Section A – Problem Solving
The last five nucleotides of the recognition site for a restriction endonuclease which recognises a
10 nucleotide palindromic sequence is 5’-GATCT-3’.
a) What is the complete recognition sequence (both strands)?
b) Approximately how many recognition sites are there for this enzyme in the human genome (for simplicity, use 3 billion bp as the size of the human genome)?
A plasmid that carries selectable marker genes for resistance to kanamycin (KanR) and to chloramphenicol (CamR) was incubated with a restriction enzyme that cuts within the CamR gene but leaves the KanR gene intact. Plasmids that had been digested with the enzyme ligated with human DNA restriction fragments (digested with the same enzyme). Following transformation with the resultant DNA molecules E. coli cells were plated on nutrient media and allowed to form colonies. Replica plates of these colonies were then made on selective antibiotic media to determine which bacteria hosted recombinant DNA. The results are as shown below:
- Kanamycin and + Chloramphenicol indicate replica plates containing the respective antibiotics.
Colonies are indicated be a • ; numbers below the colonies indicate the colony number.
Giving a brief explanation answer the following questions:
a) Which colonies carry intact plasmid DNA but no recombinant DNA?
b) Which colonies carry recombinant DNA?
SECTION B – Short Essays Questions – Answer all questions (18 marks each) Word limit for each of the questions is 350 words
You are working in a neurodegenerative disease research lab and have been instructed to generate nerve cells from adult skin cells from a patient with a rare inherited neurodegenerative condition in order to treat and research the disease. Explain two different approaches of how you would generate the nerve cells and what ex vivo research you might carry out on the cells to aid
You are asked to produce a large amount (over 500 mg) of a recombinant protein. You are provided with the cDNA, and you will need to subclone this into the vector of your choice. Design a work plan to produce sufficient soluble protein using a yeast system. The target protein is 15 kDa protein and has one disulphide bond. In your answer you should include details of how the cDNA will be incorporated into the expression vector and how transformants will be selected for.
You are developing new RNAi therapeutics. What are the different challenges for the small interfering RNA (siRNA) stability, delivery and targeting to the correct tissue? Give some examples of how you can overcome these challenges. Comment on any specific adverse effects related to RNAi therapeutics. Provide an example of a successful RNAi therapeutic, which has been recently approved.
Factor IX (FIX) is a plasma glycoprotein that plays an essential role in the blood clotting pathway. It is normally synthesised in the liver in a vitamin K-dependent manner. A deficiency in Factor IX leads to the bleeding disorder haemophilia B. A number of posttranslational modifications are made to Factor IX, including phosphorylation of Ser158, sulphation of Tyr155, and both N- and Oglycosylation. Design a strategy to produce recombinant Factor IX for replacement therapy for haemophilia B patients. Include details of the features of the DNA construct (promoters, selection
markers and other possible sequence elements) that you need and of the choice of cells you will
use. Justify your choices.
You are studying the biological processes occurring during learning and memory in the adult brain. The gene of your interest has a mice homolog, however, the gene is also essential for early development and mutations in the gene result in pre-adult lethality in mice, preventing further studies of its functions in adulthood. Explain a way to make an inducible gene-knock out in mice,
which would allow you to overcome the developmental lethality. Provide details of the molecular
tools you need and the mechanisms of how the system works.