3-6.1 Introduction

Mutation of the genes in higher organisms can be carried out by site- specific or random variation in the gene expression cassette using the model microbes (eg. E.coli , S.cerevisiae etc). The modification of the sequence of the gene is done by introducing an alteration in the sequence. Mutagenesis is powerful genetic tool to study and characterize functional elements of gene structure.

3-6.2 Classification

Mutagenesis is classified under different category depending on the target site:

1)  Site directed/ site specific mutagenesis- A specific site or stretch of some nucleic acids is altered in a predetermined way by exploiting this approach. This is accomplished by specific nucleic acid deletion or substitution to the targeted site of mutation.

2)  Mismatch mutagenesis- Mismatch mutagenesis is applied to create a desired point mutation in a unique site precisely by introducing a mismatched nucleic acid base. This method is generally used to determine the functional groups of a protein encoded by that gene.

Several methods have been developed till now to obtain a successful site directed mutation in the targeted site. Some of them are discussed below-

3-6.2.1 Kunkel's method

In the early 1980's, Thomas Kunkel developed a technique which reduced the need of selection of the mutants. Candidate DNA fragment to be mutated is inserted into a phagemid such as M13mp18/19 and then transformed into an E. coli strain deficient in dUTPase (dut ) and uracil deglycosidase (ung). These enzymes are part of a DNA repair pathway that protects the bacterial chromosome from mutations by the spontaneous deamination of dCTP to dUTP. The dUTPase deficiency prevents the breakdown of dUTP, resulting in increased level of dUTP in the cell. The uracil deglycosidase deficiency prevents the removal of uracil from newly-synthesized DNA. As the double-mutant E. coli replicates the phage DNA, its enzymatic machinery may therefore mis-incorporate dUTP instead of dTTP, resulting in single stranded DNA which contains ssUDNA. The ssUDNA is extracted from the bacteriophage released into the medium, and used as a template for mutagenesis. An oligonucleotide containing the desired mutation is used for primer extension. The heteroduplex DNA containing one parental non-mutated strand of dUTP and a mutated strand containing dTTP is then transformed into an E. coli strain carrying the wild type dut and ung genes. Here, the uracil-containing parental DNA strand is degraded, so that nearly all resulting DNA consists of the mutated strand.