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. Before proceeding, you wish to know whether this gene is actually expressed in humans (your target market for therapeutic development). ?Describe, in detail, 2 efficient experimental approaches to answer this question. ?Discuss whether your proposed methods measure synthesis or accumulation of the product

Answer 1

Experimental approaches to know if the gene is actually expressed in humans-

• Northern blot technique will determine the expression of the gene- Northern blotting is a technique that studies gene expression by detecting the RNA/mRNA in the sample. The technique involves gel electrophoresis of the isolated RNA samples by size, transfer on the membrane followed by hybridization of this to a labeled probe which is complementary to the target. Finally visualized on X-ray film using labeled probes.
• Real-time PCR analysis of mRNA - RNA (mRNA ) is isolated from the tissue or blood sample. Then the RNA is converted to cDNA using specific primers followed by real-time amplification of the cDNA using TaqMan probes or SYBR green assay which will give the expression pattern of the gene. The experiment should be normalized with housekeeping gene expression
• in situ hybridization for RNA detection will reveal the expression of the genes( If there is a tissue from the patient which is known to express the gene)
• PCR amplification of the specific gene followed by nucleic acid sequencing will provide if the gene DNA is actually present or not. The sequence can be compared with the database to check for percentage identity/ homology.

Real-time PCR will measure the synthesis of the gene as it gives a real-time quantification of the gene. Northern blotting is used to observe particular gene expression and check if the gene is upregulated or downregulated.