Question

Suppose you are working on a set of experiments using Western blotting. Using an antibody that recognizes amino sequences encoded by exon 3 of a five-exon gene, you have demonstrated that a protein is highly expressed in a particular cell line. To assess the mRNA, you design reverse transcriptase polymerase chain reaction (rtPCR) primers to exons 2 and 4, which flank exon 3. Despite all your efforts, you cannot amplify an mRNA. You know that it is indeed expressed, since the protein is detectable. You also know that your controls are working and your experimental procedure is sound, because you are able to detect an mRNA for the other cell lines. Given what you know about the “central dogma” and gene structure, how might you explain this inability to identify the mRNA encoding this exon 3 protein?

Answer 1

1. The gene of eukaryotes contains both exons and introns . Exons are the coding sequences and introns are the non coding sequences. Introns are spliced out and exons are added together to give a processed mRNA. In rt PCR , complementary DNA is synthesized from mRNA and to that c DNA forward and reverse primers are synthesized and by using these primers DNA is synthesized opposite to cDNA. If primers are synthesized to exons 2 and exon 4 only those regions only amplified but not exon 3 because introns are present in between the exons. So you cant amplify the mRNA. If you want to amplify a particular exon in mRNA gene specific primers can be used to amplify. But once the protein is synthesized due to complementarity probe can detect the protein. The genes in eukaryotes are monocistronic . So primers cant amplify the the all exons as they are flanked by introns.