Day 1: Planning

The problem:

A valuable diamond tiara has been stolen from a country mansion. The room containing the tiara had remained locked all night and there was no evidence of an intrusion. However, an eye witness saw a cat scaling the trellis on the wall of the house and cat hairs were found at the scene. You have been called in as a scientific expert to determine the identity of the cat burglar.

Your brief:

As it was dark, the witness did not see the colour of the cat burglar but a decent number of cat hairs were retrieved from the scene. You will be able to analyse these in the lab. As you will not be able to extract enough DNA for nuclear STR profiling, you should identify the suspect using mtDNA analysis. In advance of your work, the local cats have been rounded up and their mtDNA has been sequenced. Many of the cats have been ruled out of the picture due to the existence of alibis, or due to a lack of hair in the case of the neighbour’s sphynx. Only four suspects remain.

<note to teacher: insert details and photos of four real cats of your acquaintance>

You need to:

1. Make an experimental plan that will enable you to determine which of the suspects is the cat burglar. You will have two three-hour sessions to do the experimental work and can only use the resources listed. Use the questions below to structure your plans.
2. If you finish 1 and have time left, determine the possible haplotypes of each of the four suspect cats 1-4

Available resources:

• Background reading: Ottolini et al., 2017 and Grahn et al., 2010
• Sequences from a set of cats of known haplotype together with sequences from the four suspect cats: Suspect and haplotype seqs
• This Restriction Mapper tool
• Multalin alignment tool. You could also install BioEdit.
• The internet! You might want to use Phylogeny.fr for part 2.
• DNA extraction reagents
• Primers JHmtF3 and JHmtR3 (check out the Grahn paper for details)
• PCR reagents
• Restriction enzymes:
  • AlfI
  • ApoI
  • BccI
  • BsaA1
  • BseYI
  • BsmA1
  • ClaI
  • DraII
  • HaeII
  • HindIII
  • PmeI
  • SmlI
  • XhoI
• Gel electrophoresis equipment
• Laboratory protocols to tell you HOW to do everything will be provided after you have shown us your plans but you should work out WHAT you need to do.

Why have I not told you exactly what to do?!

You may feel that you don’t know where to start but problem-based learning is a really effective tool for encouraging deep thinking and therefore learning. When you are stretched out of your comfort zone a little, you build on your creativity, resilience and problem-solving skills. Get stuck in and give things a go. We are here to help you when you get stuck! I expect that many of you will initially feel confused or frustrated; overcoming these challenges builds confidence and resilience. Ask as many questions as you like!

Hint: Before doing a phylogeny map, you need to align and trim the sequences (for task 2)

Day 1. Questions to guide student planning:

• Initial brainstorm. What information do you have? What possible methods could be used to match the cat hair with the four suspect cats?
• What tools and resources have been listed that might be useful? What are you going to do with them?
• Outline the experimental steps needed for your plan and consider how long each one may take. What will you use the two experimental lab sessions for?
• What are your predicted results?

Day 2 (first lab session)

For risk assessment information for this section please refer to Appendix: Risk Assessment Info. See the sections headed “Centrifuges” and “Hot blocks”.

Please note that the following reagents are hazardous:

• Proteinase K – May cause allergy or asthma symptoms if in contact with skin or inhaled, so wear nitrile gloves when handling.
• Ethanol – Flammable, causes serious eye irritation. Therefore, keep away from heat, hot surfaces. sparks and open flames, and other sources of ignition. Wear nitrile gloves and safety glasses when handling.

DNA extraction

1. Using tweezers, add 3 hairs (thickish ones rather than the wispier ones) to a 1.5 ml eppendorf
2. Then add the following:

• 250 μl Digsol (Digestion solution: 50 mM Tris, 120 mM NaCl, 20 mM EDTA, 1% SDS, pH 8); as provided
• 20 μl Proteinase K
• 5 μl 1M DTT

3. Push the hairs down into the solution using a gilson tip, vortex well, then incubate at 55 ^oC for 30 min

4. Add 300 μl 4 M Ammonium acetate, vortex, then incubate at room temp for 15 min, vortexing every 5 min or so.

5. Centrifuge for 10 mins at 13,000 rpm, then aspirate (transfer using a pipette) supernatant into a fresh 1.5 ml eppendorf. You can discard the pellet and old tube.

6. Add 1 ml 100% ethanol to your supernatant, gently invert tube, then centrifuge 10 mins at 13,000 rpm.

Tip: If you position your tube in the centrifuge with the hinge facing outwards, you will know whereabouts your DNA pellet will form (slightly up the side in the direction of the hinge).

Note: you will need to prepare some 70% ethanol for the next step whilst your centrifugation is happening

7. Remove and discard the ethanol. You should ensure that your pipette tip doesn’t touch the part of the tube containing your DNA pellet as you may accidentally suck it up!

8. Add 500 μl of 70% ethanol to the tube and gently invert several times

9. Centrifuge for 10 mins at 13,000 rpm, then remove the 70% ethanol, pipetting any remaining droplets off with a P200, then allow to dry with caps open (20 minutes, room temperature).

Note: It is important to remove all visible ethanol from the tube before drying as residual ethanol will prevent the PCR reaction from working.

10. Add 25 μL 10 mM Tris pH 8, using the pipette to run it multiple times down the side of the tube where the pellet will have formed. Flick the tube to ensure thoroughly mixed, then incubate in a 55 oC hot block for 15 mins. During this incubation, you can be getting things ready for your PCR e.g. labelling tubes, calculating how much water you need to use and writing the details in your lab books.

PCR reactions

Template: 5 μl of purified cat hair DNA

Primers: 1 μl each of 100 μM JHmtF3 and JHmtR3

Master mix: 10 μl 2X Q5 Hot Start master mix

Total volume: 20 μl (you will need to add water to bring the total volume to 20 ul)

PCR programme:

• 1 x 94 ^oC / 5 min
• 30 x (94 ^oC / 30 s, 54 ^oC / 30 s, 72 ^oC / 40 s)
• 1 x 72 ^oC / 10 min

Day 3 (2nd lab session)

For risk assessment information for this section please refer to [Risk Assessment Info]. See the section headed “Preparation of agarose gel and gel electrophoresis”. If you need a reminder on how to make a gel, please refer to [general resources].

Digests

2 μl PCR reaction

1 μl 10X CutSmart buffer

1 μl enzyme (provided; you can add the other components to these tubes)

6 μl dH2O

(10 μl total volume)

37 ^oC for 1 h (but you can get away with 30 min if time is an issue)

Gels

Prepare two gels per bay (2% agarose)

Please rinse out your gel flasks as soon as you have poured your gels (otherwise they’re a pain to clean)

Add 2 μl 6X loading dye (it’s in your drawer) to each digest, then load all of the reaction onto a 2 % agarose TAE gel; run at 110 V for 50 minutes

The molecular weight marker provided is GeneRuler 100 bp-Plus (see below).  Load 5 μl per gel.