Chemical mutagenesis and Tilling

Prior to the implementation of more sophisticated genetic techniques, mutagenesis was often performed using simple chemical mutagens including N-methyl-N'-nitro-N-nitroso-guanidine (MMNG) and ethyl methane sulfonate (EMS) . The basic approach involves exposing a population of cells to a controlled dose of mutagen, to generate progeny that carry random genetic mutations The application of this technique has traditionally been limited by two important considerations First, it often proves difficult to strike a balance between efficiency and selectivity; exposure to the mutagen must be carefully controlled in order to maximize the frequency of single mutations Second, it should be noted that chemical mutagenesis is random; the experimenter needs to select or identify mutants of interest from a large pool of random mutants

For more detailed information on the background and procedures involved in chemical mutagenesis, we refer the reader to Miller (1992) The basic protocols that have been described for E. coli can easily be adapted for other bacteria, including insect symbionts Because various species and strains of bacteria are known to respond in different ways to mutagens and treatment regimes, it is of great importance to establish an experimental approach that yields an optimal level of mutagenesis and survival for a given strain Mutagenesis is typically quantified by measuring the conversion of the wild-type strain to a readily identifiable mutant phenotype Typically, we measure conversion to rifampicin and ciprofloxacin resistance, mediated by mutations in the RNA polymerase P-subunit (RpoB) and DNA gyrase (GyrA) enzymes respectively (Vila et al ., 1994; Xu et al ., 2005) . Using both selectable markers, it is possible to determine the frequencies of both single and double mutants to more accurately optimize the dose of mutagen

The major challenge associated with the use of reverse genetic strategies lies in the identification or characterization of mutants that have no obvious, selectable phenotype Fortunately, high-throughput methods have recently been developed to ameliorate this problem. TILLING (Targeting Induced Local Lesions in Genomes) is one such technique that has been developed to use a mismatch-specific enzyme to identify mutations in a gene of interest through high-throughput heteroduplex analysis (Till et al , 2003) Although the basic technique has been developed for use with higher organisms, it can readily be adapted for use with bacteria, as outlined in Figure 18 . 3 .

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