A linear DNA of 2200 bp carries a gene. The coding region runs from 150 to 2000 bp…
Contributed by StemPy LabTry it on paper first. A hint, the key idea and the worked solution are here when you need them.
A linear DNA of bp carries a gene. The coding region runs from to bp. The DNA has sites for HindIII at bp, for BamHI at bp, for EcoRI at bp and for KpnI at bp. The cloning region of the vector holds sites for HindIII, BamHI and EcoRI, in that order. The workers want to clone the whole gene in one direction.
- Which enzyme can not be used, and why?
- Which two enzymes should be used?
- How long is the fragment that is released from the DNA by the two enzymes?
Need a reminder? The key ideaShow
Cut inside the cloning region of the vector, and not inside the gene. Two different enzymes give directional cloning and stop the vector from closing on itself. Recombinant vector stuffer insert. A diagnostic digest with an off-centre site shows the direction of an insert.
The worked solution
Have you tried it?
Work it out on paper first. It is fine to get stuck or make a mistake: that is how we learn. When you are ready, go through the solution one step at a time, and check each step against your own.
Given. Coding region to . Sites: HindIII , BamHI , EcoRI , KpnI .
Plan. Choose two sites outside the coding region that the vector also has.
Step 1. (a) BamHI cuts at , which lies inside the coding region ( to ). It would cut the gene in two, so it can not be used.
Step 2. (b) HindIII at lies before the gene. EcoRI at lies after the gene. The vector has both sites. So HindIII and EcoRI are the pair. Two different enzymes give directional cloning. KpnI is not in the cloning region of the vector.
Step 3. (c) The fragment runs from to : bp.
Answer: (a) BamHI, because it cuts inside the gene. (b) HindIII and EcoRI. (c) bp.
Check. The fragment holds the whole coding region, to , with bp before and bp after it.
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