymerase motif in HIV-1 reverse transcriptase.