I would like to discuss an exam question on Molecular Genetics and Biotechnology, which appeared in the HKDSE Biology paper in 2018. The bold parts below are from the original question and I will provide answers and explanations.
The question is about Recombinant DNA Technology, which involves the Polymerase Chain Reaction (PCR) and transformation. The following two diagrams should be great schemes to explain the key points of these useful techniques.
Here comes the question:
The diagram below shows the change in temperature during a PCR cycle.
Which stage corresponds to DNA denaturation? Explain your answer.
Answer : The DNA denaturation happens when the hydrogen bonding interactions that govern complementary base pairing is broken down. It requires a high temperature and therefore stage 1 corresponds to DNA denaturation.
Mary planned to amplify a fragment of DNA using PCR. The following diagram shows the annealing of primers during PCR. The sequence of DNA strand X is shown below and the corresponding sequences of regions I and II are highlighted:
Mary designed the following primers for PCR:
(1) There is one type of mistake in each primer. Write the correct primers to be used.
(2) What is the predicted size (in terms of number of base pairs) of the PCR product.
Answer :
(1) Primer 1 is wrong because the primer is DNA-based and there should not be uracil (U) – they should be replaced by thymine (T).
Primer 2 is wrong because the direction of the base sequence is inverted.
Therefore, the 2 correct primers should be:
Primer I : CGGTAGTGGG ATACGACGAT
Primer II : TGTTATCCGC TCACAATTCC
(2) The predicted size of the PCR product should be 10 x 38 = 380 base pairs.
That leads us to the more challenging part of the question, for which students have not performed well enough.
Mary used the following plasmid as a vector to carry the PCR product to transform bacteria. The plasmid contained (a) an ampicillin resistance gene; (b) a Z gene encoding an enzyme that converts substance X to blue compounds; (c) some restriction site within the Z gene.
After the transformation of the bacteria, Mary grew the bacteria on agar plates containing both ampicillin and substance X. Blue and white bacterial colonies were formed.
(1) What is the purpose of adding ampicillin to the agar plates? Explain your answer.
(2) Explain which type of colony (blue or white) contains non-recombinant plasmids, i.e. without DNA insert.
Answer:
If we make sense of the 3 components of the plasmid one-by-one, then the whole logic behind the transformation will become clear and we will be able to answer both sections.
Ampicillin resistance gene :
This is a productive application of antibiotic resistance. As a selectable marker, it can be used to distinguish (screen) whether a given bacterium has taken up the plasmid or not during transformation. If a bacterium has successfully taken up the plasmid, it will confer the ampicillin resistance gene, and therefore it will survive on an agar plate with ampicillin.
Restriction site within the Z gene:
Let’s start with this before going to the action of the Z gene. Because the restriction site is present within the Z gene, therefore if a transformation has taken place, the restriction enzyme will cut through the restriction site in the Z gene. This will disrupt the Z gene and it will lose its phenotypic function as a result.
The Z gene:
It originally encodes an enzyme (through the Central Dogma : DNA > mRNA > Protein) which can convert substance X to a blue compound. Thus, the color change can only take place in those where the Z gene is intact - implying a successful expression of the enzyme, and hence a failure in the transformation because the restriction enzyme has not successfully cut through the Z gene. These are of course the non-recombinant plasmids. By using the blue/white screening, we can pick out non-recombinant plasmids visibly because the colonies should be blue in color.
Questions about Molecular Biology are getting more and more common in exams. While they may appear abstract at first sight, they can be easily tackled with a sound understanding of the concepts. Hope you find this article useful!
by Ed Law
