PCR, gel electrophoresis and DNA profilingEdexcel International A Level Biology: Revision notes

The polymerase chain reaction (PCR) makes millions of copies of a chosen DNA sequence in a few hours, so a tiny sample (a few cells from a crime scene, a hair, a seed) gives enough DNA to analyse.

The reaction mixture contains:

• the template DNA containing the target sequence
• two primers, short single-stranded DNA sequences complementary to the ends of the target region
• free nucleotides (the four bases) as building blocks
• Taq polymerase, a thermostable DNA polymerase from the bacterium Thermus aquaticus
• a buffer to hold the pH steady

The mixture is heated and cooled repeatedly in a thermal cycler.

Each cycle has three stages:

1. Denaturation at about 95 °C: hydrogen bonds between the complementary bases break and the double helix separates into two single strands.
2. Annealing at about 55 °C: the mixture is cooled so the primers form hydrogen bonds with their complementary sequences on each template strand.
3. Extension at about 72 °C: Taq polymerase adds complementary nucleotides to the 3' end of each primer, building a new strand.

The number of copies doubles each cycle, so after $n$ cycles one molecule gives $2^{n}$ copies. After 30 cycles this is $2^{30}$ = 1.07 × 10⁹.

DNA can be cut into fragments by restriction enzymes, or the fragments can be made by PCR. Gel electrophoresis separates fragments by size.

• The DNA samples are loaded into wells at the negative end of an agarose gel covered in buffer.
• A direct current is applied. The phosphate groups of DNA make it negatively charged, so the fragments move towards the positive electrode (anode).
• The gel has pores: smaller fragments pass through more easily and travel further in a given time; larger fragments move less far.
• A DNA ladder (fragments of known size) is run alongside so sizes can be estimated.
• The DNA is invisible, so it is made visible with a fluorescent dye under UV light, or with labelled probes.

Most of the human genome is non-coding. Within it are short tandem repeats (STRs), short sequences of 2–6 bases repeated many times in a row. The number of repeats at a given locus varies between individuals.

To make a DNA profile:

1. Extract DNA from a sample (blood, saliva, hair root, tissue, leaf).
2. Amplify selected STR loci by PCR with labelled primers.
3. Separate the fragments by electrophoresis; more repeats make a longer fragment that travels less far.
4. Visualise the pattern of bands and compare it with other samples.

A single locus is shared by many people, but the chance of two unrelated individuals matching at many loci is very small, because the probabilities multiply.

Identification: a profile from a crime scene is matched with a suspect's profile. A match at enough loci strongly links the sample to the suspect, and a mismatch at any locus excludes them.

Relationships: a child inherits one copy of each STR from each parent. Every band in a child's profile must therefore be present in the mother or the father. If neither a possible parent has a band the child has, that individual is excluded.

Worked example: a foal has 9 and 14 repeats at a locus; its mare has 9 and 12. The 9 came from the mare, so the 14 must have come from the sire. A stallion with 11 and 13 is excluded; one with 14 and 11 could be the sire.

Profiles of close relatives are more similar, because they share many inherited alleles, so a match with a relative must be considered.

DNA profiling is used with plants and animals, as well as in human forensics:

• Pedigree and parentage in animals: checking the sire of racehorses, dogs and livestock, and managing captive breeding programmes by avoiding closely related pairings that would reduce genetic variation.
• Wildlife crime: identifying the species or population of origin of ivory, rhino horn or timber to detect illegal trade.
• Plants: identifying crop varieties, checking seeds are the variety sold (for example rice or wheat), and tracing the origin of plant material found at a scene, such as pollen, seeds or leaves.
• Genetic relationships between populations and species, by comparing how similar their profiles are.

Evidence is stronger when more loci are used and when the laboratory avoids contamination, which PCR's sensitivity makes a real risk.