Clearly, I know enough now to feel like I can explain how it all works it the intricate world of molecular lab work…
Some time ago I promised I would write a post explaining how DNA extraction, PCR and the rest of the lab mumbo-jumbo we do in the lab work.
This is not a technical post where I list a bunch of stuff to make everyone, especially myself, feel stupid for not understanding.
This is meant to be a simple guide to the very basics of DNA extraction and PCR reactions. Just so we are all on the same page and I don\’t just keep dropping words that no one can make sense of. So we can all feel one spec of dust more clever together!
Let\’s start at the beginning:
Literally, the beginning. This is where it all starts: the coding language that is the blueprint of all living organisms. DNA is a molecule, like water, only it\’s a super long one. So long in fact, that if you stretched all the DNA in one cell (and we are talking tiny little human cells), it would be about 2m long. If that\’s not astonishing enough, think that if you then stuck the DNA molecules from all your cells together, it would be more than a round trip from sun to Pluto*. aha.
So next time you are feeling small and vulnerable, think that in your cells you have the power to lasso all the planets in our Solar System. Ιncluding the dwarf planet.
Ok, so DNA is a molecule. Its ingenious recipe consists of two complementary strands of only 3 components: A phosphate group, a sugar group and a nitrogen base. These bases come in four types: adenine (A), guanine (G), cytosine (C) and thymine (T).
The order to which these four letters arrange themselves in is what determines whether we are a mountain goat or a nasty virus, a vanilla orchid or a walrus, and whether our noses are big or our eyes are so far apart, they might as well have been a feature of someone else\’s face. And since each one of us is a result of their parent\’s magic gene combo**, we inherit our DNA from them. I, for example, have my grandfather to thank for my good hearing, whose ears were so enormous we got better satellite service when he was around.
*there are about a million different estimates on this. The numbers I used for this: If you uncoiled the DNA in your body, it would be about 10 billion miles long. Pluto is 3.67 billion miles away from earth (on average, it varies a lot).
**disclaimer: we shall not be talking about the birds and the bees here, you google that stuff up if you need to clarify how you were brought to this world. We shall also be using the term \’parents\’ pretty loosely here as some creatures can fertilise themselves, while others just split in half and create a copy of themselves. Gross but amazing.
DNA extraction: washing cells and keeping the good bits
So what if you wanted to squeeze that amazing molecule out of its cell and into a tiny little vial? All is possible in the magic world of science. This one also happens to be fairly simple.
There are 3 steps to achieving \’DNA Collector\’ status: Lysis, Precipitation and Purification. Ya. You essentially break open the cells to free the DNA, remove anything that isn\’t DNA, keep the DNA.
In my wonderfully artistic graphs (#toddlerart) below you can see the process in a bit more detail:
It is a glorified form of laundry: You take the basket of muddy, smelly clothes, throw it in the washing machine, add chemicals to get the dirt to come off, blast it all with water, rinse to remove dirt, spin to remove water – tadaaa!
How do you know it worked? Well, you don\’t at this stage. In the extraction kits I am using, every single component seems to be transparent and nothing indicates success. If you like instantly rewarding results, you are in for a disappointing experience.
There is plenty of material out there explaining every little detail of the process and if this has inspired you to read more, branch out, google that stuff, live a little.
Now you know how I make my fly smoothies. Since I am looking for what the fly has been eating rather than the fly itself, the main ingredient in my DNA extraction recipe is fly bottoms (abdomen). I have now become a prime fly butt chopper and am somewhat struggling to see what section of my CV that fits into.
Everything in my life has been leading to this sole quest: finding treasures in a fly\’s digestive tract. My dad would be so proud.
Polymerase Chain Reaction (PCR) – cooking the world\’s tiniest spaghetti alphabetti.
Real magic always happens in the kitchen and PCR is the coolest form of cooking I have encountered so far. Here is where a bit of colour comes in. My PCR mix is fluorescent pink, which never before has been my colour of choice, but I have sacrificed worse in the name of science.
This is the point in this recipe blog where I am done doing all the irrelevant talking and will finally give you the god damn recipe. We have all scrolled down countless photos of cakes from all aspects and angles and different serving styles. Does anyone ever read that bit at the top?
Here we go!
PCR is a spaghetti alphabetti with style and has five ingredients:
1. Deoxynucleotide triphosphates dNTPs (the letters A, C, T, G we talked about before) (pasta bit, only you might struggle buying this one from Aldi).
2. Primers (a DNA sequence you have designed to stick to your targeted species and no other DNA sequence).
3. Taq polymerase (an enzyme that acts as the glue to add your pasta letters to the matched sequence).
4. Buffer (creates optimal conditions for the Taq to do its thang).
5. Your extracted DNA sample.
You also need a thermocycler (an elaborate world for a fancy oven).
Step 1. Denaturation: After adding all ingredients together, the elaborate oven does all the job for you. First it raises the temperature to allow for the double-stranded DNA to break apart and form single strands. Cruel, but necessary.
Step 2. Annealing: Fancy oven lowers the temperature and the primers attach to the target sequence. This step creates starting points so that the rest of the ingredients can do their job in the next step.
Step 3. Extension: The Taq polymerase adds your spaghetti alphabetti letters onto the primers to create a double-stranded DNA.
Think zippers – you start with one set of two-sided zippers and a load of single right or left sides. Then, when the normal zipper opens, a right side attaches to the left and a left to the other side of the initial pair and now you have two sets of zippers. HA!
This is it. Are you still with me?
Everything in science feels like magic and I never understood why magic needed to be unexplainable to be fascinating. Granted, this branch of magic is more tedious and doesn\’t -always- produce colourful explosions, materialises the 10 of spades we were concentrating on out of a deck of cards, or stupefies our opponents in a duel. But unlike other branches of magic, this one has allowed us to cure diseases, land on the moon and, more importantly, learn how chameleons change colours.
See you in the wizarding world.