Random Scanning Mutagenesis

Abstract

In vitro oligonucleotide and polymerase chain reaction (PCR)-based mutagenesis is generally used for altering the nucleotide sequence of genes to study their functional importance and the products they encode. A thorough approach to this problem is to systematically change each successive amino acid residue in the protein to alanine (i.e., alanine-scanning mutagenesis) 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. “Random scanning mutagenesis” was developed to examine the functional importance of individual amino acid residues in the conserved structural motif of human immunodeficiency virus (HIV) reverse transcriptase, and this protocol is adapted from that method. This strategy is an oligonucleotide-based method for generating all 19 possible replacements at individual amino acid sites within a protein.