2. It is highly unlikely that
any two individuals have
the exact same pattern
of DNA, unless they are
twins, of course!
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.
3. 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 miniand micro-satellite sequences.
4. 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.
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.
5. 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.
6.
7. Each probe is
labelled, either with a
radioactive element or with
a fluorescent molecule.
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.
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.
8. Polymerase chain reaction
The 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.
