Question

Various experiments have shown that the SARS CoV-2 is an RNA virus that enter the human cells through a transmembrane receptor called angiotensin converting enzyme 2 (ACE2). Polymerase chain reaction (PCR) and genome sequencing have been cardinal in both diagnosis and research into the COVID-19 pandemic. Currently, DNA sequences of both SARS CoV-2 virus and susceptible human hosts are available. As a student of molecular biology and biotechnology, use the above information to answer the following questions.

9.In the study of the proteome of human cells infected with SARS CoV-2 virus, demonstrate how “hybrid release translation (HRT)” or “hybrid arrest translation (HART)” could be employed to differentiate between protein products of SARS CoV-2 and human genomes.

Answer 1

The above two techniques are used to identify the mRNA and the translated protein coded by a particular DNA sequence.

In covid virus infected cells these techniques can be used to differentiate the proteins produced by our cell and covid virus. This technique use a cell free translation system. The cell free translation system will have all molecules necessary for protein synthesis such as ribid, aminoacid, Mg, tRNA etc.

Now in HRT system what we does we will isolate the total mRNA from the covid infected cell. At the same time the single stranded cDNA of our interest ( viral or our protein) will be coated in the nitrocellulose membrane. Now the isolated mRNA from the virus infected cell will be added to the nitrocellulose membrane and only the specific mRNA corresponding to the complimentary DNA will undergo hybridisation. The unbound mRNA will be removed and the mRNA from cDNA- mRNA hybrid will be removed by denaturing the hybrid complex. And this specific mRNA will be added to cell free translation system and it will be translated to the specific protein.

Now in HART system the mRNA will be isolated from virus infected cell. This isolated total mRNA will be treated with the specific cDNA of our interest ( which can be either viral or human). Now what happens is that cDNA will get hybridised with specific mRNA. Now when added to cell free translation system except the bounded mRNA, rest of them will be translated to the protein. Thus in this technique a translation arrest is happening to the specific protein. Then the untranslated protein can be identified from autoradiograph.