Since genomic data are widely available, many strategies have been implemented to reveal the function of specific nucleotides or amino acids in promoter regions or proteins, respectively. One of the methods most commonly used to determine the impact of mutations is the site‐directed mutagenesis using the polymerase chain reaction (PCR). There are different published protocols to develop single or multiple site‐directed mutagenesis. In this chapter, we reviewed the enzymes commonly used in site‐directed mutagenesis, the methods for simple and multiple site‐directed mutagenesis in large constructs, mediated by insertion of restriction sites. Other methods reviewed include high‐throughput site‐directed mutagenesis using oligonucleotides synthesized on DNA chips, and those based on multi‐site‐directed mutagenesis, based on recombination. Software tools to design site‐directed mutagenesis primers are also presented.
Part of the book: Polymerase Chain Reaction for Biomedical Applications
Tuberculosis (TB) remains to be a serious health problem worldwide. There is an increased transmission of Mycobacterium tuberculosis strains with drug resistance, hence complicating TB control. The deciphering of the M. tuberculosis genome, together with the implementation of new molecular biology tools, has allowed the identification of changes in nucleic acid sequences with a functional impact. These mutations have become important in the design of early‐diagnostic kits to identify the resistance profile of M. tuberculosis. Since the conventional methods to determine the identity of M. tuberculosis strains based in cultures are laborious, time‐consuming and performed by specialized technicians, the result is generated until 4 months after receiving the samples. During this time, patients with TB are not adequately treated, and resistant strains may be transmitted to the rest of the population. In this chapter, we describe the most relevant mutations in genes associated with drug resistance in M. tuberculosis, the analysis of gene expression to identify new markers of drug resistance strains, and the development of new antituberculosis drugs against drug‐resistant strains.
Part of the book: Mycobacterium