Question

Question 3

You have been given two respiratory samples suspected with COVID-19. Your lab member did the RNA extraction and isolated the mRNA. Luckily, a collaborator from Wuhan had also given a sequence encoding for gene E of COVID-19 to your lab.

Your lab also has equipped with the items below:
• Agarose
• formaldehyde
• Ethidium bromide
• TBE buffer
• RNA ladder
• Nylon membrane
• DIG-High Prime DNA Labeling and Detection Starter Kit II

By having all the above items, what type of experiment you can plan to execute in order to confirm both samples.

Name the experiment and explain the methods involve together with the expected result.

Name

…………………………………..

Methods •

Expected result • (20 marks)

Answer 1

The method to be used is agarose gel electrophoresis followed by its detection using DIG-High Prime DNA Labeling and Detection Starter Kit II. As the collaborator from Wuhan has already provided the sequnce for gene E of COVID-19, the presence of this sequence in the two respiratory samples can be used to confirm the presence of COVID-19.

The gel is prepared using agarose, TBE buffer and ethidium bromide. Ethidium bromide is used to view nucleic acids under UV irradiation. As the mRNA is already isolated from the two samples, they are loaded separately on the prepared gel along with the RNA ladder. The wells are labelled properly. The gel is then run and the observations are noted. As the sequence of gene E of COVID-19 is already known, its size is also known. The two samples are compared with RNA ladder for the presence of a band matching to the size of gene E of COVID-19. If the size matches it gives a preliminary idea about the presence of COVID-19 in the sample.

For further analysis, DIG-High Prime DNA Labeling and Detection Starter Kit II can be used. The RNA bands are transferred to a nylon membrane and the RNA probes complimentary to the gene E sequence is synthesized. They are then allowed to hybridize with the bands on the nylon membrane. Then chemiluminescence detection by enzyme immunoassay is carried out. If the probes hybridize chemiluminescence will be observed indicating the presence of COVID-19 in the sample.