Once we identify the sequences to target, the aim was to synthetise them as well as guide RNA, we used Integrated DNA Technologies (IDT) for this. = METTRE LIEN + REFORMULER. You may ask yourself why use DNA if we are studying RNA viruses. Well, the main reasons are :
Once we identify the sequences to target, the aim was to synthetise them as well as guide RNA, we used Integrated DNA Technologies (IDT) for this. You may ask yourself why use DNA if we are studying RNA viruses. Well, the main reasons are :
<listyle="margin-left: 10%;"> DNA synthesis is cheaper </li>
<listyle="margin-left: 10%;"> DNA is more stable than RNA </li>
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<pstyle="margin-top: 20px;"> Afterward, we employed PCR (Polymerase Chain Reaction) to increase yield of our target, and guide sequences. Different forward and reverse sets of primers were used for this purpose (see tables below) :</p>
<p><u><b>Fig 2 :</b></u> Scheme of the primers we used to synthetize WNV target sequences</p>
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<p>The forward primer starts with the enhancer, followed by the T7 promoter, an upstream sequence (-20 nucleotides <br> before the target), 28 nucleotides of the actual target sequence, and finally a downstream region of the target site <br> of 20 nucleotides. Those 20 nucleotides upsteam and downstream are used to keep the general spacial context of </p>
<listyle="margin-left:10%">The forward primer starts with the enhancer, followed by the T7 promoter, an upstream sequence (-20 nucleotides <br> before the target), 28 nucleotides of the actual target sequence, and finally a downstream region of the target site <br> of 20 nucleotides. Those 20 nucleotides upsteam and downstream are used to keep the general spacial context of </p>
<p> The reverse primer is composed of the reverse complement of the +20 nucleotides sequence and the reverse <br> complement of the 28 nucleotides to target.</p>
<listyle="margin-left:10%"> The reverse primer is composed of the reverse complement of the +20 nucleotides sequence and the reverse <br> complement of the 28 nucleotides to target.</p>
<p> You will find below the target DNA sequence after amplification</p>
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<p><b><u> Guide :</u></b>
<h4><b><u> Guide :</u></b></h4>
<p> The synthesis of primers for the guides use the same principle as the target sequences. The difference is that the guide need to be able to hybridize with the 28 nucleotide sequence site of the target, also it contain a scaffold that is specific to the type of Cas used: CasRX or LwCas13a (see Fig 3).
<figcaptionstyle="margin-top: 20px;margin-left: -10px;text-align: center;"><b><u>Fig 3: </u></b>Scheme for the synthesis of LwCas13a and CasRX guides primers</figcaption>
<b><u>Tab 1.4: </u></b> Table of the CasRX guide sequences
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<p> Forward primer composed of an enhancer, T7 promoter, and the stem loop of the Cas used (LwCas13a or RfxCas13d). This primer stay the same for all guide sequences of the same Cas. </p>
<listyle="margin-left:10%"> Forward primer composed of an enhancer, T7 promoter, and the stem loop of the Cas used (LwCas13a or RfxCas13d). This primer stay the same for all guide sequences of the same Cas. </li>
<p> Reverse primer consisted of the 28 nucleotides target sequence, accompanied by the reverse complement of the Cas stem loop. This primer change as it is specific to target sequence.</p>
<listyle="margin-left:10%"> Reverse primer consisted of the 28 nucleotides target sequence, accompanied by the reverse complement of the Cas stem loop. This primer change as it is specific to target sequence.</li>
