Preparation for “WDYTYA” practical 3

Recap

So far – have taken Buccal swab – extracted the DNA and amplified the D-loop with use of PCR. Once this was purified –
checked sample with Gel electrophoresis, we then took our sample and set up sanger sequencing. We must now purify
the products of the sanger sequence.

Cleaning Sequencing Product

Add in various reagents, starting with EDTA – this has been added previously to protect DNA. This is because EDTA
chelates divalent cations – stopped DNase enzymes from working. However, if too much is added – DNA pol cannot work
– doing this on purpose causes halt in proceedings stopping DNA pol from working (this is what we now want). Then add
ethanol to the mix – precipitates out DNA allowing formation of pellet with centrifugation. This is then washed in 70%
ethanol before being resuspended in 50ul of sterile distilled water – this is an artificial step – if done in lab you resuspend
in formamide – this a nasty chemical, just to transfer it in a tube – we use sterile water.

ABI 3500 Sequencer

Once you’ve transferred your resuspended DNA – transferred into a 96 well microtiter plate and vacuum dried – this puts
DNA straight back into a precipitated form as there will be no solvent for it to be dissolved in and all the water will be
evaporated. This is important as water and formamide do not mix – creates formic acid – therefore you need to
completely dry the sample before resuspending in formamide itself.

Formamide is used to capillary electrophoresis because of its chemical properties. Formamide maintains the quality and
conductivity of the solvent – good for stabilizing single strands of denatured DNA. Once this is all done – plates ran on
sequencer over next few days – can download data digitally

ABI 3500 sequencer – glorified gel electrophoresis. Inside machine is the capillary in which the DNA fragments will be
passed – does exact same job as gel electrophoresis – separating fragments by size. Amplified DNA is injected – current is
applied. DNA pulled towards positive electrode – large fragments travel slower. Colour of fragments detected/recorded
as it passes the detector plates – this will detect differences of 1bp – in the end you can read the sequence.