Welcome to the second part of this series. This excerpt is also from Book Two, Chapter 7 – Polymerase Chain Reaction in The Sex Tourist:
After changing to our lab gears I rub the buccal swab vigorously to my inner cheek. I ask Nicky to do the same. I could have started with loading all the samples at once into the PCR, but I want to get my own DNA profile first. I don’t want to waste even a tiny bit of the samples I have from Lily’s body. I’m a novice and doing this process for the first time means many things can go wrong due to my inexperience. Later I will compare Lily’s DNA profile to mine. This way I would know for sure that those samples are valid and belong to her. Even if we’re not identical twins, our DNA profiles should have some similarities. Nicky’s DNA is useful as well, because later we should be able to discover straight away if she inadvertently contaminates a sample by adding her own DNA to the mixture.
Even blindfolded I would know where I am. The smell of chemicals and the “white noise” from the air conditioning system are so typical of our epidemiology lab. My nose and ears quickly adapt to them. Nicky is a great help, but we are both beginners, so it still takes more than a couple of hours just to isolate the DNA in Nicky’s and my own sample. We’re using the so called solid-phase extraction method with one of Qiagen’s extraction kit.
“Quantitation” is the next step. Too much is as bad as too little DNA. The literature says that about a billionth of a gram is the ideal quantity for PCR. Since I can’t weigh this amount on a kitchen scale, I have to use another method. Our real-time quantitative PCR equipment is going to be my kitchen scale. For this to work I have ordered another assay called TaqMan. We add it to our first PCR mix. Despite the air-conditioning I have to take off my goggles and wipe the mist off from the inside. I press the mask under my nose to wipe the sweat, and my palms are getting wet under my latex gloves too. We change gloves.
It’s time to prepare the PCR master mixtures. I have the pre-prepared mixture for the so called allelic ladder which came in the kit. The allelic ladder is like a tape measure or a ruler for measuring length. More precisely the COfiler kit I’m using now is like six rulers for the six different STR loci. Without it I wouldn’t be able to measure the length of Nicky’s or my own STRs, the lottery numbers. This allelic ladder has to go into another row of tubes in the PCR equipment and would act as a so called “positive control” too. The last two rows are the strip-tubes with my own and Nicky’s purified DNA mixtures.
It takes us another hour, but I’m learning a lot as I set up the PCR program for the second round and turn on the equipment. This PCR run is making millions of copies of seven different sections of our DNAs. Six of them are STR markers. The seventh section, a gene called ameloginin, is going to prove that we’re females. Not that we need any proof of that, but it’s going to come in handy when I do the same with my other samples. Right now certain fragments of my DNA are being replicated millions of times. In an hour and a half my PCR products are ready for the next stage. For that I need the other box. Paul is bringing it from the Netherlands tomorrow. I carefully remove my treasures, the PCR strip-tubes, and pack them in a special cooler bag for the short trip home. I wipe the last PCR run off the box and the laptop.