Site-directed mutagenesis of gene sequences in cells of plants, Essay

Site-directed mutagenesis of gene sequences in cells of plants, insects and animals can be achieved in situ used engineered zinc - Essay Example The ability to generate new DNA molecules and the process of inserting it into the genome to correct the diseases are the biomedical research nowadays. This type of modification of the cellular phenotype has become the back bone for the biomedical research. Knowing the position and the reading frame of the genes will help us to identify the nonsense mutants by the ochre suppressors. These non sense mutants are highlighted and this will help us for an efficient screening for silent mutants. The small oligonucleotides containing 7 nucleotides in length can be used to form stable duplexes with the single stranded DNA. (Jensen et al. 2011). These duplexes are stable even with a mismatch. This led to the introduction of oligonucleotide mutagenesis. The emergence of site directed mutagenesis as an analytical tool in the in the year 1985 was a mile stone in the field of molecular biology. (Herzog and Zolotukhin 2010). Site directed mutagenesis is widely used for the study of the protein fun ctions. The mutation at a defined site of the DNA leads to the change of an amino acid specific to a protein and this leads to the alteration of the protein molecules. Two methods of site directed mutagenesis can be used. The first one is overlapping method and the second one is called as whole round second round PCR. They create modified DNA sequences that are used to study the importance of the specific residues in protein structure and function. Protein engineering and altered protein substrate activity can be achieved using Site directed mutagenesis. PCR based Site directed mutagenesis is the most common method where the desired mutation is carried in one of the primer and annealed to the site of interest. Two cycles of PCR are carried out for insertion of mutation. (Lloyd 2005). Fig 1: Methods available to site specifically modify a genome target. (Herzog and Zolotukhin 2010). Zinc finger nuclease (ZFN) is a dimeric protein with each monomer having a zinc finger array (contains three or four zinc fingers). They are artificial restriction enzymes that have a lot of application in plants and animals. These zinc fingers (Cys 2 Hys 2) are arranged in the ? ? ? – fold ( beta- beta- alpha folding ) which is coordinated by the Zn+ ion and has the non specific cleavage site. (Gupta et al. 2011).These zinc finger arrays are bound to the cleavage site of the type II restriction endonuclease enzyme, Fok I. Fok l is responsible for the cleavage function of the Type II endonuclease. (Gupta et al. 2011).The dimerization of the cleavage domain is much essential for the enzyme activity. If the zinc finger array is engineered to bind to some specific site of the genome, then Fok l will perform the cutting action at that site. This double stranded break can be utilized for the site directed mutagenesis for protein engineering. This break is then repaired by using either homologous recombination (HR) or non-homologous end joining (NHEJ) technique. The HR requires ext ensive homology between the strands whereas NHEJ does not require any homology and the repair at the targeted site usually results in the mutation. (Herzog and Zolotukhin 2010). Because of this reason NHEJ is used in the site directed mutagenesis associated with the ZFNs. ZFN mediated gene modification will produce cell lines to contain biallelic knock outs. If ZFN targeted gene modificat