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+ | Our software simplifies the primer design process for target-specific mutagenesis via reverse transcriptase (RT). We called it tRNA primer designer because 5' end of the tRNA was used for the RT initiation, and the 3' end must match the mRNA encoding the target. The software consists of 4 parts: the reverse transcriptase selection, target sequence input, designed-tRNA visualization and primer output. Although, we only test MMLV RT experimentally, the software is highly flexible as it can adjust its designing method based on the properties of 3 well-studied RT, MMLV, HIV1 or RSV, which makes it possible for users to design their tRNA primers even for eukaryotic experiments. In addition, we calculate the acceptor stem annealing temperature, which could be used as an indicator for likelihood to success. | ||
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− | Previous studies have shown that a tRNA primer is required for the initiation of reverse transcription (<a href="#Ref1">Dahlberg et al.</a>). In our system, we express the tRNA primer in E.coli by cloning it onto the plasmid that is used for generating the tools for mutation, i.e. | + | Previous studies have shown that a tRNA primer is required for the initiation of reverse transcription (<a href="#Ref1">Dahlberg et al.</a>). In our system, we express the tRNA primer in E.coli by cloning it onto the plasmid that is used for generating the tools for mutation, i.e. P<sub>mutant</sub>. However, designing the primer sequence according to different target sequences is time-consuming and needs many adjustments to find the perfect match. This motivates us to build a software for tRNA primer designing. |
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Revision as of 09:29, 20 October 2019