How PCR Turns One Copy of a Gene Into a Billion

Knowing the tools of biotechnology is only the start. To make a useful protein, scientists must isolate DNA, cut out the right gene, make many copies, insert it into a host and then grow that host on a huge scale.

This part covers isolating DNA, cutting and separating DNA fragments, the polymerase chain reaction, and insertion, bioreactors and downstream processing.

DNA is released by breaking open cells with enzymes such as lysozyme for bacteria, cellulase for plant cells and chitinase for fungi, then freed from RNA and proteins with ribonuclease and protease, and finally precipitated as fine threads by adding chilled ethanol.

Breaking cells open:

- Lysozyme — bacterial cell walls
- Cellulase — plant cell walls
- Chitinase — fungal cell walls

Removing other molecules:

- Inside cells, DNA is wrapped with histones and mixed with RNA, proteins, polysaccharides and lipids
- Ribonuclease removes RNA
- Protease removes proteins
- Other molecules are removed by suitable treatments

Precipitating DNA. Adding chilled ethanol makes purified DNA come out of solution as a mass of fine threads, which can be spooled out.

An everyday example. Salt, detergent and cold alcohol can pull visible white DNA threads out of crushed onion in a school laboratory.

The substance. The enzyme must match the cell wall — lysozyme cannot break down a plant cell wall made of cellulose.

Purified DNA is incubated with a restriction enzyme to cut it at specific sites, and the fragments are separated by agarose gel electrophoresis, in which negatively charged DNA moves towards the anode and smaller fragments travel farther; the desired bands are cut out of the gel and the DNA recovered by elution.

Cutting:

- DNA is incubated with a restriction enzyme under optimal conditions
- Progress of digestion is checked by agarose gel electrophoresis
- The vector is cut with the same enzyme, the gene is joined to it, and DNA ligase seals the recombinant DNA

Gel electrophoresis:

- DNA is negatively charged, so it moves towards the anode in an electric field
- The agarose gel, obtained from seaweed, acts as a sieve, so smaller fragments move farther
- DNA is stained with ethidium bromide and seen as orange bands under UV light

Elution. Bands containing the desired fragment are cut out of the gel, and the DNA is extracted, ready for joining to vectors.

An everyday example. A kitchen sieve lets fine flour through faster than coarse grains — the agarose gel lets small DNA fragments move faster than large ones.

The substance. Separation depends on size, not charge — every DNA fragment carries negative charge in proportion to its length, so size decides how far it travels.

PCR makes huge numbers of copies of a chosen DNA segment in a tube by repeating three steps — denaturation, annealing of primers, and extension by a thermostable DNA polymerase — and Taq polymerase from Thermus aquaticus is used because it survives the high temperature of denaturation.

Ingredients: template DNA, two primers (short, chemically synthesised stretches of DNA complementary to the ends of the target region), deoxynucleotides and a thermostable DNA polymerase.

The three steps of each cycle:

- Denaturation — heating separates the two DNA strands
- Annealing — cooling lets the primers bind to their complementary sequences
- Extension — the polymerase adds nucleotides from the primers, copying the target region

Why Taq polymerase. It comes from the bacterium Thermus aquaticus, which lives in hot springs, so it stays active through the repeated high-temperature denaturation steps.

Worked example. Each cycle doubles the number of copies, so one DNA molecule gives $2^n$ copies after $n$ cycles. After $30$ cycles:

$$2^{30}\approx 1.07\times 10^9\text{ copies}$$

An everyday example. Diagnostic PCR tests for viral infections amplify tiny amounts of viral genetic material until it can be detected.

The substance. The primers decide what gets copied — PCR amplifies only the stretch between them.

Recombinant DNA is introduced into competent host cells, transformed cells are selected with markers such as antibiotic resistance, and the cells are grown on a large scale in bioreactors such as stirred-tank and sparged designs, before the product is separated, purified, formulated and quality-tested in downstream processing.

Insertion and selection:

- Competent host cells take up the recombinant DNA
- If the DNA carries an antibiotic-resistance gene, only transformed cells grow on medium containing that antibiotic
- A protein made from a foreign gene in the host is a recombinant protein

Bioreactors:

- Large vessels, often 100 to 1000 litres, providing optimal temperature, pH, substrate, salts, vitamins and oxygen
- Stirred-tank reactor — usually cylindrical with a curved base, and a stirrer that mixes the contents and spreads oxygen evenly
- Sparged stirred-tank reactor — air is bubbled in, greatly increasing the area for oxygen transfer
- Fitted with foam control, temperature and pH control and a sampling port

Continuous culture. Used medium is drained out while fresh medium is added, keeping cells in their most active log phase and raising yield.

Downstream processing:

- Separation and purification of the product
- Formulation with suitable preservatives
- Clinical trials for drugs and strict quality control

An everyday example. Tall steel fermentation tanks in Indian vaccine and enzyme factories are bioreactors running under tightly controlled conditions.

The substance. Making the protein is only half the job — without downstream processing it cannot be safely used as a medicine.