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The first property is that the 3'-terminal of the tRNA primer should be complementary to the PBS on mRNA template <a>(Kosloff et al.)</a>(Kosloff et al.). The second one is that different viruses prefer specific type of tRNA primer for reverse transcription <a>(Kulpa et al.,</a><a> Kosloff et al.)</a>. What should also be noted is that for different viruses, the lengths of PBS as well as the types of tRNA primer are different. The PBS lengths and the preferred tRNA types of 3 most well-studied retroviruses are listed in Table I.<br /><br /> | The first property is that the 3'-terminal of the tRNA primer should be complementary to the PBS on mRNA template <a>(Kosloff et al.)</a>(Kosloff et al.). The second one is that different viruses prefer specific type of tRNA primer for reverse transcription <a>(Kulpa et al.,</a><a> Kosloff et al.)</a>. What should also be noted is that for different viruses, the lengths of PBS as well as the types of tRNA primer are different. The PBS lengths and the preferred tRNA types of 3 most well-studied retroviruses are listed in Table I.<br /><br /> | ||
These discoveries serve as the theoretical basis for our tRNA primer designer. So basically, the function of our tRNA primer designer is to change the tRNA template in order to suit the basic properties of the reverse transcriptase (MMLV RT/ HIV-1 RT/ RSV RT) selected by the user as well as to replace several nucleotides (17 or 18) on 3'-terminal of the tRNA templates to match with nucleotides at the 5'-terminal of the GOI which users input. Also, to make sure that the RNA sequence is a tRNA sequence, the secondary structure should be revealed. We achieve this goal by using the similar tRNA secondary structure prediction scheme as the one implemented in the opensource software tRNAfinder <a>(Kurokawa et al.)</a>.<br /><br /> | These discoveries serve as the theoretical basis for our tRNA primer designer. So basically, the function of our tRNA primer designer is to change the tRNA template in order to suit the basic properties of the reverse transcriptase (MMLV RT/ HIV-1 RT/ RSV RT) selected by the user as well as to replace several nucleotides (17 or 18) on 3'-terminal of the tRNA templates to match with nucleotides at the 5'-terminal of the GOI which users input. Also, to make sure that the RNA sequence is a tRNA sequence, the secondary structure should be revealed. We achieve this goal by using the similar tRNA secondary structure prediction scheme as the one implemented in the opensource software tRNAfinder <a>(Kurokawa et al.)</a>.<br /><br /> | ||
− | Studies have shown that the primary factor guiding the selection of tRNA primer for MMLV RT is the PBS sequence instead of the inherent nature of reverse transcriptase <a>(A. H. Lund et al.,</a><a> S. P. Goff et al.)</a>. So, by making mutations on both the PBS and tRNA sequence, the researchers have found that reverse transcription could still successfully take place while the virus’ titer is not greatly affected. Also, after several cycles of replication, the mutated sequence is not changed back to its original version <a>(Pedersen et al., 1997)</a>. Even though it is found that the primer is not stringent for MMLV, studies have revealed that the tRNA-like structure is necessary. A study that the inclusion of one single non-Watson-Crick base pair between PBS and tRNA primer would improve the replication efficiency (F. S. Pedersen et al., 1993), but we didn’t adopt this construct as the one base pair mismatch would often be changed to the full-complementary version after the first cycle of replication <a>(Pedersen et al., 1997)</a> | + | Studies have shown that the primary factor guiding the selection of tRNA primer for MMLV RT is the PBS sequence instead of the inherent nature of reverse transcriptase <a>(A. H. Lund et al.,</a><a> S. P. Goff et al.)</a>. So, by making mutations on both the PBS and tRNA sequence, the researchers have found that reverse transcription could still successfully take place while the virus’ titer is not greatly affected. Also, after several cycles of replication, the mutated sequence is not changed back to its original version <a>(Pedersen et al., 1997)</a>. Even though it is found that the primer is not stringent for MMLV, studies have revealed that the tRNA-like structure is necessary. A study that the inclusion of one single non-Watson-Crick base pair between PBS and tRNA primer would improve the replication efficiency (F. S. Pedersen et al., 1993), but we didn’t adopt this construct as the one base pair mismatch would often be changed to the full-complementary version after the first cycle of replication <a>(Pedersen et al., 1997)</a>, making this addition unnecessary. |
</div> | </div> | ||
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<div class="col"> | <div class="col"> | ||
Our tRNA primer designer is a web tool for potential users of our mutagenesis system to design their own tRNA primers according to their experimental setups. Here we provide a step-by-step guide to using this software.<br /><br /> | Our tRNA primer designer is a web tool for potential users of our mutagenesis system to design their own tRNA primers according to their experimental setups. Here we provide a step-by-step guide to using this software.<br /><br /> | ||
− | Step 1. Select the type of reverse transcriptase (RT) that you want to use based on your experimental design. Note that this software only allows you to choose from MMLV RT/ HIV-1 RT/ RSV RT.<br /><br /> | + | Step 1. Select the type of reverse transcriptase (RT) that you want to use based on your experimental design, as shown in the figure below. Note that this software only allows you to choose from MMLV RT/ HIV-1 RT/ RSV RT.<br /><br /> |
− | + | ||
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="row legend"> | ||
+ | <div class="row"> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/5/53/T--Fudan-TSI--SFig2.gif" style="width:30%; margin:auto;"> | ||
+ | </div> | ||
+ | <div class="row legends"> | ||
+ | <b>Figure 1. Model for the whole process of reverse transcription.</b><br /> | ||
+ | Legend waiting to be added. | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="row para1"> | ||
+ | <div class="row"> | ||
+ | <div class="col"> | ||
+ | Step 2. Input a DNA sequence that you want to mutate, as shown in the figure below (upper panel). You can find a demo for it if you click on the icon on the right side of the web page, as shown in the figure below (lower panel). The last 17/18 nucleotides (nt) of the sequence are selected to be PBS, depending on the type of RT that you have chosen in <b><i>Step 1</i></b>. The length of PBS is 18nt if MMLV RT/ HIV-1 RT is selected, and is 17nt if RSV RT is selected. Note that this sequence should be longer than 17/18 nucleotides based on your selected RT. Besides, it shouldn't contain any characters other than A/T/C/G.<br /><br /> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="row legend"> | ||
+ | <div class="row"> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/2/29/T--Fudan-TSI--SFig3.gif" style="width:30%; margin:auto;"> | ||
+ | </div> | ||
+ | <div class="row legends"> | ||
+ | <b>Figure 1. Model for the whole process of reverse transcription.</b><br /> | ||
+ | Legend waiting to be added. | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="row para1"> | ||
+ | <div class="row"> | ||
+ | <div class="col"> | ||
+ | Step3. Click on the "DESIGN FOR ME!" button and see the result, as shown in the figure below (upper panel). The result is composed of two parts. The first part shows you the secondary structure of the template tRNA that you will be using as well as the designed tRNA primer. The fragment that can be annealed to PBS of the input DNA sequence is shown in red. The second part will give you the DNA sequence encoding the tRNA primer that satisfies your need. You can just copy it and use it elsewhere. These two parts are demonstrated in the figure below (lower panel). | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="row legend"> | ||
+ | <div class="row"> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/2/29/T--Fudan-TSI--SFig4.gif" style="width:30%; margin:auto;"> | ||
+ | </div> | ||
+ | <div class="row legends"> | ||
+ | <b>Figure 1. Model for the whole process of reverse transcription.</b><br /> | ||
+ | Legend waiting to be added. | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="row legend"> | ||
+ | <div class="row"> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/2/29/T--Fudan-TSI--SFig5.gif" style="width:30%; margin:auto;"> | ||
+ | </div> | ||
+ | <div class="row legends"> | ||
+ | <b>Figure 1. Model for the whole process of reverse transcription.</b><br /> | ||
+ | Legend waiting to be added. | ||
+ | </div> | ||
</div> | </div> | ||
Revision as of 07:52, 16 October 2019