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- 35) A researcher inserted DNA fragments from an organism into expression vector plasmids and introduced the modified plasmids into bacterial cells. Which of the following methods would be an effective means of identifying which clones contain a specific gene of interest?1. Why would multi-gene families complicate things in terms of being sure of which member of the family was responsible for a particular phenotype? 2. How could multi-genes families complicate things in terms of using CRISPR to knock out a target gene and achieve a target phenotype?7. You are preparing a library to sequence two samples, one from a WS and one from a FS, via Illumina. After tagmentation, you perform the PCR using a pair of sequencing primers, which consists of a forward and a reverse primer, as described in lecture. Unfortunately, you used the same pair of sequencing primers to both samples, instead of a unique pair to each of them. Briefly explain how and why this change is going to affect the outcome. Hint: in other words, what it will happen when the software will try to align the reads after sequencing?
- 1. The polymerase chain reaction (PCR) is used by scientists to amplify DNA, particularly when the quantity of DNA is very small, mixed, or contaminated with other organisms. Explain (with words) the how PCR works using a diagram to help illustrate this (show a minimum of three cycles to illustrate your point). 2. How would one go about designing primers (I am interested in the concepts you would use to accomplish this task) that would amplify a specific sequence of the "16S rDNA," that would be found in the Clostridium family? Be sure you consider the importance of both "highly conserved" and "highly variable" regions during the amplification process. 3. How many molecules (target sequence copies) will be produced by 30 PCR cycles? Assume you start with only 1 copy of the target sequence (very unlikely)? Show your work! 4. Explain how gel electrophoresis works? Why are DNA samples that are to be separated by gel electrophoresis always loaded at the cathode end of the power source? The…1. What two restriction sites are you going to use to clone your PCR product into the pL4440 plasmid? What are their DNA sequence? 2. State the primer sequence you will use to amplify the F27C1.7 gene ready to be cloned into the pL4440 plasmid? 3. How would you go about cloning this amplified DNA into pL4440? Using your knowledge of cloning list 5 important aspects of the method.16. Which of the following enzyme repairs the DNA backbone during molecular cloning (that is you are inserting the DNA fragment into a plasmid and the bases in the sticky ends are aligned but the backbone needs to be repaired). a) reverse transcriptase b) restriction enzymes c) DNA ligase d) polymerase
- 5. After extracting your DNA from your cheek cells, we will add a solution called Master Mix to your DNA sample. One important component of the Master Mix are the "oligonucleotide primers" (or "primers" for short). What is the importance of these primers to the PCR process? 6. How did Taq DNA polymerase acquire its name? What is the job of this enzyme? Why aren't we using human DNA polymerase? briofly summarize inFigure 17.7 You are working in a molecular biology lab and, unbeknownst to you, your lab partner left the foreign genomic DNA that you are planning to clone on the lab bench overnight instead of storing it in the freezer. As a result, it was degraded by nucleases, but still used in the experiment. The plasmid, on the other hand, is fine. What results would you expect from your molecular cloning experiment? There will be no colonies on the bacterial plate. There will be blue colonies only. There will be blue and white colonies. The will be white colonies only.CRISPR One of the more recent advances in biotechnology is the development of the CRISPR gene modification tool. Match the following descriptions the the key players in this technology. NOTE: If you want to change your selection, you'll need to delete the one you already chose. After you delete it, the list of choices will pop back up and you can make a different choice. CRISPR technology Specialized stretches of DNA with nucleotide repeats and Cas9 protein spacers Enzyme that cuts DNA crRNA Guides the enzyme to the target site CRISPR DNA Adapted from the natural defense mechanism of bacteria and archaea
- 30) In the CRISPR system, the targeted DNA sequence is12. Which of the following are not the functional components of a simple retroviral genome? gag, pol, and env genes 5' Untranslated regions 3' Untranslated regions transgene 13. One of the major drawback of using Microarray over RNA sequencing for high throughput sequence analysis is allows quantification of transcripts over five orders of magnitude used to identify new transcripts and alternative isoforms RNA-Seq is sensitive and offers a way of profiling transcripts of single cells a significant amount of input RNA is required15. The CRISPR/Cas9 system provides a form of adaptive immunity in bacteria. We discussed in class that it can be used in human cells to: A: Protect cells from infection with viruses B: Kill cells that carry a specific deletion C: Give cells a memory of the viruses that have infected them D: Greatly enhance the frequency of homologous recombination at a chosen I locus E: Kill cells that have not undergone the desired genome edit
