PCR & Agarose Gel – Sample Solved Question

bWe’ve discussed PCR and Agarose Gel in GAMSAT Sec 3 Biology in a previous article. In this post, we shall take a look at a sample question based on this topic, and find out the correct answer.

Information

Gene of a plant degrading enzyme was amplified with PCR. PCR stands for Polymerase Chain Reaction. It is a method used in molecular biology that amplifies sequences of DNA via the following steps:

  1. The genomic DNA and the primer were denatured by being heated to 95°C for 5 minutes.
  2. The temperature was lowered and retained at 40°C for 1 minute to let the primers anneal to their corresponding sequences on the genomic DNA.
  3. The temperature was then raised and retained at 72°C for 1 minute to let Taq polymerase replicate the genomic DNA. Taq polymerase then extended the primers at the 3′ end.
  4. The temperature was raised and retained at 95°C for 1 minute to denature the newly synthesised PCR product.
  5. Steps 2-4 were repeated 20-30 times. It is ensured that the DNA is not denatured at the end of the final cycle.
  6. Finally, the PCR products obtained were isolated.

PCR requires two sequence specific primers, a 5′ (forward) and 3′ (reverse) primer, to be synthesised for each gene because Taq needs these small sequences to initiate elongation. The 5′ primer consists of a sequence that is identical to the 5′ end of the gene. The 3′ primer consists of a sequence that is the reverse complement of the 3′ end of the gene. Thus, the 5′ primer binds to the 3′ end of the template strand and the 3′ primer binds to the 3′ end of the gene. The following figure shows different domains of plant degrading enzyme genes, and primers which were designed to amplify these genes.

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Question

Which of the following lanes show that the PCR product, if expressed, will form an intact enzyme?

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A. Lane 3

B. Lane 4

C. Lane 5

D. Lane 6

 

Breaking Down the Information

Now let’s cut down on the science jargon and translate the information to human language

 

Genetic stuff was replicated many times=PCR

  1. The genomic DNA and the primer were denatured by being heated to 95°C for 5 minutes.
    • Genetic stuff was uncoiled.
  2. The temperature was lowered and retained at 40°C for 1 minute to let the primers anneal to their corresponding sequences on the genomic DNA.
    • The primers attach to genetic stuff.
  3. The temperature was then raised and retained at 72°C for 1 minute to let Taq polymerase replicate the genomic DNA. Taq polymerase then extended the primers at the 3′ end.
    • The replicator thing replicated the genetic stuff
  4. The temperature was raised and retained at 95°C for 1 minute to denature the newly synthesised PCR product.
    • Then it was uncoiled again.
  5. Steps 2-4 were repeated 20-30 times. It is ensured that the DNA is not denatured at the end of the final cycle.
  6. Finally, the PCR products obtained were isolated.

 

Instructions on replicating DNA

  1. Attach forward primer (ATG)
  2. Attach reverse primer (ATT)
  3. Set the taq polymerase loose, and biochemistry will do all the hard work for you!

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Remembering why DNA is negatively charged.

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Correct Answer

Option A

 

Explanation

From the information above, we can see that Lane 3 product has moved least through the gel which means that it is a long fragment. Smaller fragments have moved down in the gel, but the fragment which has moved slowest on account of being the longest is expected to be the full length gene fragment, which on expression will give a fully functional enzyme. An enzyme will be functionally active only if all its fragments are intact including the different domains involved. So, the band that has migrated the least will represent the intact, and hence, the functionally active enzyme. Thus option A is the correct answer.

A huge thanks to our tutor, Cary Wang, to whom we are indebted for a lot of help on the material. Thank you so much, Cary! 

Image source: Cary Wang, Wikipedia

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