PC Arrrggghh!

PCR (see what I did there?) isn’t as terrifying as the title of this post suggests – although it has been known to induce screams of frustration in poor hard working students and researchers!

IMG_1014

So what is PCR?

The ‘Polymerase Chain Reaction’ (don’t worry – the meaning of this will become completely clear!) is one of the most commonly used lab techniques. Briefly, it is a technique to ‘bulk up’ (or Amplify) a certain piece of DNA that you are interested in from a sample of mixed bits of DNA. This makes it easier to find the interesting piece among all the boring bits you aren’t interested in.

There are several reasons why you might want to do this, but most frequently it is used to check whether a certain piece of DNA is present in your sample.

Why would that be useful?

  • You would want to do this if you tried to change the DNA in a cell line or animal model and you need to check if it worked.
  • Alternatively, PCR can be used to see if there are any natural mutations between, say, the DNA from ‘healthy’ people and the DNA from a group of people with a particular disease
  • Along the same lines as above, it can be used to test for genetic diseases (where the cause of the disease is known to be in the DNA and is passed down through families)
  • Out of the research lab and hospitals, it is also the technique used for paternity testing (as appears frequently on The Jeremy Kyle Show etc) and in forensic science (g. as seen in CSI) – in these cases, two DNA samples are compared for their similarity to each other, or in the case of forensic science, it can also create a much larger sample for testing from an initially very small trace of DNA left at a crime scene.

IMG_1015

Finding what you want in the DN-hAystack! (LOL)

But what actually is the polymerase chain reaction?

 

It’s a tricky one to explain as there are several stages, so first I’ll note the two important things you need to start with, followed by the process itself. A word of warning – it’s one of those things where looking at the pictures really helps!

You need to:

  1. Collect your DNA sample

This might be from a cell line, from a lab rat sample or from a sample taken from a patient or volunteer. Typically, the DNA is then ‘extracted’ from the cells (as animal/human samples will consist of cells – which is also where DNA is stored). By extracting the DNA and getting rid of the other bits of the cell, you should get a ‘cleaner’ sample that is less likely to fail during the PCR. This sample is called the ‘DNA template.’

  1. Prepare your primers

DNA is made up by a chain of ‘bases’ (or ‘nucleotides’) – Cytosine, Guanine, Adenine and Thymine (C, G, A and T), which pair together to form a double stranded DNA helix. In order for the PCR technique to amplify the part of DNA you are interested in, you need to tell it what part of the DNA to pay attention to. This is done with ‘primers’ (as they prime the reaction). Primers are short, single stranded chains of bases that are designed to match (‘complement’) the sequence of bases on the interesting region of DNA.

Steps for PCR:

  1. Denaturation

This is basically just separating the two strands of DNA from each other to form single strands. This is so that the primers are able to pair with their complementary sequence on the DNA strand. Denaturation is done by briefly heating the DNA to 94-98˚C.

pcr1

  1. Annealing

The temperature is dropped to 50-65˚C to allow the primers to pair (‘anneal’) with the DNA

pcr2

  1. Extension

An enzyme called DNA Polymerasehence the ‘P’ in ‘PCR!) recognises the primer-DNA pair, and recruits spare bases/nucleotides from the surrounding area (these are added by the researcher, along with the DNA polymerase).

The DNA polymerase is typically taken from a bacteria called ‘Thermus Aquaticus’ and is referred to as ‘Taq’ – this is used because it can withstand the high temperature used in step 1, whereas polymerase from any other source would break down and stop working.

The polymerase then synthesises a new strand of DNA that matches the original strand of DNA. Primers are designed to match both strands of DNA (as the sequence of the second strand will be reversed compared to the first), so during the extension phase, both the ‘forward’ and ‘reverse’ strands of DNA are synthesised to form a copy of double stranded DNA.

pcr3

  1. And repeat!

The previous three stages are repeated between 20-40 times. Each time it is repeated, the amount of DNA is doubled, so that there is an exponential increase in the number of copies of the DNA sequence you are interested in (until the spare nucleotides and DNA polymerase run out).

Now you have loads of a specific sequence of DNA! Yay!

The resulting DNA can be passed through a gel and separated by size in the same manner as the protein described in Western Whats?‘ A difference in size indicates a different nucleotide sequence, and therefore a potential mutation, or mismatch between samples. Large amounts of amplified DNA are also required for other biochemical techniques such as sequencing (which reads the whole sequence of the DNA strand one nucleotide at a time) or for inserting into the DNA of another organism, such as yeast or bacteria (with the purpose of seeing the effect this region of DNA may have on a cell).

So that seems pretty straightforward, right?

Well, yes, PCR is one of those things that can be very easy – but only when it works! Unfortunately, every stage of PCR is very sensitive to disruption, and many different sequences of DNA will need slightly different conditions for the PCR to work. For example, both too much and too little template DNA can completely ruin a PCR, and if the primers are not specific enough to the region of interest, they can pair with the wrong section of your template DNA and cause all kinds of rubbish to be amplified!

It therefore takes an experienced/skilled/lucky researcher to get a perfect PCR first time; otherwise you start to hear those screams….

 

The Biocheminist

 

Did this post make sense to non-scientists? I’d love some feedback on how understandable my posts are and if I’m managing to explain biochemistry and neuroscience to you!

Is there more about PCR you would like to know? Or are there any other lab techniques or neurological diseases you’d like to learn more about? Comment below!

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