Transcript 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



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)
DNA profiling using probesprocedure

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)
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
DNA profiling using probes –
procedure cont…

Gel electrophoresis

Used to separate the different sized fragments
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
DNA profiling using probes –
procedure cont…
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)
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
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…….
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