DNA profiling

DNA Introns – the regions of the chromosomes

which are used in DNA profiling. Mini-satellites – 20-50 base sequence

repeated from 50 to several hundred times.

Micro-satellites – 2-4 bases repeated between 5 and 15 times.

The more closely related the two individuals, the more similar the DNA patterns are.

It is highly unlikely that any two individuals

have the exact same pattern of DNA, unless

they are twins, of course!

The process DNA is extracted from a blood or cell

sample. It is split into fragments using restriction

endonucleases. These cut the DNA at certain points in the

intron sequences. Each type cuts a DNA molecule into

fragments at different recognition sites.Using restriction enzymes that cut

either side of mini- and micro-satellite units leaves the repeated sequences intact, giving a mixture

of DNA fragments made up largely of mini- and micro-satellite sequences.

Gel electrophoresis The fragments are placed in wells in an agarose gel

medium in a buffering solution, with known DNA fragments.

The gel contains a dye which binds to the DNA fragments in the gel.

A dye is also added to the DNA samples. An electric current is passed through the apparatus

and the DNA fragments move towards the positive anode.

The plate is placed under UV light. The DNA fluoresces so it can be identified.

A dye is also added to the DNA samples. It moves through the gel faster than the DNA so that the current can be turned off

before all the samples run off at the end.

Southern blotting An alkaline buffer solution is added to

the gel after electrophoresis. A nylon filter or (nitrocellulose paper)

is placed over it. Dry absorbent paper is used to draw

the solution containing the fragments from the gel to the filter, leaving ‘blots’ on it.

The alkaline solution also denatures the fragments so the strands separate and the base sequences are exposed.

Gene probes These are short DNA sequences that are

complementary to specific sequences which are required.

Hybridisation - large amounts of the probe are added to the filter and bind with the complementary DNA strands.

Excess probes are washed away. X-ray pictures are taken of the filter or the filter is

placed under UV light.

Each probe is labelled, either with a radioactive

element or with a fluorescent molecule.

In forensic science, they are used for picking out short tandem repeats – micro-satellite regions which are now widely used in DNA identification . The more micro-satellites are used to make up a profile the more accurate it will be.

Polymerase chain reactionThe reactants are placed in a vial in a PCR machine.

The reactants:• DNA sample

• DNA polymerase (enzyme)

• Primers• The four nucleotide

bases

Mixture is heated to 90-95°C – which causes the DNA strands to separate as the hydrogen bonds

between them break down.

Mixture is cooled to 50-60°C – the primers bind (anneal) to the single DNA strands.

Mixture is heated to 75°C – the optimum temperature for the enzyme to build the

complementary strands of DNA.