1. DNA Profiling

2. DNA Profiling There are two methods used to carry out DNA profiling
DNA profiling using a probe
DNA profiling using PCR
Knowledge of this is not required
It is the more common method now days

3. DNA profiling using a probe
This procedure relies on Minisatellites or Variable Number Tandem Repeats (VNTR)
VNTR are sequences of DNA 10 – 100bp long that repeat
The number of repeats is inherited resulting in sections of DNA of different length (can be between 0.5kbp – 40kbp)
Cf to restriction mapping which relies on microsatellites which are 1-4 bp repeats

4. DNA profiling using probes- procedure
Isolate DNA
DNA is isolated from sample using organic solvent which dissolves proteins leaving DNA
If DNA quantity is insufficient PCR will be performed (note this is different to the method of DNA profiling using PCR)

5. DNA profiling using probes – procedure cont…
Restriction enzyme digestion
Restriction enzyme which doesn’t cut in the minisatellite region is chosen
Creates DNA fragment of different sizes

6. DNA profiling using probes – procedure cont…
Gel electrophoresis
Used to separate the different sized fragments

7. DNA profiling using probes – procedure cont…
Blotting DNA onto a filter (Southern Blotting)
DNA is denatured using, for example, a strong alkali – this creates single stranded DNA
Single stranded DNA (still in the electrophoresis pattern) is transferred to a filter
Named after the scientist who first carried out the procedure – Edward M Southern working at Edinburgh University in 1970s

8. DNA profiling using probes – procedure cont…

9. DNA profiling using probes – procedure cont…
Hybridisation with probe
Labelled (either radioactively or fluorescent) probe which binds to a specific base sequence in the target fragments.
Multi-locus probes bind at more than one site. Used where certainty needs to be very high.
Single locus probes – bind to 1 sequence (giving 2 bands for each sequence)
Discuss the use of multi – locus probes being difficult to analyse due to the complex profiles given. Note students are expected to be able to analyse single – locus probe cases. Explain the origin of the two bands

10. DNA profiling using probes – procedure cont…
Create autoradiograph
Creates permanent record of the filter (usually using x-ray film)
If using multi-locus, the filter is then washed and another probe is used to add to the autoradiograph

11. Interpreting Autoradiograph – to establish paternity
After using a single locus probe 2 bands are present
As the VTNR forming the ‘band’ is inherited, 1 band is from maternal DNA, the other from paternal DNA.
Therefore, a person that has the same band as another person is potentially related.
A child’s bands should match one of the mothers and one of the fathers.
If this is not the case…….

12. M = maternal bands, F = paternal bands
M = maternal bands, F = paternal bands. Go through the process of determining paternity

13. Learning Activities Monograph pg 84 – 90 DART pg 87 – 88 Scholar 9.4
DNA profiling using Probes worksheet
DNA profiling using PCR worksheet (extension)
Assignment questions 2001 Q11, 2002 Section C 1B, 2003 Q12, 2006 Q12