Question

Given a. whole cell protein extracts, b. nuclear protein extracts - derived from cells infected with a virus and c. the molecular DNA clone of the virus.
What experiments would you recommend to determine where the genes are? where the promoters are? and what proteins bind to the promoters?

Answer 1

Biomolecules such as whole protein extracts, nuclear extracts, those from infected cells or clones can be used to determine the location of genes. In order to perform the experiment, following simple steps can be taken:

1. Label 4 tubes, each containing all these samples.
2. Prepare the 1:1 dilution of these tubes.
3. Add protease to one set and RNase/DNasesto other set.
4. Prepare appropriate forward and reverse primers against the operator site, gene site and any other DNA target.
5. Perform an 8-plex PCR for all the tubes.
6. Analyse on an agarose gel. The gels giving positive results for either genes or their promoter sites suggest their binding sites. Negative results show the proteinaceous nature of the sample.
7. Finally, conduct a CHIP assay by using appropriate antibodies against the gene product and the same primers.
8. Analayse the results on agarsoe gel.
9. The location on the gel showing absence of the bands shows the binding site of the protein for the promoter.

Thus, these simple steps can be performed to investigate the appropriate sites on the biomolecules.