DNA profiling and PCREdexcel A-Level Biology A: Revision notes

A DNA profile is a pattern of bands that is specific to an individual (except identical twins). It is built from short tandem repeats (STRs), also called variable number tandem repeats, which are non-coding sequences of DNA repeated a different number of times in different people. Because the number of repeats varies, the fragments produced vary in length.

Steps: DNA is extracted from cells; the STR regions are amplified by PCR; the fragments are separated by gel electrophoresis; the pattern is stained or detected with a fluorescent or labelled probe and compared.

The chance that two unrelated people share the same profile is tiny because several STR regions are compared.

Identification: matching DNA from a crime scene with a suspect, or identifying remains. Genetic relationships: half of each person's bands come from each parent, so bands in a child that are absent from the mother must come from the father. The more bands two individuals share, the more closely related they are.

In animals and plants profiling is used to identify species and the origin of wildlife products, to avoid inbreeding in captive breeding programmes (choose pairs sharing the fewest bands), to check the parentage of livestock and to identify crop varieties.

PCR amplifies a tiny DNA sample into millions of copies in a thermal cycler. The mixture contains the DNA template, two primers, free nucleotides (dNTPs), a buffer and Taq polymerase.

Each cycle:

• Denaturation, about 95 °C: hydrogen bonds between the strands break, giving single strands.
• Annealing, about 55 °C: primers bind (anneal) to the complementary ends of the target sequence.
• Extension, about 72 °C: Taq polymerase adds nucleotides to the 3' end of each primer, making a new strand.

The number of copies doubles every cycle, so after $n$ cycles one molecule gives $2^n$ copies. For example $2^{30}$ is about $1.1 \times 10^{9}$.

Gel electrophoresis separates DNA fragments by length. DNA samples, mixed with loading dye, are placed in wells at the cathode (negative) end of an agarose gel covered with buffer. A potential difference is applied.

DNA is negatively charged because of its phosphate groups, so it moves towards the anode (positive). The gel is a mesh, so smaller fragments move faster and further. A ladder of known fragment sizes is run alongside. After the run the gel is stained (or a fluorescent dye is used) and viewed under UV light.

To estimate the size of a fragment, measure how far it travelled and compare it with the ladder. Sizes outside the ladder range cannot be estimated reliably.

Safety: wear gloves, because many stains are hazardous, and avoid direct UV exposure.

In eukaryotes the gene is first transcribed into a primary transcript (pre-mRNA) that contains exons (coding sequences) and introns (non-coding sequences). This is modified after transcription: the introns are removed and the exons joined by splicing to make mature mRNA, which leaves the nucleus for translation.

In alternative splicing different combinations of exons are joined, so one gene gives different mature mRNAs, with different codon sequences. These are translated into proteins with different amino acid sequences and tertiary structures, so one gene can give rise to more than one protein with different functions. This is why humans have more proteins than genes.