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.

B) Another student mentions that her should have cloned the gene sequence using mRNA instead of genomic DNA. Is this a good suggestion? How does one go about doing this?

Answer 1

yes. in bacteria there are no introns, so there is no splicing machinery in the bacteria, but 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, so the protein produced will be longer than normal protein and it will be non-functional.

to avoid this start with mRNA from the cell, isolate mRNA and make cDNA using reverse transcriptase enzyme, since mature mRNA lacks introns, cDNA made from mRNA also lacks introns,so cDNA has only exons which codes for proteins, after making cDNA, amplify the gene of interest with proper primers having restriction sites to clone into a plasmid, then digest the amplified gene with proper restriction enzymes and then ligate this gene of interest to the plasmid digested with same set of restriction enzymes and then transsform the bacteria using recombinant plasmids.