DNA Profiling: Polymerase Chain Reaction

We're back to the murky world of DNA forensics, the first technique on the agenda is the polymerase chain reaction.  The polymerase chain reaction is an enzymatic process that facilitates the isolation and amplification of a specific sequence of DNA, without affecting the surrounding regions. When the quality of the DNA sample available to you is so limited in both quantity and quality,as it so often is in forensics, the importance of this technique sky rockets.

Without further ado, here's the protocol:
 1. Sample is incubated at 94-97˚c to separate the DNA helix into two separate strands by denaturing the DNA.
 2. Reduce the temperature to 50-60˚c to allow primers to 'anneal' (recombine into) to the DNA.
 3. Raise the temperature to 70-72˚c to initiate a polymerisation step, where an enzyme called Taq DNA polymerase utilises the DNA template identified by the primers to create a complimentary strand to the template.
 4. This sequence is repeated so that the original sequence is quickly amplified a thousand fold.
 5. After amplification the PCR products (amplicons) are separated on the basis of their length. The technique used to do this is known as capillary electrophoresis. Capillary Electrophoresis separates samples based on the velocity of their attraction to an oppositely charged electrode. Since DNA has a slight negative charge, the velocity of attraction increases with chain length.

The primer design for this process is extremely important. They a short strands of DNA that will hybridise onto the beginning and end of the are you want to analyse. The primers are also labelled with fluorescent dyes. This allows PCR products to be detected when exposed to a fluorescence inducing laser. This is recorded by a camera and allows the DNA produced to be quantified in real time. Once sufficient DNA has been produced it can be used for analytical techniques such as sequencing or southern blotting.

Short Tandem Repeat Marker analysis is the cornerstone of DNA forensics, however it can only be used with sufficient amounts of a specific genome. This can be determined using the fluorescence analysis technique described above, in combination with a TaqMan probe. A TaqMan probe is a protein that displaces part of a DNA strand, but cleaves when the synthesis enzyme reaches it, cleaving into a reporter dye and quencher dye. Now that the reporter and quencher die are no longer in close proximity (i.e. bound to each other), the reporter die fluoresces.

Given the comparatively hardcore science nature of this post I'm going to give your brains a rest and leave the fun that is STRs,sequencing and southern blotting for another time! Over and Out.