Introduction to Site-Directed Mutagenesis     (biotechnology)

Mutations are the sudden heritable changes that occur in the genome of an organism. They may occur at the gene or chromosomal level and can be natural or induced. 

Mutants are generated by treating the test organism is with chemicals or physical agents. Such agents are called mutagens.

 Instead of isolating and mutagenizing many cells of an organism and analyzing multiple numbers of cells, it is now possible to study mutation at a single nucleotide of the DNA sequence. 

When such a mutation is brought about at the specific site it is termed as site-directed Mutagenesis. In 1978, Michael Smith studied sitedirected mutagenesis by using oligonucleotides in a primer extension method with DNA-Polymerase enzyme. He was awarded a Nobel Prize in 1993, for the development of this technique. These mutations occur at the specific targets in the double-stranded DNA at a defined site.

 Thus, site-directed Mutagenesis widely serves as a molecular biology technique for generating amino acid changes in the polypeptide chain. It is the most commonly used technique in molecular biology. Due to advancements in the field of recombinant DNA technology in manipulating the regulatory elements and gene products in addition to site-directed mutagenesis, there has been larger progress in protein structural and functional studies

When specific alterations in the genetic information of an organism are required site-directed mutagenesis is used. So, with the help of PCR desired mutations in the DNA can be achieved. Further, we shall study the varies steps involved in this process; Different methods of site-directed mutagenesisinclude the single primer-based method, Oligonucleotide directed mutagenesis, Cassette mutagenesis, and PCR-based mutagenesis

These target specific changes in DNA help us to study the protein activity, or to screen for mutants with desired characteristics, and to remove or insert a restriction endonuclease site. In the previous block you have studied about invitro DNA amplification and sequencing, now in this unit you will learn the basics of site-directed mutagenesis, let’s think and answer what kind of changes can be made to the double-stranded DNA to have enhanced benefits of gene manipulation (Fig. 11.2). The basic mechanism of site-directed mutagenesis involves: 1. Denaturation of the gene with the targeted site mutation. 

2. Annealing with primers containing desired mutations. 

3. Using non-strand displacing polymerase action, nicked circular strands are obtained

Genetic modification in plants, animals, and microbes for the production of foods with desired traits began about 10,000 years ago. Scientists have studied gene manipulation techniques since the early 1970s with the beginning of the recombinant DNA revolution. The first artificial genetic modification was accomplished using biotechnology employing transgenesis. Herbert Boyer and Stanley Cohen in 1973,first carried out the process of transferring genes from one organism to another. This was made possible due to the discovery of restriction enzymes, DNA ligases, and the ability to design plasmids and technologies like polymerase chain reaction and sequencing. Thus, changes have resulted in increased food production, reliability, and yield; with enhanced taste and nutritional value. The objective was mainly to identify, select, and analyze individual organisms that possess genetically enhanced features. To investigate the structural and functional features of biomolecules the changes in the sequence of a gene or gene products must be done. Earlier, attempts were made using radiation or chemical mutagens to bring about nonspecific mutations. But, later sitedirected mutagenesis was achieved in 1974 in the laboratory of Charles Weissmann, using a nucleotide analog N4-hydroxycytidine, for inducing transitions from GC to AT. However, due to its limited specificity, later site-directed mutagenesis methods employing oligonucleotides, emerged as a more flexible and viable tool. With the advancement of recombinant DNA technology and site-directed mutagenesis, protein engineering has found a wide range of applications.  Introduction to Site-Directed Mutagenesis Have you ever thought about how mutations occur and what are its causes? Let us understand the concept related to mutations. Mutations are the sudden heritable changes that occur in the genome of an organism. They may occur at the gene or chromosomal level and can be natural or induced. Mutants are generated by treating the test organism is with chemicals or physical agents. Such agents are called mutagens. Instead of isolating and mutagenizing many cells of an organism and analyzing multiple numbers of cells, it is now possible to study mutation at a single nucleotide of the DNA sequence. When such a mutation is brought about at the specific site it is termed as site-directed Mutagenesis. In 1978, Michael Smith studied siteIn 1993, the Nobel Prize in Chemistry was shared by Michael Smith a British-born Canadian biochemist and Kary Mullis for his work in developing site-directed mutagenesis.  The first genetically modified animal was a mouse created in 1974 by Rudolf Jaenisch.  In 1976 the technology was commercialized, with the advent of genetically modified bacteria that produced somatostatin hormone, followed by insulin in 1978. When a mutation is brought about at the specific site it is termed as “Site-Directed Mutagenesis.”.