DNA Profiling

(STRs) are short DNA sequences repeated many times. They're found within (intragenic regions) that are non-coding. They're inherited in the same way as genes in the coding regions - (expressed regions). STRs occur at the same on both chromosomes of a pair. The number of repeats on each of the homologous chromosomes can be different. The number of repeats at a locus varies between individuals. There's a large amount of variation in the number of repeats at each locus. Therefore 2 individuals are highly unlikely to have the same combination of STRs.

The tissue sample is broken down in a solution (includes salt and detergent) to disrupt the cell membranes. The small suspended particles, including the DNA, are separated from the rest of the cell debris by filtering or . enzymes are incubated with the suspension to remove proteins. Then, cold is added to precipitate out the DNA. Several stages of washing the DNA in buffer solution follow.

Polymerase Chain Reaction: The sample is placed in a reaction tube with DNA , DNA primers and . The reaction tube goes into the PCR thermal cycler. It's heated to degrees C which separates the double stranded DNA. It's then cooled to degrees C which optimises the binding of the to the target DNA sequence in the sample. It's then heated to degrees C which is the temperature for the heat stable DNA polymerase. The polymerases attach and are added, extending the DNA from the primer. After 2 cycles, copies that are just the sequence fragment are produced. As the cycle continues, huge numbers of the targeted DNA fragments are produced.

Gel Electrophoresis: DNA placed on or polyacrymalide, which provide a stable medium through which the fragments can move (porous). The gel is submerged in a solution, and connected to that produce a potential difference (voltage across the gel). The charged DNA fragments migrate through the gel according to their overall size and . In a given time, smaller fragments end up closer to the electrode. A reference sample with fragments of known length may be added to the gel. This is known as a ladder or marker. The fragments are measured in base pairs.

Southern blotting is used to transfer the fragments to a more resilient or nitrocellulose membrane. The membrane is placed directly onto the gel and a wad of dry absorbent is placed on top. This acts as a wick to draw up through the gel, carrying onto the membrane. During this process, the fragments maintain their positions relative to each other and are denatured into strands, exposing the base sequences. The membrane is then incubated with an excess of DNA . After allowing time for the probe to bind to any complementary sequences (hybridise), any unbound probe is washed away.