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DNA Profiling. There are two methods used to carry out DNA profiling There are two methods used to carry out DNA profiling DNA profiling using a probe.

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Presentation on theme: "DNA Profiling. There are two methods used to carry out DNA profiling There are two methods used to carry out DNA profiling DNA profiling using a probe."— Presentation transcript:

1 DNA Profiling

2 There are two methods used to carry out DNA profiling There are two methods used to carry out DNA profiling DNA profiling using a probe DNA profiling using a probe DNA profiling using PCR DNA profiling using PCR Knowledge of this is not required Knowledge of this is not required It is the more common method now days 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) This procedure relies on Minisatellites or Variable Number Tandem Repeats (VNTR) VNTR are sequences of DNA 10 – 100bp long that repeat 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) 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 Isolate DNA DNA is isolated from sample using organic solvent which dissolves proteins leaving 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) 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 digestion Restriction enzyme which doesnt cut in the minisatellite region is chosen Restriction enzyme which doesnt cut in the minisatellite region is chosen Creates DNA fragment of different sizes Creates DNA fragment of different sizes

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

7 DNA profiling using probes – procedure cont… Blotting DNA onto a filter (Southern Blotting) Blotting DNA onto a filter (Southern Blotting) DNA is denatured using, for example, a strong alkali – this creates single stranded DNA 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 Single stranded DNA (still in the electrophoresis pattern) is transferred to a filter

8 DNA profiling using probes – procedure cont…

9 Hybridisation with probe Hybridisation with probe Labelled (either radioactively or fluorescent) probe which binds to a specific base sequence in the target fragments. 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. 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) Single locus probes – bind to 1 sequence (giving 2 bands for each sequence)

10 DNA profiling using probes – procedure cont… Create autoradiograph Create autoradiograph Creates permanent record of the filter (usually using x-ray film) 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 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 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. 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. Therefore, a person that has the same band as another person is potentially related. A childs bands should match one of the mothers and one of the fathers. A childs bands should match one of the mothers and one of the fathers. If this is not the case……. If this is not the case…….

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


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