The in vitro transcription process
The starting template of in vitro transcription is a linear DNA template that includes a promoter region together with the usual ingredients for a successful transcription process: RNA polymerase, nucleotides, and an appropriate buffer system. Since every reaction is different, the conditions may vary depending on your DNA template, expected RNA yield, length, and purity.
Make use of our In Vitro Transcription Troubleshoot for any experimental issues.
You need a double stranded DNA (dsDNA) template, but its origin is irrelevant. The template can be anything from normal PCR reactions, plasmids, or even cDNA templates that themselves were generated from RNA molecules. However, different kits may be suitable for different templates, so take care when choosing your kit.
- In order to use a PCR product in an in vitro transcription reaction, you have to add a promoter to your fragment so that it is recognized by RNA polymerase. The promoter is added at the 5’ end of the fragment.
- Plasmid vectors must first be linearized before the transcription reaction can ensue. This is (fairly) easily done by using restriction enzymes. Purification should be carried out after such a digest as the restriction enzymes may inhibit the downstream in vitro transcription process.
- One of the latest developments in this field is using in vitro transcription in RNA amplification reactions. Here, the reaction template is created from RNA during reverse transcription, generating cDNA. A second strand synthesis reaction is then performed to convert the cDNA to dsDNA for the in vitro transcription to be carried out.
Other applications also exists such as the use of oligonucleotides as templates.
In Vitro Translation
Following you’re in vitro transcription procedure you may want to take it a step further and translate your newly formed RNA into the protein in which it encodes. Like in vitro transcription, in vitro translation or ‘cell-free protein synthesis’ is carried out using cellular machinery in a cell-free system. In vitro translation boasts many advantages over cell-based translation as it is not constrained by homeostasis or a cell wall so it enables the molecular biologist to have direct control over the environment in which translation is taking place. It also allows for the overexpression of proteins that would normally be toxic to the host cell and prevents proteolytic degradation by proteases normally present in the host.
The Applications of In Vitro Translation
Due to these advantages, the in vitro process has many applications. The identification of protein products can be achieved much more rapidly due to the direct access one has to the reaction. This then enables the subsequent determination of protein structure, folding dynamics and function through the use of proteomics. It also allows for the incorporation of non-natural amino acids and the synthesis of truncated or mutated gene products for functional and kinetic modifications of the protein.
How is it Achieved?
Only a few cell-free systems have been developed for the in vitro production of proteins, however, in theory, it should be possible to do this using any type of cell. Common cell-free systems used today are made from E. coli (ECE), wheat germ (WGE), rabbit reticulocytes (RRL) and insect cells. The components for cell-free reactions are obtained by lysing the cell of interest and removing unwanted cell debris by centrifugation. The leftover cell extract should include 70S and 80S ribosomes, elongation and translation factors, aminoacyl-tRNA synthetases, amino acids and an energy source such as ATP or GTP.
There are two approaches to in vitro translation: the standard approach or the coupled approach. The standard approach is used when you have an RNA template as your starting material while the coupled approach is used if you have DNA. The coupled approach incorporates in vitro transcription so that an RNA template can be produced for translation.
Here at ZAGENO, you have the unique possibility to compare in vitro transcription & translation kits from different suppliers to find the most suitable one for your experiment.
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