You have cloned the gene sequence of the novel eukaryotic protein you have named

Question

You have cloned the gene sequence of the novel eukaryotic protein you have named Protein Bob (2,100 bp in length). You have used PCR to amplify up the Protein Bob gene from your isolated genomic DNA. After growing up colonies and testing the plasmid you discovered that your cloning protocol has gone according to plan and you did every step correctly, there was no human error. Yet, there is no functional protein production. Explain the reasoning behind this problem. How would you rectify the problem?

Explanation / Answer

The probable reasons behind this problem might be -

Eukaryotic genes have introns and exons, interpersed with each other. Before translation, the pre-mRNA is spliced to remove the introns. This splicing machinery is absent in prokaryotic cells (bacterial cells where the protein is tried to exprssed). The genomic DNA will have the entire gene of protein Bob along with its introns- which is probably messing up the translation step.

This can be rectified by- taking the cDNA of the protein bob and using it to make the clone.

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