All aboard the PCR train! Experience the attraction of the Polymerase Chain Reaction! Polymerase chain reaction or polymerase *train* reaction? In PCR, a molecular “train” copies pieces of DNA by traveling along it & laying down track ahead of it as it goes.
We saw http://bit.ly/2CWPRil how we can take advantage of bacteria’s ability to make copies of circular DNA called plasmids -> we can design plasmids to serve as vectors for genes we want to study -> stick our gene in the plasmid, stick that plasmid in the bacteria, and let the bacteria go to work
But there’s a way to amplify (make copies of) short stretches of DNA “in vitro” without using anything “living”
Genetic instructions are written in DNA (deoxyribonucleic acid). It’s usually double-stranded, like it’s zippered up, which offers protection as well as an easy copy mechanism. Its alphabet only has 4 nucleotide letters (the unique part of which are called bases): A, T, C, & G & each letter can only zip up across from 1 other letter : A across from T and C across from G. (kinda like having a puzzle with only 4 kinds of pieces) So if you know the sequence of one zipper strand you can figure out the sequence of the complementary strand. And if you were to take that complementary strand and figure out it’s complementary strand, you’d be back at the strand you started with.
This way, before dividing, cells can unzip their DNA and make fresh new zipper strands to complement the unzipped zippers -> you end up with 2 copies of fully zippered zippers
The machinery that looks at one strand & adds the complementary base is called DNA POLYMERASE (DNA Pol). It works kinda like a train laying down tracks as it goes. Imagine a train track. Split it in half down the middle (length-wise). The train can start going in either direction, but it can only travel if there’s a full-width track. So in order to move forward, it has to complete the track in front of it as it goes. It knows what track it needs to add because it has to complement the 1/2 track on the other side (e.g. see an A, lay down a T; see a C, gotta put in a G). Like the tracks it’s laying down are puzzle pieces with 1 unique side sticking out. As long as there are track pieces (nucleotides) available, it can build the track and keep moving.
But it does have limitations. Just like it can only travel on a full track, it can only start on a full track – you have to provide a start station – a short stretch that’s double-stranded (full-tracked). You can do this by putting in a PRIMER – a short piece of 2nd strand complementary to where you want the copying train to latch on and start.
In order to start copying, you first need to unzip the zipper (split the tracks) – you do this by heating up the DNA so the strands come apart (MELTING) -> this gives the primer a chance to sneak in and bind (ANNEAL). This provides starting stations for the DNA Pol train to latch on & you give it time to lay down track (EXTENSION).
DNA Pol can travel on either strand, but only 1 direction on each (like it can only travel if it’s adding the right half of the track, so if it’s on the “top strand” it can only travel to the right & if it’s on the “bottom strand” it can only travel to the left – hard to explain in words, so check out the pic
In order to rezip the unzipped zippers you need to copy BOTH strands, so you need to provide a start station (primer) for each strand.
PCR is performed in “rounds” of melting, annealing, & extension -> The train will keep laying down tracks until it runs off the track, runs out of track pieces, runs out of fuel, or runs out of time -> Then you reheat the DNA to split the tracks again but now you have more 1/2 tracks so you can make more full tracks -> each round you double each strand so you copy exponentially – like a molecular pyramid scheme
During the 1st round, the train will go until it runs out of fuel (or time). The strands have defined start points but you don’t know precisely where they’ll end.
Now when you remelt and reanneal you have 4 separate strands – your 2 original & your 2 new ones & each can act as a template for a train. When the new tracks are used, the new trains are traveling in the opposite direction the tracks were laid down in, so they’ll run out of track to move on when they reach the “start station” for the other strand
During the subsequent rounds, the start station for 1 becomes the stop station for the other.
So by defining the start sites for each strand, you define the start & stop of the region you copy so that instead of copying the entire cross-continental railroad you can just copy the track between a couple cities
PCR is really useful for lots of things, not just copying genes -> in order to sequence DNA (read out its letters) you need lots of it, so PCR helps amplify it so you have enough to sequence -then you can check for “typos” like genetic mutations that might cause disease (or mess up your experiment) or you can look for evidence of microbial invaders (like bacteria)
To physically carry out PCR, we mix template DNA (what we want to copy) with the “forward” and “reverse” primers that bookend the sequence were interested in and provide the “train stations”, a DNA Pol (the track-laying train), nucleotides (the track’s puzzle pieces) & a solution of things like salts to keep DNA Pol happy. We mix them in a tiny little tube & stick them in a thermal cycler, a machine that rapidly heats (for the melting steps) & cools (for the annealing).