Author’s note: This is part of an installment on how many methods and techniques in biological sciences work, written for educational purposes! Hopefully, this blog will rise to be something more than silly short stories.
If you’ve ever worked in a biology lab, you’ve probably heard professors, researchers, or even other students talking about PCR. But what in the world is this strange acronym?
PCR stands for Polymerase Chain Reaction, and is a method for amplifying DNA! This process can take a few strands of DNA, or even a single strand, and turn it into thousands of (nearly) identical copies! Although it has a few limitations, it is a very rapid, powerful tool, and has many different uses.
So, you may be wondering, how does PCR work? It’s simple, and all depends on temperature. But first, a little refresher on DNA.
DNA is made up of two strands, each of which is complementary to the other. Because they are perfectly complementary, they fit together, bonding perfectly. Think of it like ripping a piece of paper in half – the two pieces of paper will perfectly match up together, unlike any other ripped piece of paper! Same thing with DNA. Once enough complementary bases are in a strand of DNA, it will only fit perfectly with its exact match.
Even though DNA normally sticks together, it does separate – when heated up! In order to separate the DNA, the solution containing the DNA is heated up to between 95 to 98 degrees Celsius, near-boiling. At this point, the DNA peels apart into the two different strands, but doesn’t fall apart quite yet.
Now, the enzyme that copies DNA is known as polymerase, and it’s a big, complex protein. Polymerase attaches on to an exposed single DNA strand, and builds the complementary strand of the DNA. Bam, DNA copied! Unfortunately, it’s not quite that easy – there are a couple requirements of polymerase, however, that make it a little tricky to work with.
The first issue, which made PCR impossible for a long time, is the fact that polymerase is heat-sensitive. If you heat the protein up too much, it acts a bit like a spaghetti noodle – it falls apart, and no longer performs its DNA-copying function. Even if you cool it back down, it is still broken. Now, when you have to heat up the DNA to separate the strands, this becomes an issue.
The way to solve this problem is actually rather ingenious, in terms of biology. In some hot vents, such as those found in hot springs in Iceland or at the bottom of the ocean, certain bacteria (known as thermophilic bacteria) are able to thrive. In order to survive in the boiling-hot water, they have evolved special forms of enzymes that remain stable at very high temperatures. The polymerase used in PCR comes from one of these bacteria, T. aquaticus, and is thus known as Taq polymerase. It doesn’t break down in hot water!
The other issue with polymerase, however, is that it can’t start on just a single strand – it needs to start on a double strand. This means that if you chop a bit out of a double-stranded piece of DNA, the polymerase can copy that area missing its complementary strand. If you have a single strand by itself, however, there’s no place for the polymerase to attach.
Now, in order to get around this problem, two different short pieces of DNA, known as primers, are added to the mix for PCR. These primers are usually 8-10 bases each in length, and are specially selected to perfectly fit the DNA strands at either end. This means that the primer forms a very short section of double-stranded DNA, allowing the polymerase to attach!
The last thing needed for a PCR to work successfully is the raw ingredient – the bases that DNA is built from! These are easily synthesized, and are added to the mixture so that the polymerase has raw materials to use to build its strands.
So, PCR goes through three steps – denaturing (where the DNA separates into single strands), annealing (where the DNA is cooled off enough for the primers to bond to either end of the single DNA strands), and elongation (where the polymerase attaches to the DNA at the double-stranded primer, and then builds the second strand down the rest of the length of the strand). Each of these steps is performed at a different temperature, so a PCR machine, also called a thermocycler, rotates a sample between the three temperatures. Although temperature can vary, denaturing is usually around 96 degrees, annealing happens around 58 degrees, and elongation is generally at 72 degrees. The thermocycler simply keeps on cycling the temperature of the sample between the three programmed temperatures.
Because the primers/Taq polymerase combination makes a copy of every single-stranded DNA molecule, each cycle should, in theory, double the amount of copied DNA! Here’s a quick example:
Start with 1 strand.
After 1 cycle, you have 2 strands.
After 2 cycles, you have 4 strands.
After 3 cycles, you have 8 strands.
After 4 cycles, you have 16 strands.
After 5 cycles, you have 32 strands.
As you can see, the amount of DNA grows very rapidly! Of course, this growth stops if you run out of primer pieces of DNA or raw bases, but in general, this method allows for millions of copies of DNA to be synthesized in a half hour or less.
PCR has a couple other limitations. Because polymerases don’t copy DNA instantly, there is a limit on how long the copied DNA strands can be. The maximum length of DNA that can be copied by PCR is about 10,000 base pairs, although some methods can go up to 40,000 base pairs. PCR also has occasional errors, as the Taq polymerase has an error rate of about 1 in 10,000 bases. Sometimes, the copied DNA strands aren’t perfect – and that, of course, means that future strands copied from those are also flawed.
Despite this, however, PCR is a very powerful tool, and is used all the time in biology labs! And now, you know how it works!