Site Directed Mutagenesis

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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 then initiated with a polymerase and is then transformed into a host like E.coli were the clones are recovered and checked for appropriate mutagenic change by DNA sequencing. Once the mutagenic clone is identified the fragment is subcloned into a vector for further analysis. In detail the procedure employs two mutagenic oligonucleotide primers, one primer contains the desired mutation and the second contains a mutation with a unique, nonessential restriction site. The two primers are annealed to circular single-stranded DNA and direct synthesis of a new second strand containing both primers. The resulting DNA is transformed into a mismatch repair defective (mutS) E. coli strain, which increases the probability that the two mutations will segregate together during the first round of DNA replication. Even though the site directed mutagenesis is considered as one of the important discovery in molecular biology, it also got risk as the results are mostly unpredicted and surprising. Also, size of template plays a major role in determination of success of site directed mutagenesis. (Noll,Kranz 2009)

METHOD:

The experiment was performed in five steps and the procedure was started with synthesis of the second strand of DNA.

In the first part, pUC19M DNA was supplied which was denatured by heating at 1000C for 3 minutes directly by the technician in an eppendorf tube. To another eppendorf tube 14 μl of denatured pUC19M DNA was transferred to which 2 μl of 10x annealing buffer (to help the primers to get annealed to the single stranded DNA),2 μl of mutagenesis primer (to disable the mutation and make the LacZ gene functional) and 2 μl of selection primer (to alter the restriction site which was found on the plasmid) was added. The sample was then heated to 600C for 5 minutes to facilitate the annealing process to take place and was chilled on ice. The tube was later spun to collect the liquid.

To 20 μl of sample in the tube 3 μl of synthesis buffer (to provide...
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