problem sheet in gene technology

You are employed at a biotechnology company that is seeking to use human proteins for therapeutic applications. People in the protein purification lab have purified a protein to homogeneity and it is your job to work out how to clone the genetic sequence for the protein. A couple of details about the protein, (i) it is a single polypeptide protein, is soluble, i.e. not membrane associated, and has been isolated from cultured human fibroblast cells. (skin cells) that are maintained in tissue culture. (ii) It appears that the expression of the protein can be induced in the fibroblast cultures by briefly exposing the cells to 40oC for 30 minutes and waiting 2 hours and (iii) an homologous protein from pigs has been purified and characterised. The pig protein appears to carry out the same function as the human protein and a cDNA and antibodies raised against the pig protein are available.

Briefly outline two separate strategies that you could use to isolate the gene (or cDNA) for the human version of this gene.

Question 2. – 4 marks

When cloning a foreign DNA fragment into a plasmid there are a number of methods that allow the selection of recombinant clones, such as cloning into a selectable marker ( in the case of pBR322) or cloning into and interrupting the ß-galactosidase gene (in the case of pUC derived vectors). The loss of function of these genes can then be used to identify those clones that have a foreign DNA fragment insert. However with bacteriophage lambda (?) vectors it is not necessary to do this, yet one can readily distinguish the vectors that have incorporated the large fragments of foreign DNA from those that have not. How are the recombinant vectors identified?

Question 3. – 8 marks

The plasmid cloning vector pBR322 is cleaved with the restriction enzyme PstI. An isolated DNA fragment from the eukaryote genome (also produced by PstI digestion) is added to the prepared vector and the mixture is ligated. The mixture of DNA is then transformed into competent E.coli cells and the plasmid containing bacteria are selected by plating on tetracycline.

(a) in addition to the desired recombinant plasmid, what other plasmids might be found among the transformants that are tetracycline resistant? How might these be distinguished from the desired plasmids?

(b) The cloned DNA fragment is 1,000 bp in length and has an EcoRI site 250 bp from one end. Three different recombinant plasmids are purified and cleaved with EcoRI and analysed by gel electrophoresis, giving the pattern shown below, the molecular weight markers are shown in lane 4. What does each pattern say about the cloned DNA. Note in pBR322 the PstI and EcoRI sites are about 750 bp apart. The entire pBR322 plamid without any cloned insert is 4,361 bp.