Kath Crawford, Paul Beaumont, Kate Andrews, Tricia Geraghty DNA profiling - a practical exercise Kath Crawford, Paul Beaumont, Kate Andrews, Tricia Geraghty

Aims Offer hands-on experience of carrying out DNA restriction and electrophoresis suitable for use with post-16 students Simulate exercises in the use of DNA profiling in biodiversity applications Explore how such practical work may support Curriculum for Excellence

I have extracted DNA and understand its function I have extracted DNA and understand its function. I can express an informed view of the risks and benefits of DNA profiling [SCN 3-14b]

Advanced Higher Biology (current) Unit: CELL AND MOLECULAR BIOLOGY d) Applications of DNA technology Techniques used include: isolation of overlapping DNA segments obtained by cutting two samples of the original DNA with restriction endonuclease enzymes

Revised Highers: Biology; Human Biology Practical work is essential in providing the contexts for the development of science skills. Through practical work, candidates develop a deeper understanding of biological knowledge and acquire skills of scientific experimentation, investigation and enquiry.

Revised Higher Biology Unit: DNA AND THE GENOME Introduction Through the study of DNA and the genome, this Unit explores the molecular basis of evolution and biodiversity.

Revised Higher Biology (ii) Recombinant DNA technology Plasmids and artificial chromosomes Extra-chromosomal DNA molecules can be transferred to microorganisms. They contain marker genes and restriction sites in addition to genes for self-replication and regulatory sequences to allow the control of gene expression. Restriction endonucleases cut target sequences of DNA leaving sticky ends. Treatment of vectors with the same restriction endonuclease forms complementary sticky ends that are then combined using DNA ligase to form recombinant DNA.

Revised Higher Biology Enzyme action The activity of enzymes depends on their flexible and dynamic shape. The affinity of substrate molecules for the active site of an enzyme and induced fit. The role of the active site in orientating reactants, lowering the activation energy of the transition state and the release of products with low affinity for the active site.

Revised Higher Biology (ii) Measuring biodiversity Measurable components of biodiversity include genetic diversity and species diversity and ecosystem diversity

The activity Dispensing DNA samples Incubation Preparing the gel Part 1 Dispensing DNA samples Incubation Preparing the gel Part 2 Loading the gel Running the gel Part 3 Staining/de-staining the gel Identification of the bands

The activity Part 1 Dispensing DNA samples Incubation (Restriction digest)

The activity 5 DNA samples: Sample from potato to be tested Morene Pentland Dell Maris Piper DNA ladder

Using the microsyringes Diagram: Dean Madden NCBEE

Diagram: Dean Madden NCBE Dispensing the DNA sample Clean tip on the microsyringe 20 μL of P1 into tube containing dried restriction enzyme Mix by drawing liquid up and down a few times until even blue colour produced Cap tube tightly with a lid Repeat for other DNA samples (P2, P3 and P4 but not the DNA ladder) Samples into a floating rack Diagram: Dean Madden NCBE

Incubation Check tubes are firmly capped Incubate at 37°C (30-45 min) Diagram: Dean Madden NCBE Optional stopping point

Image courtesy of Dean Madden, NCBE

10 September 1984 08:55 - “my forensic thoughts were precisely zero” 09:05 - “I realised that the "rather fuzzy-looking barcode-like patterns" were the genetic fingerprints of my technician… 10:30 - brainstormed – paternity disputes, crime scene DNA, identical twin testing, conservation biology, biodiversity monitoring”

Maternity testing?

10 September 1984 22:00 - “in discussion my wife suggested immigration disputes” May 1985 "I was there at the immigration tribunal when his mother was told that the case against the boy had been dropped because of the DNA evidence. And the look in that woman's eyes... that was my magic moment.”

Maternity disputes?

Pouring a gel Place 6-well comb into gel tank Pour melted agarose into tank Allow gel to set Cover with TBE buffer Diagram: Dean Madden NCBE

Science and Advice for Scottish Agriculture (SASA) Scotland provides 75% of UK seed potatoes

DNA profiling and biodiversity SASA: Maintains quality of Scottish potato crop Monitors disease in the field Ensures varieties are ‘true’ Maintains a DNA database of all known potato varieties

New varieties may have a premium value Need to establish ’ownership’ and veracity of new variety Need to check that it is what ‘it says on the tin’ More Maris Piper potatoes are sold in the UK than are grown!

Article - ‘Retailers cash in on foreign potato fraud’ Principal buyer for a supermarket chain Are the new organic potatoes, recently offered to you at a good price, Maris Pipers? The morphological data seems OK - can you confirm this?

Diagram: Dean Madden NCBE Using the microsyringes Diagram: Dean Madden NCBE 20 µl sample per well (2 x 10 µl)

Diagram: Dean Madden NCBE Loading the gel Diagram: Dean Madden NCBE Is gel covered with TBE buffer? Gently ease comb from the gel Clean tip on 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 loading dye/ DNA mixture into one of the wells, holding the tip above the well but under the buffer solution. Try not to puncture the well! Mark on the tank which DNA you have put into the well Repeat for other DNA samples Diagram: Dean Madden NCBE

Diagram: Dean Madden NCBE Running the gel Diagram: Dean Madden NCBE Fit one electrode at each end of the tank

Running the gel

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 Optional stopping point Rinse the surface of the gel with cold distilled / 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 Optional stopping point

DNA profiling and biodiversity 4 scenarios: Japanese knotweed Characterisation of British orchids Reforestation project Potato variety identification

Acknowledgements: 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 - Postgraduate science communication team

The scenarios Reforestation Japanese knotweed New species of orchid? Japanese Knotweed growing through a pavement. Photo courtesy of Environment and Heritage Service, Cornwall. Epipactis youngiana © Christopher A Fields New species of orchid? Which potato?

The practical - materials Materials from NCBE - 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 truth about the DNA samples The practical – the truth about the DNA samples 3 bacterial plasmids of different sizes Plasmids mixed to give three DNA preparations Each plasmid - single site for BamH1 - treatment with BamH1 cuts circular DNA to form a linear fragment Diagram: Dean Madden NCBE

Japanese knotweed Native to Japan, Taiwan, N China Introduced to UK in 1800s Now widespread in British isles - reproduces asexually in Britain - overruns British native plants - causes damage - difficult to eradicate Categorised as invasive, non-native species and deliberate spread prohibited; classified as controlled waste Important to understand genetics Japanese Knotweed Photo courtesy of Michelle Hollingsworth Fallopia japonica syn, Polyganum cuspidatum

Japanese knotweed Lane 1: Scotland Lane 2: Ireland Lane 3: Wales 6 5 4 3 2 1 Lane 1: Scotland Lane 2: Ireland Lane 3: Wales Lane 4: England Lane 5: Giant knotweed Lane 6: Ladder

Conservation of British orchids Epipactis youngiana © Christopher A Fields Conservation of British orchids 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? Epipactis helleborine © Tim Rich

Conservation of British orchids 1 2 3 4 5 6 Lane 1: E. helleborine Lane 2: E.youngiana Lane 3: E.youngiana Lane 4: E. helleborine Lane 5: E.youngiana Lane 6: E.youngiana

Reforestation projects Oak species - major component of European forest resource FAIROAK project - create map of European oak genetic resources Sampled ctDNA of oaks Provided information for use conservation policies Which seed to choose for reforestation project?

Reforestation projects 6 5 4 3 2 1 Lane 1: Area 1 Lane 2: Area 2 Lane 3: Area 3 Lane 4: Area 4 Lane 5: Ladder

Potato - The results → Lane 1 → Lane 2 → Lane 3 → Lane 4 → Lane 5