Team:REC-CHENNAI/projects

Projects

1.What is mutagenesis?

The process in which mutations arise in the genetic material of an organism is called mutagenesis. Mutations are changes in the base sequences of the genetic material. Mutagenesis alters the genetic information of an organism. Mutagenesis may occur either spontaneously or may be artificially induced. One of the most common examples cited is of UV rays or other ionising radiations that induce mutations.

2.Why do we need mutagenesis?

The applications of mutagenesis can be twofold – investigative and commercial. In investigative mutagenesis, the alterations are studied in terms of their pre and post mutation effects. Extensive studies of effects of specific amino acids on post translational modification and protein folding are studied in investigative mutagenesis. It is also an emerging field in diagnostics. Commercial mutagenesis sees practical applications. Improvement of crop nutrition, inducement of sustained resistance to pests and pathogens are the most commonly found applications today.

3.What are the methods to accomplish mutagenesis?

Mutagenesis may be random or site directed. Of the preferred latter method, PCR based and oligo based methods are employed. The major disadvantage of PCR based in vitro mutagenesis is the low stability of Taq DNA polymerase that results in various undesired mutations. In oligo based site directed mutagenesis, an oligo with the mutation polymerises in the presence of a complementary strand and hence, the mutations are induced.

4.Why do we need a new system?

Currently practised oligo based mutagenesis procedures involve screening for mutants using Ampicillin and Tetracycline resistant genes. The mutants see a reversal in resistance and sensitivity to the corresponding antibiotics on account of the mutations induced. This confirms successful mutagenesis. Although this technique guarantees that 90% of the transformants possess the specific mutation, it is time consuming. Specific problems include the two antibiotic resistance genes, time involved and the complexities of replica plating.

5.What is our system?

Our orthogonal system replaces the antibiotic resistance genes with a gene coding for Dual Fluorescence Protein as the marker. This mutant form of green fluorescent protein (GFP) is proven to emit both red and green fluorescence. Mutations in our system are induced by an oligo primer and further polymerization facilitated by Klenow is carried out in both the gene of interest and the marker gene.

6. How do we know if mutations have occurred?

Upon successful mutagenesis, the DFP gene mutates to express a GFP. Mutants glow in green colour whereas the non-mutants express emit both green and red fluorescence. This system completely eliminates the need for replica plating. The amount of time used to check for mutations in antibiotic resistant genes is significantly reduced. This is because a detection of green fluorescence and absence of red fluorescence will confirm successful occurrence of mutation within the gene of interest. Hence the system stands largely simplified.