You are a researcher at a small biotech company and your company has just obtain

Question

You are a researcher at a small biotech company and your company has just obtained the license for use of a human GENOMIC DNA fragment putatively encoding a potentially novel protein, which is thought to regulate p53, the known tumor supressor protein. The scientists who originally cloned this GENE fragment HDM5 "claim" that HDM5 shares 90% DNA sequence homology with one of the HDM2 genes (refer to the review Levine & Oren, 2009). They propose that HDM5 may have HDM2-like properties and may be involved in regulating cell proliferation, and thus a good target to potentially develop as a cancer therapy. Your company has asked you to characterize the gene and gene products, as well as to provide an opinion as to its potential human therapeutic uses.

1. You now want to determine the tissue distribution (expression pattern) of your protein HDM5 and compare that to the expression of other HDM2-like proteins.

Explain how you might use one method to determine the relative human tissue expression levels and patterns.

Explanation / Answer

Ans: Simplest method would be real-time PCR or quantitative PCR.

First step: Identify a set of DNA sequences that are highly specific for HDM5 only and HDM2 only (taking care that there is no overlap) that can be used as a primer for quantitative PCR (using various bioinformatics tools available online)

Second step: once we have procured the primers, by real time or quantitative PCR, we can analyse the levels of expression of these genes (HDM2 and HDM5) in various human tissues. A good idea would be to first compare the tissue expression in normal human tissues isolates and then in various cancer tissue isolates. This will directly give us an idea whether the expression pattern of HDM5 is similar to HDM2 and whether or not it is good target for cancer therapy.In case we want to be doubly sure, we can include another protein upstream or downstream of HDM2 pathway and measure its expression levels simultaneously.

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