PCR and DNA profiling

PCR: copying DNA

The polymerase chain reaction (PCR) makes millions or billions of copies of a chosen stretch of DNA. It was invented by American chemist Kary Mullis in 1983, an idea he said came to him while driving at night, and won him the 1993 Nobel Prize in Chemistry. PCR is one of the most used techniques in biology: it is how COVID-19 PCR tests detected the virus's genetic material.

A PCR tube contains the sample DNA, a supply of free nucleotides, a DNA polymerase enzyme, and two primers: short single strands of DNA, about 20 bases long, made to match the two ends of the section to be copied. A machine called a thermocycler then repeats three steps, each taking a minute or less:

1. Denature (about 95 °C): the heat breaks the hydrogen bonds and separates the two strands.
2. Anneal (about 50–65 °C): the mixture cools and the primers pair with their matching sequences on each single strand.
3. Extend (72 °C): DNA polymerase starts at each primer and adds nucleotides to build a new complementary strand.

Each cycle doubles the number of copies of the target section. After n cycles there are 2ⁿ copies of each starting molecule: 2¹⁰ ≈ 1000 after 10 cycles and 2³⁰ ≈ 1 billion after 30. Most enzymes would be destroyed by the 95 °C step. The trick that made PCR practical is Taq polymerase, from a bacterium, Thermus aquaticus, discovered in a hot spring in Yellowstone National Park. Its enzymes survive near-boiling temperatures.

Cutting DNA with restriction enzymes

Restriction enzymes are made by bacteria to chop up the DNA of invading viruses. Each one recognises a particular short sequence, usually 4 to 8 bases, and cuts the DNA there. For example, the enzyme EcoRI, from E. coli, finds the sequence GAATTC and cuts between the G and the A. Because the sequence reads the same on the opposite strand (in the opposite direction), the cuts are staggered, leaving single-stranded overhangs called sticky ends that can pair with any other DNA cut by the same enzyme. Hundreds of restriction enzymes are known, and they are basic tools for cutting DNA in gene technology (1J).

Gel electrophoresis

Gel electrophoresis separates pieces of DNA by size.

• A slab of agarose gel, a jelly made from seaweed, is set with a row of small wells at one end and covered in a salt buffer solution.
• DNA samples are loaded into the wells, with a ladder of fragments of known sizes in one lane for comparison.
• An electric current is passed through the gel. DNA is negatively charged, because of its phosphate groups, so all the fragments move towards the positive electrode.
• The gel is a mesh of fibres. Short fragments slip through faster and travel further; long fragments are held back.
• The gel is stained with a dye that glows under UV or blue light, showing each group of same-sized fragments as a band.

Fragment sizes are measured in base pairs (bp). The distance a fragment travels is roughly proportional to the logarithm of its size, so the bands of a ladder get closer together near the top of the gel.

DNA profiling

Since 99.9% of human DNA is the same in everyone, profiles focus on places where people differ a lot. Short tandem repeats (STRs) are short sequences, such as GATA, repeated over and over: one person might have 8 repeats at a particular place on chromosome 5, another 13. Each person has two alleles at each STR site, one from each parent. PCR copies about 20 STR sites at once, and the lengths are measured, today usually by a machine that pushes them through a thin gel-filled tube (capillary electrophoresis) and reads them with a laser. The result is a list of numbers: the person's DNA profile.

British geneticist Alec Jeffreys invented DNA fingerprinting in 1984. It was first used in a criminal case in England in 1986–87, where it cleared an innocent suspect who had confessed, and then identified the real killer. Uses now include:

• forensic science: matching crime scene samples to suspects, and freeing people who were wrongly convicted
• paternity and family testing: a child's alleles must each come from one parent
• identifying human remains after disasters, or of soldiers from past wars, by comparison with relatives
• conservation and wildlife crime: identifying poached ivory or illegally traded animals, and checking how related individuals in breeding programs are, such as Tasmanian devils kept free of facial tumour disease.

Limits and safeguards

• A match shows that DNA came from a person, not how or when it got there. Skin cells and hair can be transferred by touch or carried on clothing, so innocent people's DNA can turn up at crime scenes.
• Tiny samples can be contaminated, so forensic labs use strict procedures, protective clothing and "elimination" profiles of their own staff.
• Close relatives share many alleles, and identical twins share all of them.
• DNA databases raise privacy questions: who is stored, for how long, and who can search them. In Australia, laws in each state and territory control when police can take samples and how long profiles are kept.