Application of SSCP to Identification of Resistance Mutations
There has been a significant increase in the number of genes associated with antibiotic resistance that have been described. For many antimicrobials all of the principal genes associated with their action have been identified (1). There is increasing interest in the epidemiological distribution of resistance mutations of these genes and research into their origin and routes of transmission. At the more fundamental level, there is interest in the impact of such mutations on the fitness/survivability of the pathogen (2). We have described the strategies for selection of mutants in the mycobacteria (3) and also a polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) approach to investigation of the distribution of such mutants. In this method PCR amplimers are denatured to form single-stranded nucleic acids and then submitted to gel electrophoresis to identify sequence polymorphisms. Sequencing of clones remains relatively expensive and time consuming for investigating a large number of isolates from clinical practice or strains from mutation experiments. This chapter outlines a method for screening large numbers of PCR amplimers, which can then inform rational selection for cloning and sequence analysis, or for identifying novel mutations for detailed sequence. Alternatively, this approach can be used for rapid screening where the SSCP profile relating to each mutation is already known.
KeywordsSterile Deionized Water Drug Resistance Mutation Conformational Polymorphism Vertical Format Short Plate
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