Gene Modification Application
Gene Modification refers to numerous gene editing techniques that can introduce mutations into double stranded DNA. Whilst the CRISPR-Cas9 system induces site-specific mutation in vivo, other genetic engineering techniques can introduce such changes during your sub-cloning steps or in vitro. Such gene modification techniques can also be used to change the specificity of RNA interference approaches.
Site Specific Mutagenesis
Site-specific mutagenesis (SSM) inserts, deletes, or substitutes a single base within the DNA sequence of a plasmid. Doing so allows for the investigation of the biological activity of selected genes and proteins.
SSM procedure starts with the synthesis of a short DNA primer, which contains your desired mutation. This synthetic primer should also be complementary to its DNA template strand so that it can hybridize with the gene of interest. Next, this primer is extended via a DNA polymerase, which inadvertently produces the gene with the desired mutation. This vector is then introduced into a host cell and cloned.
Kits for SSM are based on PCR amplification and ligation to create a modified single strand of DNA. SSM kits differ in the type of mutation they induce, differentiating between insertion, deletions or base changes.
Kits express the efficiency of the mutation process through the percentage mutated sequences of the total final product. High-fidelity polymerases are specially engineered for increased accuracy, and thus will accurately copy your fragment, and in turn, increase the efficiency of mutations.
When methyl groups are added to a DNA sequence, it essentially ‘turns off’ a gene. Methylation modifies cytosine bases with the addition of a methyl group, which causes the repression of gene transcription. In nature, DNA Methylation is utilized for cellular differentiation and development. In the lab, it can be used to identify the function of a gene and its downstream effects.
The efficiency of this technique is related to the methods used to select the mutated sequence over the original template. The most common techniques are selection against DNA methylation, a signature of “naturally” amplified DNA, or by base uracil substitution in the template strand that will result in the destruction of the template itself. Bisulfite conversion is the most popular method; converting unmethylated cytosines to uracil, therefore enabling identification of methylation patterns.
Transposons are sequences of DNA which are able to move around the genome and insert themselves at certain positions. This method makes use of the enzyme transposase, which binds to the end of transposable DNA elements, (so-called transposons); catalyzing their movement in the genome by a cut and paste method. This method often results in duplication events and may thus alter the cell’s genome significantly. Transposons can also be inserted into a plasmid. This technique can mutate an existing gene and subsequently silence it by insertion within the gene. Alternatively, it can activate a gene by inserting upstream, where its promoter initiates transcription of the transposon and the target gene.
For further guidance see our Gene Modification Troubleshoot.
Compare Sited-Directed Mutagenesis Kits
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The ZAGENO comparison engine does not show the best kit, but instead helps you, the researcher, choose the most suitable kit
for your experiment.
Go to our How it Works page for a guide to using the ZAGENO comparison engine.
Video: Garvan Institute of Medical Research/YouTube