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Constructed Mutants Using Synthetic Oligodeoxyribonucleotides as Site-Specific Mutagens

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Genetic Engineering

Part of the book series: Genetic Engineering ((GEPM))

Abstract

Classical genetics provides a bridge between biology and the chemical structure of the gene. In the past, isolation of spontaneous or induced mutants by screening or selection for a changed phenotype was followed by genetic mapping and, more recently, by determination of the structure of the gene of interest. The advent of rapid methods for DNA sequence determination (1,2) has created a new situation whereby whole genomes, or parts of genomes, can have their sequences determined in advance of detailed genetic analysis. On occasion it is possible to assign genetic function to a DNA sequence by inspection. For example, in the case of genes J and K of the small coliphage øX174 and G4, knowledge of the protein sequences allowed assignment of gene function to DNA in the absence of mutants (3,4). Assignment of tRNA genes to particular tracts of mitochondrial DNA sequence was achieved by using computer analysis of possible secondary structure (5). However, such parallel information is not usually available. This has led to the development of methods for constructing mutants in vitro by modifying DNA of known sequence. Examination of the in vivo properties of the modified DNA allows assignment of particular functions to specific DNA sequences. An early example of this approach is the construction of specific deletions in simian virus 40 (6). Allied methodologies include strategies for introducing deletions of variable length at specific or random sites (7,8) and specific or random insertion of short segments of duplex DNA (9,10). These all are powerful techniques for demonstrating the location of genetic functions and establishing their approximate boundaries. However, full understanding of genetic functions requires mutations that modulate gene activity, e.g., mutations that increase or decrease the efficiency of origins of replication, of transcription or of translation and mutations that establish the reading frame for the triplet code which determines the sequence of a protein. In general, this requires point mutations.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Medicine University of British Columbia, 2075 Wesbrook Place, Vancouver, B.C., V6T 1W5, Canada

    M. Smith & S. Gillam

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  1. M. Smith
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  2. S. Gillam
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Editors and Affiliations

  1. Brookhaven National Laboratory, Upton, New York, USA

    Jane K. Setlow

  2. Associated Universities, Inc., Washington, D.C., USA

    Alexander Hollaender

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Smith, M., Gillam, S. (1981). Constructed Mutants Using Synthetic Oligodeoxyribonucleotides as Site-Specific Mutagens. In: Setlow, J.K., Hollaender, A. (eds) Genetic Engineering. Genetic Engineering. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-7075-2_1

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  • DOI: https://doi.org/10.1007/978-1-4615-7075-2_1

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-1-4615-7077-6

  • Online ISBN: 978-1-4615-7075-2

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