Summary
Highlights
Recombinant DNA technology has broad applications in medicine, agriculture, and biotechnology. The initial step involves preparing the DNA by selecting a gene of interest, which can be chemically synthesized if its sequence is known. Complimentary DNA (cDNA) libraries are used to collect the desired gene sequence. cDNA libraries are constructed by isolating mRNA, annealing an oligo-dT primer to its polyA tail, and using reverse transcriptase to synthesize a cDNA strand. DNA polymerase then synthesizes a second DNA strand, forming a double-stranded cDNA molecule. This cDNA sequence is then incorporated into libraries for future use to synthesize specific proteins.
The next step is preparing a transfer vector, a DNA molecule that transports a specific DNA segment into a host cell. Vectors can be plasmids, cosmids, viral vectors, or artificial chromosomes, with plasmids being the most common, especially E. coli plasmids like pUC8. After vector preparation, restriction enzyme digestion is performed. Restriction enzymes cut DNA at specific sites, creating either 'sticky ends' or 'blunt ends'. The gene of interest and the plasmid vector are treated with the same restriction enzymes to create compatible ends. Plasmids typically have an origin of replication, a drug resistance gene (like ampicillin resistance), and a region for DNA insertion like the lacZ gene, which contains multiple cloning sites.
Following restriction digestion, the gene of interest and the cut plasmid vector are combined, and DNA ligase enzymes are added. DNA ligase facilitates the joining of DNA strands by catalyzing the formation of a phosphodiester bond, requiring ATP as a co-factor. This process integrates the gene into the plasmid, physically attaching the insert DNA to the plasmid backbone.
The recombinant plasmid is then introduced into host cells, typically E. coli, using chemical transformation. This method involves using calcium chloride to neutralize the negative charges of both the bacterial cell membrane and the DNA, reducing repulsion. The mixture is incubated on ice, then heat-shocked at 42°C for 90 seconds to create temporary pores in the cell membrane, allowing the plasmid DNA to enter. Afterward, the tube is placed back on ice for two minutes to help the cells recover and secure the plasmid. A recovery medium is then added, and the cells are incubated at 37°C for one hour to facilitate recovery.
After transformation, bacterial cells are cultivated by spreading them on a culture medium and incubating overnight. Three outcomes are possible: non-transformed cells (that won't survive due to ampicillin in the medium), transformed cells with unaltered vectors, and transformed cells with recombinant vectors. Only transformed cells survive due to the ampicillin resistance gene in the plasmid. To differentiate between unaltered and recombinant vectors, x-gal substrate is included in the medium. Unaltered vectors allow the lacZ gene to function, producing beta-galactosidase which cleaves x-gal, resulting in blue colonies. Recombinant vectors, where the gene of interest disrupts lacZ, result in white colonies because beta-galactosidase is not produced. White colonies are then isolated and transferred to an enriched culture medium.
The isolated recombinant cells are grown on a small scale in a laboratory or on a large scale using bioreactors. Bioreactors provide optimal conditions for bacterial cell multiplication, ensuring efficient production of the desired protein.
Once the desired protein is produced, it needs purification. If the protein is intracellular, cell disruption is necessary, often by centrifuging cells and then using an enzymatic lysis buffer. This breaks open the cells and releases the protein. Affinity chromatography is then used for separation. A stationary phase with a specific support medium binds the target protein while other molecules pass through. A wash buffer removes non-specifically bound impurities. Finally, an elution buffer disrupts the specific binding, releasing the purified protein into a new collection tube. The purified protein is then ready for use.