Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Random Scanning Mutagenesis

(Protocol summary only for purposes of this preview site)

In vitro oligonucleotide and PCR-based mutagenesis is generally used for altering the nucleotide sequence of genes to study their functional importance and the products they encode (Hutchison et al. 1978; Botstein and Shortle 1985; Kunkel 1985; Higuchi et al. 1988). 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 (Cunningham and Wells 1989). Although these strategies can provide useful information, it is sometimes desirable to test a broader spectrum of amino acid changes at the targeted positions. Recently, Smith and colleagues developed an approach called random scanning mutagenesis 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 their method (Smith et al. 2004, 2006). This strategy is an oligonucleotide-based method for generating all 19 possible replacements at individual amino acid sites within a protein. Figure 1 illustrates the major steps of this protocol.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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