Into the laboratory II

Day two of three was split in half.
1. Another method of DNA extraction, using the two cricket legs from Friday
2. PCR (https://www.youtube.com/watch?v=mvvP90Cpdfc)

So, last time we extracted DNA we ground up samples and soaked them in various chemicals, then used centrifuging to separate the liquid containing the DNA from the rest of the mixture. This time, we used a method which keeps the sample more or less intact. The museum does this to analyse its collection specimens, for which grinding up is discouraged. The cricket legs had been stored in a solution containing enzymes over the weekend. These released small amounts of DNA into the solution without changing the morphology of the leg, so if we really wanted to, we could put the legs out on display and nobody would know what we'd done.

The downside of this method is that it can be difficult to get enough DNA out to work with. Fortunately, my two leg samples turned out to be fine. After washing away lots of the protein and other stuff, I ran them through a NanoDrop again which showed that DNA was present and fairly uncontaminated, and then through another instrument called a Qubit which quantified the amount more accurately. I do not remember the number but I'm sure the average reader won't mind.

So, with DNA from both extraction attempts, it was time for PCR. The ultimate goal of most DNA extraction (though not ours because we only had three days) is to sequence it, and sequencing needs relatively large amounts of very pure DNA, of just the parts you want to sequence. For example, the cricket leg was probably covered in bacteria, and bacterial DNA probably got into the tube, but I wouldn't be interested in sequencing the bacterias' DNA. The solution, to purifying and increasing quantity, is the polymerase chain reaction: PCR.

Without going into terrifying bichemistry, PCR takes advantage of how DNA naturally replicates. Recall that DNA is ladder-shaped, with two long backbones connected by many rungs. Each 'rung' is actually two bases, the parts that code the information. The sequence of bases is equivalent to the strings of 0s and 1s that define a computer program. There are four types of base in DNA: A, T, C and G.
 
To copy itself, DNA splits lengthways, separating the backbones, each of which takes the bases it's attached to. Each side can then collect new bases and build a new second backbone. The key is that each base can only join to one other base: base A joins with T, and C joins with G. So, when the missing half of the molecule is reconstructed it forms a perfect (in theory - mistakes do happen and we call them mutations) copy of the one that was lost.

(See that the incoming bases bring a bit of backbone with them)

PCR is essentially this process in a test tube, with one important addition: rather than allowing the entire molecule to replicate, it starts and stops replication in specific places. So, you can have a whole genome in the tube but only replicate the gene you're interested in. There are so many copies of that gene that by the end, the concentration of the original DNA molecules is nearly zero, and the sample of that gene is extremely pure.

I spent most of the afternoon preparing the PCR tubes. Every one (of twenty) needed the right amount of PCR 'mastermix', which provides the ingredients for DNA replication. The quantities are so very tiny, even an extra half a microlitre could stop the reaction from happening at all. The pipettes are very clever but you still need to be very organised. You can't just look at the amount in the tube to see whether you added the last ingredient! Finally, I added my samples of DNA to their own individual tubes and loaded them into the thermocycler. This machine would carefully control the temperature, stopping and starting the PCR reaction while I left to catch my train. The results would have to wait until tomorrow...