CRITICAL THINKING QUESTIONS: You are studying a novel protein. The gene encoding

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CRITICAL THINKING QUESTIONS: You are studying a novel protein. The gene encoding this protein of interest is written in Figure 1 of your supplementary material. Your goal is to perform site-directed mutagenesis on this gene of interest, clone the mutated version of the gene into a GST-fusion vector (pGEX-5X-2), over- express and purify the fusion protein and ultimately characterize the protein. Note: To make reading easier, write nuclcotides ACT in capital letters and g in lower case. You- want to use PCR to amplify wild-type and mutated versions of the gene of interest. For the mutated form, you want to convert the valine (V 111) to a tryptophan (W). The valine is indicated by an asterisk in supplemental material figure 1. A codon table is provided in figure 4 of supplemental material. (a) The wild-type primers corresponding to the 5' and 3' ends of the gene should cover the regions underlined in Figure 1. In other words, your primer will be longer than nucleotides underlined, but only those nucleotides are specific to your gene of interest. Wild-type primer 1 (WtP1) should cover the start codon, while Wild-type Primer 2 (WtP2) should include the stop codon. Design these primers for optimal cloning of your PCR product into the cloning vector pGEX-5X-2 (see figure 3 in supplemental material below) with the ultimate goal of expressing your fusion protein. Write the sequence of these primers including restriction sites and any other nucleotides-put parenthesis () around restriction sites and any other nucleotides. e Wild-type Pimer 1 (WEPI)S GAATTCnnatat ata casaaauta g ta gua wild-type Pinner 2 (WtP2):5. CTC GAGnnttatagatatecccatette ?tagat eat ecceatcffe 31 3' (b) The mutagenic primers should be 27 nucleotides long (see figure 1 in supplemental material). Mutagenic Pimer l (MPI) will be paired with wild-type Pimer l (wtP?) while Mutagenic Piner 2 (MP2) will be paired with Wild-type Pimer 2 (WtP2) during the first round(s) of PCR Write the sequence of your primers below: Put a box around the mutagenic nucleotides. Mutagenic Pimer 1 (MP1):5 Mutagenic Pimer 2 (MP2):5 CANCATACTOCAAGTETG AT ACTTA -??6TCATCA GRC TB6AGTA ATGTTA 3' term

Explanation / Answer

For wild-type Forward primers pick first 21 nucleotides and add EcoRI site ie. GAATTC before the first nucleotide. Sometimes we need to add 2 nucleotides so that enzyme efficiently cleaves the DNA.

For reverse primer take last 21 nucleotides and add the site of XhoI i.e. CTCGAG. Add 2 nucleotides so that enzyme efficiently cleaves the DNA. make the reverse complement the sequence. This will be our reverse primer

If we add two nucleotides after the site of enzyme then the whole frame of the sequence will change and we will not get the protein.

Forward primer - 5' (gCgAATTC)ATg Tgg AgA gAT ACA gAA ATA 3'

Reverse Primer -

Original sequence - 5' gAA gAT ggg gAT gAT CTA TAA CTCgAg GC 3'

When we reverse complement this sequence we will get 5' (gCCTCgAg)TTA TAg ATC ATC CCC ATC TTC 3'

For mutation, we need to mutate GTT to TGG.

The mutagenic primer 2 will be the same as the sequence in which GTT is mutated in TGG.

Mutagenic primer 2 - 5' AgT CCT CAg gAC Tgg gAg TAA TgT TgT 3'

The mutagenic primer 1 will be the reverse complement of mutagenic primer 2 the sequence in which GTT is mutated in TGG.

Mutagenic primer 1- 5' ACA ACA TTA CTC CCA gTC CTg Agg ACT 3'

I have bold and underlined the mutated nucleotide

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