A student sets up a PCR with the DNA polymerase of Escherichia coli instead of Taq polymerase…
Contributed by StemPy LabTry it on paper first. A hint, the key idea and the worked solution are here when you need them.
A student sets up a PCR with the DNA polymerase of Escherichia coli instead of Taq polymerase. The tube goes through cycles in a thermocycler. At the end, she finds that very little new DNA has been made.
- What is the likely reason?
- What would she have to do to make the experiment work with this enzyme?
- Which bacterium gives the right enzyme, and why does it survive?
Need a reminder? The key ideaShow
PCR needs a template, two primers, nucleotides and Taq polymerase. Each cycle has denaturation, annealing and extension. With a perfect efficiency, . With an efficiency , .
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. A PCR with an enzyme from Escherichia coli.
Plan. Follow the enzyme through one cycle. The first step is heating to about degrees Celsius.
Step 1. (a) The enzyme of Escherichia coli is destroyed by the high temperature of the denaturation step. After the first cycle, no working enzyme is left, so almost no new DNA is made.
Step 2. (b) She would have to add a fresh dose of enzyme after every denaturation step, which is slow and costly.
Step 3. (c) The enzyme of the bacterium Thermus aquaticus, Taq polymerase, is thermostable. The bacterium lives in hot springs, and its enzyme is not destroyed by the heat.
Answer: (a) The enzyme is destroyed at the denaturation temperature. (b) Add fresh enzyme after each denaturation. (c) Thermus aquaticus, because its polymerase is thermostable.
Watch out. Taq is not a special enzyme for copying DNA. It does the same job as other polymerases. Its advantage is that it survives the heat.
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