Molecular Biology 101: qPCR when you need to detect duplication events

Why are you in work so late?! asked my housemate’s boyfriend, when I explained via Facebook chat that that was where I was.

I got really into writing. And now I’ve been here so long that the qPCR I planned to run over night is done, so I may as well do the analysis! I replied

Dedication to the cause! Came the reply. But I have no idea what qPCR is…

I pride myself on talking about science far too much and boring everyone around me, especially housemates and their boyfriends. So how have I possibly avoided explaining what qPCR is?!

DNA Replication

 DNA exists in relatively small quantities compared to how much we need to do molecular biology. In order to work with it – or, for that matter, check whether it is there – we need to make more of it. A particularly awesome feature of DNA makes this possible. It is a double stranded molecule: if you could straighten out the helix that it works itself into it would look like a ladder, and the two sides are inverse copies of one another. (Once you have finished marvelling open-mouthed at my non-existent artistic skills you will, I’m sure, spot that pink always pairs with orange etc).

This means that if you split the molecule in two, it’s possible to rebuild the other side from the side that you have. This happens inside your cells all the time and is called semi-conservative replication.

The Polymerase Chain Reaction

We can simulate this in the lab through a reaction called PCR (Polymerase Chain Reaction). PCR can best be summed up by my favourite geeky advert of all time. (A geeky advert so immense that for my first 4 months as a PhD student I drank from a BioRad mug for no other reason).

In case that was all a bit much for you, here’s a quick recap:

A polymerase is an enzyme that finds one strand of a DNA molecule and adds nucleotides (the little molecules that are added together to make a whole DNA strand) to make the other strand. The chain reaction refers to the fact that you exponentially increase the amount of DNA. You begin with one strand, and then you have two. Then four, then eight, sixteen and so on.

This is achieved in the lab by a series of heating and cooling steps. At 94C the DNA melts and splits into two strands. At 60C or so it begins to reanneal. At 72C the polymerase can quickly work to extend the molecule. Now copying the entire DNA of an organism would be unnecessary and expensive: usually we’re just interested in a single gene. We use short lengths of DNA called primers as starting points for the PCR reaction.

So PCR can be used to amplify a gene of interest (for instance, if you want to clone it). It can also be used to check whether the gene is there. You might want to do this if you were working with deletion lines or if you were looking at a gene with variable copy number. (And there you were thinking that everyone had two copies of every gene!)

What’s the q? 

The q in qPCR stands for quantitative. Sometimes you don’t just want to know whether a gene is there. You want to know how much of it is there. For instance if you are looking for genes that allow a plant to tolerate cold, you might look for anything that is transcribed at a higher level in plants exposed to cold than plants exposed to warm temperatures.

qPCR allows you to do that. It uses fluorescence to tell you – in real time – how much of a particular transcript is present. There are two ways of doing this, known as SYBR Green qPCR and probe-based qPCR. In SYBR green the reaction mix contains a green dye that only glows when intercalated (stuck in the middle) into DNA, so the more the DNA, the more the glowing. In the probe based method a short length of DNA has a fluorophore (glowing bit) and a quencher, which sit in the middle of your gene of interest. When the polymerase comes along to build the new strand it destroys the probe, dissociating the quencher from the fluorophore and – you guessed it – making it glow more.

We use that fluorescence to compare samples. There’s semi-quantitative PCR where we just say that Sample A glows more (and therefore has more transcript) than Sample B, and also fully quantitative PCR where we compare them to a standard curve of known samples. (E.g. if 5ng comes up after 13 cycles and 0.5ng comes up after 16 cycles and 0.05ng comes up after 19 cycles and my sample comes up after 18 then how much DNA does it contain?)

So what use is this anyway?

We use quantitative PCR to see how much of a particular gene is being transcribed. That can be useful in lots of ways. It can tell us about gene duplication events (i.e. do you have more copies of a particular gene). It can tell us about genes that might be involved in a response to something (e.g. saline tolerance or immune response). It can also tell us about whether a particular allele (version) of the gene of interest is being expressed. This is especially useful if you work on an organism with many copies of every gene, like wheat, which is a  hexaploid (yep, six genomes). So we can determine that it has many copies of many genes, but rarely which ones are being used. By using qPCR we can spot which copy (if any) is the dominant copy, and whether – if we switch that one off – the others can take over.

Sorry, that was a bit more in depth than my usual 101-ing, wasn’t it? Hopefully normal service will resume soon!

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