DNA Profiling DNA Profiling n There are two

DNA Profiling

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

DNA profiling using a probe n n n This procedure relies on Minisatellites or Variable Number Tandem Repeats (VNTR) VNTR are sequences of DNA 10 – 100 bp long that repeat The number of repeats is inherited resulting in sections of DNA of different length (can be between 0. 5 kbp – 40 kbp)

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

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

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

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

DNA profiling using probes – procedure cont…

DNA profiling using probes – procedure cont… n Hybridisation with probe Labelled (either radioactively or fluorescent) probe which binds to a specific base sequence in the target fragments. n Multi-locus probes bind at more than one site. Used where certainty needs to be very high. n Single locus probes – bind to 1 sequence (giving 2 bands for each sequence) n

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

Interpreting Autoradiograph – to establish paternity n n n 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…….

Learning Activities n n n 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 Q 11, 2002 Section C 1 B, 2003 Q 12, 2006 Q 12