Abstract
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.
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Smith, R.A. (2010). Random-Scanning Mutagenesis. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology, vol 634. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-652-8_27
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DOI: https://doi.org/10.1007/978-1-60761-652-8_27
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-60761-652-8
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