What is biotechnology?
Biotechnology uses living organisms, or enzymes from them, to make useful products and processes. Modern biotechnology rests on two core techniques:
Genetic engineering: changing the genetic material (DNA or RNA) to alter an organism's characteristics.
Bioprocess engineering: keeping a sterile environment so that only the desired microbe or cell grows, in large quantities, to make products such as antibiotics, vaccines and enzymes.
The first recombinant DNA was made in 1972 by Stanley Cohen and Herbert Boyer. They cut an antibiotic-resistance gene from a plasmid of Salmonella typhimurium and joined it into a plasmid of E. coli.
Tools of recombinant DNA technology
1. Restriction enzymes: molecular scissors
Restriction endonucleases cut DNA at specific recognition sequences. These sequences are usually palindromic: they read the same on both strands when read in the same direction (5′→3′).
EcoRI, from E. coli RY13, recognises GAATTC and cuts between G and A on both strands. This leaves single-stranded overhangs called sticky ends. Because any DNA cut with the same enzyme has matching sticky ends, pieces from different sources can be joined.
DNA ligase acts as molecular glue: it seals the sugar–phosphate backbone of the joined fragments.
2. Separating DNA fragments: gel electrophoresis
DNA is negatively charged, so in agarose gel electrophoresis the fragments move towards the positive electrode (anode). Smaller fragments move faster and farther. The gel is stained with ethidium bromide and viewed under UV light, where the DNA shows up as orange bands. Bands can be cut out and purified (elution).
3. Cloning vectors
A vector carries the foreign DNA into the host and lets it multiply. Plasmids and bacteriophages are common vectors. A good vector has:
an origin of replication (ori): where replication starts, which controls the copy number,
a selectable marker: usually an antibiotic-resistance gene, to tell transformed cells from non-transformed ones,
cloning sites: preferably unique recognition sites for restriction enzymes,
a small size.
pBR322 is a widely used E. coli plasmid with resistance genes for ampicillin (amp^R) and tetracycline (tet^R).
Insertional inactivation. If foreign DNA is inserted into one resistance gene, that gene stops working. In a newer approach, the DNA is inserted into the lacZ gene, which codes for β-galactosidase. On a medium containing a chromogenic substrate, colonies with the insert stay white, and those without it turn blue. This blue–white screening makes selection easy.
4. Competent host
DNA cannot easily enter a cell. Bacteria are made competent by treating them with calcium ions, followed by a heat shock (ice, then 42 °C, then ice again). Other methods include:
microinjection of DNA directly into the nucleus of an animal cell,
the gene gun (biolistics): gold or tungsten particles coated with DNA are shot into plant cells,
disarmed pathogens such as Agrobacterium used as vectors.
The process step by step
Isolate DNA: break open cells with enzymes (lysozyme for bacteria, cellulase for plants, chitinase for fungi), remove RNA and proteins with ribonuclease and protease, then precipitate the DNA with chilled ethanol.
Cut the DNA at specific sites with restriction enzymes.
Amplify the gene of interest with PCR (polymerase chain reaction). Each cycle has three steps, denaturation, annealing of primers, and extension, and doubles the DNA. Extension uses the heat-stable Taq polymerase from the bacterium Thermus aquaticus. About 30 cycles produce over a billion copies.
Ligate the gene into a vector to make recombinant DNA.
Insert the recombinant DNA into a competent host.
Obtain the product: grow the host cells in large bioreactors, which control temperature, pH, oxygen and nutrients (stirred-tank bioreactors are common).
Downstream processing: separate and purify the product, then formulate it with preservatives. Products such as medicines must pass strict quality-control testing and clinical trials.
Why it matters
Recombinant DNA technology makes possible human insulin made by bacteria, vaccines, genetically modified crops such as Bt cotton, gene therapy, and the production of enzymes for industry.