Genetic Engineering

| June 22, 2015
  1. Explain a method by which you can efficiently clone PCR products into a plasmid vector and at the same time reduce background non-recombinants. (5 marks)
  2. During Sanger dideoxy-chain-termination DNA sequencing, a population of dideoxy-terminated DNA strands is formed, which contains a mixture of fragments that are terminated at various base-locations within the growing strand.  What feature of the reaction mixture circumvents the problem of all newly synthesised strands terminating at the first base? (5 marks)
  3. You are screening a genomic library constructed in a rare ancient sheep species (Desert Bighorn Sheep Ovis canadensis ) for a gene associated with disease resistance. The only probe you have is the sequence of the same gene from the closely related sheep species (Merino sheep Ovis aries). What level of stringency will you use in screening your first colony hybridization experiment? Explain why. (5 marks)
  4. Describe how you would prepare an RNase free 0.1 M Tris based buffer that you intend to use in a RNA purification experiment. (no calculations necessary). (5 marks
  1. Describe the radioactive labelling of double stranded DNA using the random priming method.   (5 marks)
  2. Describe, in principle, how you would prime the first-strand synthesis of cDNA of eukaryotic mRNA. (5 marks)
  1. What is meant by the term “homopolymer tailing”? How is it performed and for what use? (5 marks)
  2. Describe how you would prepare the Supercos vector for cloning of DNA fragments. (5 marks)

 

  1. A fragment of DNA has been isolated by digesting with the following restriction endonuclease BamHI (5’ G/GATCC 3’). You wish to clone this into a plasmid vector but the vector only has a multiple cloning site with the following restriction sites in its multiple cloning site. You have access to any other reagents as required. Describe one way you could achieve this and at the same time achieving minimal background non-recombinants.       (5 marks)                                                                                                               Bcl I                5′ T /GATCA 3                                                                                   Dra I                5′ TTT/AAA 3’                                                                                    Hind III          5′ A/AGCTT 3’                                                                                   Kpn I               5′ GGTAC/C 3’                                                                                   Pst I                 5′ CTGCA/G 3’                                                                                   Eco RI             5’ G/AATTC 3’

 

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  1. Use the codon table provided below to generate a DNA code for a protein with the following partial sequence: Gly-Val-Trp-Ile-Met-Pro-Leu-Val-Met. (Hint more than one possible combination). (5 marks)

Third Position                        A      C      G      U                 _____________________________             AA  |    Lys    Asn    Lys    Asn        F    AC  |    Thr    Thr    Thr    Thr        i    AG  |    Arg    Ser    Arg    Ser        r    AU  |    Ile    Ile    MET    Ile        s P  CA  |    Gln    His    Gln    His        t o  CC  |    Pro    Pro    Pro    Pro          s  CG  |    Arg    Arg    Arg    Arg        & i  CU  |    Leu    Leu    Leu    Leu          t  GA  |    Glu    Asp    Glu    Asp        S i  GC  |    Ala    Ala    Ala    Ala        e o  GG  |    Gly    Gly    Gly    Gly        c n  GU  |    Val    Val    Val    Val        o    UA  |     Z     Tyr     Z     Tyr        n    UC  |    Ser    Ser    Ser    Ser        d    UG  |     Z     Cys    Trp    Cys             UU  |    Leu    Phe    Leu    PheZ = STOP CODON 

  1. You have identified a plasmid that contains cDNA for the protein from which the partial sequence shown above was used to identify (Q17). Describe in detail how you could use site directed mutagenesis to change the proline to serine in this cDNA sequence. (5 marks)
  2. You have made an IPTG inducible plasmid gene expression cDNA library from a bacterium that produces a characteristic pink pigment, shown previously by you, to be encoded for by a single gene. Describe a method that could be used to identify the clones that contain the gene, which encodes protein responsible for producing the pigment, for which there is no DNA sequence information available. (5 marks)
  3. A short stretch of double stranded DNA sequence is shown below together with two priming sequences (shown in bold and italics) is a target sequence for PCR amplification. Write the DNA sequences of the two primers (in a 5′ to 3′ direction) used for a PCR and determine their respective Tms. (5 marks)

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