Question

A student is tasked with cloning a specific gene-coding sequence from a human cancer cell in order to produce the corresponding protein in bacteria at a concentration sufficient to conduct in vitro biochemical studies.

A) The student decides to use primers specific to the 5’ and 3’ ends of the human gene based on a genomic DNA sequence he found in the human genome database. After using a genomic library for PCR amplification, plasmid cloning, and bacterial transformation, the student discovers that only short polypeptides of the protein are produced rather than full length. Why might this occur?

Answer 1

In bacteria, there are no introns, so there is no splicing machinery in the bacteria, but the eukaryotic gene has introns. Introns are removed by splicing after the transcription if the genomic DNA corresponding to the gene of interest is cloned into the bacteria under a prokaryotic promoter the transcript produced will have both exons and introns.

The student got smaller protein than the expected one, this can be due to the presence of a stop codon in the first intron in frame with the starting codon in the mRNA, translation stops at the stop codon and small polypeptide is produced.

answered by: anonymous