DNA Profiling and
Biodiversity
Kath Crawford
Science and Plants for Schools, SSERC
DNA profiling and biodiversity
Thanks to:

Royal Botanic Garden, Edinburgh
- Michelle Hollingsworth (Japanese knotweed)
- Pete Hollingsworth (Epipactis youngiana)
- Mark Hughes (Reforestation projects)

Scottish Initiative for Biotechnology (SIBE)
Dr Jan Barfoot

University of Edinburgh
Post-graduate science communication team
This morning’s activities
Plant biodiversity through DNA profiling
09:00-09:30
09:30-10:00
10:00-10:45
10:45-11:05
11:05-11:15
11:15 – 12:00
12:00-12:15
12:15-12:30
Introduction
Set up restriction digest
Restriction digest
Demo of pouring gels
Loading wells, run gels
Theory
Coffee
Stain gels
Roslin and SASA feedback
Observing gels
Closing remarks
This morning’s activities
- aims
• Offer hands-on experience of carrying out DNA restriction and
electrophoresis suitable for use with post-16 students
• Increase awareness and understanding of the use of DNA
profiling in ecological applications
• Simulate exercises in the use of DNA profiling in biodiversity
applications
• Explore how such practical work may support a Curriculum
for Excellence
This morning’s activities
Plant biodiversity through DNA Profiling
three scenarios:
• DNA profiling and Japanese knotweed (yellow microtubes)
• DNA profiling and the characterisation of British orchids (blue
microtubes)
• DNA profiling and reforestation projects (green microtubes)
Work in teams of six people, split into pairs,
Each pair to carry out one scenario. At end,
each pair to describe the scenario, results and
any conclusions to the rest of the team of six.
Using the microsyringes
Diagram: Dean Madden NCBE
Using the microsyringes
• Never pull the plunger out of the
microsyringe
• Before loading, pull out the plunger a
little (1 – 2 mm)
• When dispensing liquid, hold the
microsyringe as near to vertical as
possible and at eye level
• Remove the droplet of liquid from the
end of the microsyringe tip by touching
the inner wall of the microtube
• Do not touch the point of the
microsyringe tip with your fingers
Dispensing the DNA sample
• Put a clean tip on the microsyringe
• Put 20 μL of G1, L, or K1 into tube
containing dried restriction enzyme
• Mix by drawing liquid up and down a
few times
even blue colour!
• Cap tube tightly with a lid
• Repeat steps above for other DNA
samples
Diagram: Dean Madden NCBE
• Put samples into a floating rack
Incubation
• Check that the tubes are
firmly capped, then incubate
them in a water bath at 37 ° C
for 30 min
Diagram: Dean Madden NCBE
Meanwhile, Anne and Gordon will
demonstrate pouring gels
Loading the gel
• Your gel has been supplied covered
with TBE buffer and with comb in
place.
• Very gently, ease the comb from the
gel.
• Put a clean tip on the microsyringe.
Add 2 μL of loading dye to the tube
containing DNA. Mix well by drawing
the mixture up and down in the
microsyringe tip.
Diagram: Dean Madden NCBE
Loading the gel
• Pipette the mixture of
loading dye and DNA into
one of the wells, holding
the tip above the well but
under the buffer solution.
Take care not to puncture
the bottom of the well!
• Mark on the tank which
DNA you have put into the
well.
• Repeat for your other DNA
samples.
Diagram: Dean Madden NCBE
Running the gel
• Place gel tank in large
electrophoresis tank and fit
wires with crocodile clips
Diagram: Dean Madden NCBE
• Fit one electrode at each
end of the tank.
The scenarios
Japanese Knotweed growing through a pavement.
Epipactis youngiana
Photo courtesy of Environment and Heritage
Service, Cornwall.
Japanese knotweed
- invasive, nonnative species
© Christopher A Fields
New species of
orchid?
Reforestation – which seed?
Japanese knotweed
• Native to Japan, Taiwan, N China
Japanese Knotweed
• Introduced to UK in 1800s as
ornamental plant
• Now widespread in British isles
- reproduces only asexually in Britain
- overruns British native plants
- causes damage
- difficult to eradicate
Photo courtesy of Michelle Hollingsworth
Fallopia japonica syn, Polyganum cuspidatum
• Categorised as invasive, non-native
species and deliberate spread
prohibited; classified as controlled
waste
• Important to understand genetics
Conservation of British orchids
Epipactis youngiana
• Biodiversity action plans to honour ‘Convention
on Biological Diversity’
© Christopher A Fields
Epipactis helleborine
• Epipactis youngiana – first described in 1980s
- found on mine spoil heaps in Northumberland
and Glasgow
- thought to be new species
- given full conservation status
• Limited resources for conservation purposes
• Is E. youngiana a new species or a variant of E.
helleborine?
© Tim Rich
Reforestation projects
• Oak species are a major component of
European forest resource
- provide habitat for other organisms
• FAIROAK project to create map of oak
genetic resources across Europe
- sampled ctDNA of oaks across
Europe
- provided solid scientific information for
use in development of conservation
policies
• Which seed to choose for reforestation
project?
The practical - materials
• Uses materials from the NCBE’s
modular kits
- gel tanks, 6-tooth combs,
microsyringes, calibrated microsyringe
tips, carbon fibre electrode material,
high-grade agarose, buffers, Azure A
DNA stain
• Uses restriction enzymes and DNA from
the NCBE’s ‘Nature’s Dice’ kit
- BamH1
- DNA
The practical – the truth about
the DNA samples
• Three bacterial plasmids of different
sizes
• Plasmids mixed to give three DNA
preparations (Mix 1, 2 and 3)
• Each plasmid
- single site for BamH1
Diagram: Dean Madden NCBE
- treatment with BamH1 cuts circular
DNA to form a linear fragment that gives
a single band after electrophoresis
The practical – the truth about
the DNA samples
• DNA negatively charged
• Gel porous
• When voltage applied, DNA molecules
move through gel towards positive
electrode
• DNA molecules separated by size
Diagram: Dean Madden NCBE
Staining the gel
• Pour ~ 10 mL Azure A stain
on to the surface of the gel.
Leave for exactly four
minutes. Pour back into
bottle.
• Remove and dispose of
electrodes. Pour off the
buffer solution.
Staining the gel
• Rinse the surface of
the gel very carefully
with cold distilled or
deionised water. Pour
the water away.
• Put the gel in a plastic
bag to prevent it drying
out, then leave to
develop.
Diagram: Dean Madden NCBE