ION 10 EcoRI BamHl Accl HindllOri pONE Accl AmpR TetR BamHl BamHi Suppose you ha

Question

ION 10 EcoRI BamHl Accl HindllOri pONE Accl AmpR TetR BamHl BamHi Suppose you have a human gene with Accl, BamHl, EcoRI and Hindlll restriction enzyme recognition sites on both sides of the gene. You want to insert your gene into the plasmid, pONE (shown above), which contains two selectable markers, TetR and AmpR, as well as the restriction enzyme sites shown. For each of the restriction enzyme(s), say whether it would be a good choice to use or not (circle yes or no), and explain your answers. (6 marks) yes /no because: ! (i) BamHl: yes /nobecause: (ii) EcoRI: yes/no because: (iv) Hindlll: yes /no because: ining two genes shown below, to

Explanation / Answer

i) Accl cannot be used as a restriction enzyme because on the given plasmid the enzyme has two restriction sites that flank the oriC. If this enzyme was to be used, the segment with oriC will be lost and the plasmid would not be able to replicate itself.

ii) BamHI cannot be used for processing this plasmid either because it has three restriction sites, two of which are present in the genes for the resistance to the antibiotics. These genes are necessary because these will help us choose the colonies that have been successfully transformed with the recombinant plasmid that has the human gene of interest. Thus, we cannot have both the resistance genes disrupted.

iii) EcorI will be a good choice since only one site is present which ensures only one insertion per plasmid and the site is not located within any important regions that disrupt normal replication and selection of the recombinant plasmid.

iv) HindIII is a bad option as a restriction enzyme for this plasmid because the restriction site is present within Origin of replication (oriC). Using this restriction enzyme would disrupt the OriC and the recombinant plasmid would not be able to replicate properly

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