3) Site-Directed Mutagenesis
Site-directed mutagenesis is a molecular method made possible by modern biochemical techniques such as PCR and gene sequencing. It involves doing exactly what it sounds like – creating a genetic mutation at a specific site in a gene. Of all the means of altering protein structure, this is the method that requires the most detailed knowledge about the gene of interest. The complete nucleotide sequence needs to be known, or at least enough to generate primers for PCR at either end, and in the region that is to be mutated.
Generally the active site of a protein is mutated (the site of substrate binding and reaction catalysis), in order to enhance substrate binding, or otherwise improve rates of reaction. Other regions that might have to do with enzyme activity and warrant attention might be those having to do with protein folding (eg. formation of disulfide bonds between cystine residues), cofactor binding, or charge (eg. acid-base catalysis) and ligand (eg. carboxy, acetyl, amino, phosphate, hydroxyl group) interactions.
To perform site-directed mutagenesis, the gene of interest is cloned by PCR, but using primers in the region of interest that introduce a single site (base-pair) mutation resulting in a codon for a different amino acid at that site on the resulting protein. Generally the site of interest will be somewhere in the middle of the gene, so the gene is cloned in 2 or more fragments, with restriction enzyme sites introduced by the primers that allow for the resulting fragments to be ligated (connected) together in the end. Another form of site-directed mutagenesis is to completely remove a section of the gene and ligating the remaining pieces to generate a fusion protein.
4) DNA Shuffling
This technique has become very popular in recent years due to some major successes in the industries for thermostable and other commercialized enzyme products. The method is dependent on mutational cloning but less specific than site-directed mutagensis. In order to perform DNA shuffling, one needs only to know some basics about the gene in question: Overall size, end sequences for primer synthesis and PCR, and how to assay for activity (i.e. using bioassays and indicators). One must be able to clone the gene using PCR techniques which allows for the introduction of mutations by "error-prone" PCR.
Error-prone PCR is PCR performed in the presence of a mixture of nucleotide bases where one particular base is present at concentrations far exceeding those of the other three (eg. 10×, 100×). The resulting product is a mixture of gene clones that might each contain a different mix of mutations. These alone could be cloned into a microorganism for expression, and tested for the desired traits, but DNA shuffling goes one step further.
The next step is to digest the PCR products with restriction enzymes generating a "soup" of pieces of mixed size and mutations. The pieces are then ligated back together, creating a mixture of many different randomly mutated gene products. These are also cloned into a test organism so the expression and activities of each product can be screened. Companies can be found that will perform DNA shuffling for you, using rapid assay techniques that allow the screening of hundreds, or even thousands of gene products, to find the one that best suits your needs.
