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DNA Profiling

DNA Profiling. 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. DNA profiling using a probe.

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DNA Profiling

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  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)

  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

  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)

  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. 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

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