22. You use the primer 5' GCCTCGAATCGGGTACC 3' to sequence part of the human DNA insert of a recombinant DNA molecule made with a plasmid vector. The result of the automated DNA sequence analysis is shown here. The height of the peaks is unimportant. (A = green; C = purple; G = black; T = red) Smaller Larger a. Write the sequence of all the nucleotides of human DNA that you can determine. Indicate the 5'-to-3' orientation of this sequence. b. Is the sequence you wrote in (a) part of the new DNA strand that was synthesized in the sequencing reaction or part of the template strand used in the sequencing reaction? c. How did you know how to design the primer you would need for the sequencing reaction? Diagram the recombinant DNA molecule to be sequenced, indicating the human and vector sequences, the position and orientation of the primer, and the position and orientation of the new DNA that would be synthesized during the sequencing reaction, using Fig. 9.7 as a guide. d. Show the full sequence of the smallest DNA molecule that would be synthesized in the sequencing reaction and that would contain dideoxyG (ddG). Indicate the 5'-to-3' orientation of this molecule and the location of the ddG. e. How would the data differ from that shown if you accidentally left the dATP out of the reaction?
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However, the sequence would be read from the bottom to the top of the graph, with the colors corresponding to the nucleotides (A = green, C = purple, G = black, T = red). The sequence would be in the 5'-to-3' orientation. Show more…
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You use the primer 5'-GCCTCGAATCGGGTACC-3' to sequence part of the human DNA insert of a recombinant DNA molecule made with a plasmid vector. The result of the automated DNA sequence analysis is shown here. (A = green, C = purple, G = black, T = red)
Josee P.
Question 1 (5 marks). In your experiments, you have discovered a DNA fragment which has the following sequence. CGCCCTCCGCAGTCC The newly discovered DNA fragment has a very interesting set of functions. Let's assume that you do not know the exact sequence and now you are very much interested in identifying the sequence of the DNA. You are going to perform the sanger sequencing, which will be performed in 4 separate tubes. Please address the following questions in detail. 1.1: List all the necessary components required to carry out the sequencing. (1 marks) 1.2: Describe the steps of sequencing each nucleotide and show how different fragments will be formed during the sequencing process. (2 marks) 1.3: Explain how electrophoresis and autoradiography can be used to make the final sequence? (1 marks) 1.4: Draw the double helical structure of the newly sequenced DNA, showing all the nucleotides and hydrogen bond between bases of the complementary strand. (1 marks) Question 2 (5 marks). During the process of transcription, DNA molecule is transcribed to RNA molecule. This process takes place in cytoplasm in prokaryotes whereas in eukaryotes it takes place inside the nucleus. After transcription, the mRNA will be translated to make protein. Let's suppose that you have obtained the following DNA sequence in which the transcription start site (TSS) is written in small letter (a) and the transcription stop is at the end of the sequence. CCGAATAAAT AGGATTGACA GCGAAGTATA TGACGCCTCA TATAATATTG aGGTGTAGGG TACGGTGCTT CGGCGTAGGT CTCCCGCGTA ATTCATCGAG 2.1: Highlight the promoter regions. (1 marks) 2.2: Explain how transcription will take place and what will be the transcribed mRNA sequence. (2 marks) 2.3: What do you think should be the translated protein sequence? (2 marks)
Adi S.
are about CRISPR/Cas9 and relate to the following diagram, which shows the complex between an sgRNA and the genomic site it targets so that Cas9 can cut the genomic DNA at the positions shown. You should note the so-called protospacer adjacent motif or $P A M$ site located adjacent to the target site. The PAM site $\left(5^{\prime}-\mathrm{NGG}-3^{\prime}\right.$ on one strand, $\mathrm{N} \text { is any base })$ must be present in the position indicated for cleavage to occur. One way to think about this situation is that the sgRNA brings the Cas9 enzyme to the adjacent PAM site to initiate cleavage. F. Port and S. Bullock at the University of Cambridge (UK) designed the elegant plasmid vector $p C F D 3$ for the expression of sgRNAs in Drosophila. The following figure shows a part of this vector. The orange sequences are part of a strong promoter (transcription from this promoter starts at the $\mathbf{G}$ in bold-which must be present- and goes from left to right). The purple sequences are a portion of the tracrRNA component of the sgRNA. After cutting the $p C F D 3$ plasmid with the restriction enzyme $B b s I$ (whose recognition site is also shown in the following figure), you will replace the blue sequences in the figure with sequences that will allow the expression of an sgRNA that targets a Drosophila gene called NiPp 1 The last part of the jigsaw puzzle you will need is the following sequence, which shows part of the $N i P p l$ gene including the triplet corresponding to the start codon. Capital letters are in the gene's first exon with the coding region in blue; lowercase letters are in the first intron. The NiPp1 protein is 383 amino acids long. Your assignment is to generate a knockout allele of this gene by inducing Cas9 to produce a doublestrand break into the gene that will be repaired imprecisely by nonhomologous end-joining (NHEJ). a. Identify the two PAM sites in this sequence. Which of these PAM sites would you want to use in order to produce a null allele of the NiPp1 gene? Why would you prefer this site? b. If you targeted Cas9 to the proper location in the NiPp1 gene, and the resultant double-strand break was repaired imprecisely by NHEJ (so that a few usually $\leq 6$ bp are deleted or added at that location), about what percentage of the imprecisely repaired genes could you say with confidence would be null alleles? Explain. c. Diagram the $p C F D 3$ vector after it has been cut with the $B b s$ I enzyme. Don't worry about the small blue fragment that will be removed; the emphasis here is to show the $5^{\prime}$ -overhangs that will be made. d. Design two 24 -nt DNA oligonucleotides that you could anneal together and clone into $B b s I$ -cut $p C F D 3$ vector so that the recombinant plasmid could express an sgRNA useful for making null mutations in the NiPp1 gene. e. Show exactly where Cas9 would cut the NiPp 1 gene. f. Briefly outline what you would do with your recombinant plasmid to make a null mutation in the fly NiPp 1 gene. g. Briefly outline how you would modify this technique to generate a knockin allele in which the first amino acid in the NiPp1 protein after the initiating Met (that is, Thr) would be changed to Ala.
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