Site Directed Mutagenesis
INTRODUCTION:
Site directed mutagenesis is molecular biology technique that is used to make specific, intentional and precise changes to the DNA sequence of a gene or any gene products. It is also used to determine the structure and biological activity of DNA, RNA and protein molecules. It is also called as site-specific mutagenesis or oligonucleotide-directed mutagenesis. This method has also found use in understanding important genetic process like DNA conformational analysis and also functioning of processes like transposition and site-specific recombination.
History/Background:
Numerous methods have been developed earlier to mutate DNA. Initially all of they approaches focused on the generation of random mutations in chromosomal DNA for example induced by X-rays and chemicals. While these provided a valuable tool for classical genetic studies, they were limited as they could not target the mutation to a specific gene. Techniques for randomly mutagenizing a genome required screening or selection from massive numbers of mutants to obtain the desired mutation. The ability to control DNA through the use of plasmid vectors became an attraction for newer technologies, which allowed specific changes in discrete, manageable segments of the genome with relatively little effort.
Site-directed mutagenesis method was first benefited from recombinant DNA technology in 1970s, when isolated genes were exposed to conditions such as chemical agents or nucleotide analogs to localize their mutagenic effects. During this time, the use of plasmid vectors for DNA replication greatly enhanced the study of mutations. (Cosby and Lesley).
Site-directed mutagenesis can be grouped generally into three categories of which oligonucleotide-directed mutagenesis is by far the most commonly used method.
Mutagenesis is carried out with one single stranded template (usually M13) by annealing a synthetic primer in which defined changes can be incorporated. Second strand synthesis is
Site directed mutagenesis is molecular biology technique that is used to make specific, intentional and precise changes to the DNA sequence of a gene or any gene products. It is also used to determine the structure and biological activity of DNA, RNA and protein molecules. It is also called as site-specific mutagenesis or oligonucleotide-directed mutagenesis. This method has also found use in understanding important genetic process like DNA conformational analysis and also functioning of processes like transposition and site-specific recombination.
History/Background:
Numerous methods have been developed earlier to mutate DNA. Initially all of they approaches focused on the generation of random mutations in chromosomal DNA for example induced by X-rays and chemicals. While these provided a valuable tool for classical genetic studies, they were limited as they could not target the mutation to a specific gene. Techniques for randomly mutagenizing a genome required screening or selection from massive numbers of mutants to obtain the desired mutation. The ability to control DNA through the use of plasmid vectors became an attraction for newer technologies, which allowed specific changes in discrete, manageable segments of the genome with relatively little effort.
Site-directed mutagenesis method was first benefited from recombinant DNA technology in 1970s, when isolated genes were exposed to conditions such as chemical agents or nucleotide analogs to localize their mutagenic effects. During this time, the use of plasmid vectors for DNA replication greatly enhanced the study of mutations. (Cosby and Lesley).
Site-directed mutagenesis can be grouped generally into three categories of which oligonucleotide-directed mutagenesis is by far the most commonly used method.
Mutagenesis is carried out with one single stranded template (usually M13) by annealing a synthetic primer in which defined changes can be incorporated. Second strand synthesis is