Processes of recombinant DNA technology
Isolating DNA, amplifying it by PCR, inserting it into a host, bioreactors and downstream processing.
The essentials
Recombinant DNA technology follows a series of steps.
Isolation of DNA: To release DNA, cells are broken open using enzymes: lysozyme for bacteria, cellulase for plant cells and chitinase for fungi. DNA is associated with RNA, proteins and lipids, so it is treated with ribonuclease to remove RNA and protease to remove proteins. Purified DNA is precipitated by adding chilled ethanol and appears as fine threads that can be spooled.
Cutting DNA: The purified DNA and the vector are cut with the same restriction enzyme, so they share complementary sticky ends. Agarose gel electrophoresis checks the progress of digestion. The gene of interest is then joined to the vector with DNA ligase.
Amplification by PCR (polymerase chain reaction): Many copies of a gene are made in vitro using two sets of primers (short chemically synthesised oligonucleotides complementary to the ends of the region) and DNA polymerase. Each cycle has three steps:
- Denaturation: heat separates the double-stranded DNA.
- Annealing: primers bind to complementary sequences.
- Extension: DNA polymerase extends the primers using nucleotides.
A thermostable enzyme, Taq polymerase from the bacterium Thermus aquaticus, survives the high temperature of denaturation. Each cycle roughly doubles the target DNA, so after n cycles one molecule can give up to 2ⁿ copies; repeating many cycles can give about a billion copies.
Insertion into the host: The ligated DNA is transferred into competent cells, which are selected on antibiotic-containing media.
Obtaining the foreign gene product: A protein encoded by a recombinant gene in a heterologous host is called a recombinant protein. For large-scale production, cells are grown in bioreactors (100–1000 litres) that provide optimum temperature, pH, substrate, salts, vitamins and oxygen. The stirred-tank bioreactor is commonly used; it has an agitator, oxygen delivery, foam control, temperature and pH control and a sampling port. Culture may be batch or continuous; in continuous culture, used medium is drained and fresh medium added to keep cells in the active log phase.
Downstream processing includes separation and purification of the product, formulation with preservatives, and strict quality control testing (clinical trials for drugs).