Using lasers to pinpoint DNA molecules

11 July 2010

Interview with

Alex Knight, National Physical Laboratory

Alex -   We're using lasers to look at single molecules, which could be DNA or proteins for example, and you can get a lot more information about molecules when you look at them as a collection of individual molecules.  You can get a lot more information about how the molecules differ from each other and how they behave.

So what we need to see single molecules is we need very bright light sources and lasers are very nice in that they're bright, they're stable, and they're a single colour.  So it's a very pure form of light that is very easy to work with and because they're parallel, and they form very nice parallel straight beams, they're very easy to manipulate and focus them exactly where you want them.  And so, we use a property of molecules called fluorescence where we make the molecules emit light by putting special chemical groups - a tag - on the parts of the molecules that we want to detect then you shine a laser of one colour at them and they emit light of another colour.  And that's a very sensitive way of detecting molecules and it's used for all kinds of applications in biology.

So, by having these tags on the molecules, you can do things like: you can count how many molecules are there, or if you use a special type of tag, you can use them to follow what the molecules are doing.

All you see of the molecules is a point of light and it's just like when you look up into the night sky and look at the stars, and it's the same with these molecules.  You just see this point of light.  But even that is enough to tell you quite a lot.  So depending on what kind of experiment you're trying to do and what kind of tag you've put on the molecule, you can learn a lot just by looking at these points of light.

Argon-ion and He-Ne laser beamsMeera -   So what have been potential applications then or techniques that have benefited from the use of lasers in this way?

Alex -   Well, one example is much faster sequencing of genomes.  So we talk about detecting single molecules but of course the nice thing is, you can actually look at many molecules at the same time.  You use these tags I was talking about and you put a different tag on each of the four letters that you find in a DNA sequence, A, T, C, and G.  And so, by using different lasers, you would see different letters and so, you could read out the sequence in that way.

Meera -   So you would scan say, for all of the As and that a certain colour would be emitted, and then all of the Ts in a certain colour would be emitted and you'd combine that all together to therefore then get the sequence.

Alex -   That's right.  By doing that for many, many millions of molecules all at the same time, you can collect a huge amount of information.

Meera -   You've got something here on the screen which does actually resemble a night sky with occasional twinkles here and there, and this is showing us a new area that you're working on which is the field of nano switches.  What are these?

Alex -   So, one type of molecule we're looking at is actually an artificial molecule which is a switch made out of DNA and this is a collaboration we have with some guys at the University of Edinburgh and these DNA nano switches are very useful for detecting specific DNA sequences.  When they bind to this sequence, they switch and you see that as a fluorescence signal that we can monitor with our microscope here.

The reason they're interesting is, you can use them for genetic testing, and the idea is, if you're looking for a particular sequence of DNA, say in a patient, these molecules only switch if they find the exact right sequence of DNA.  When you look at the individual molecules, you can actually see this switching happen.  And so, when you look at the screen, you can see these points of light and you'll see that they blink on and off, and that's the actual switching process happening.

Meera -   And having developed these switches, you can then identify if particular sequences, say the sequence for a particular disease is present then by the fact these switches are switched on.

Alex -   That's right, yes.

DNAMeera -   But this principle, using fluorescent tags or probes to attach to specific sequences of DNA has been used before, so what makes these switches better?  Are they more accurate?

Alex -   That's the essence of it.  The idea is that they're more accurate because they only switch when they bind to the exact right sequence of DNA because many of the genetic changes that people need to test for are changes of only a single letter in this genetic sequence.  A lot of the methods aren't specific enough to detect just a single change very accurately and the idea of this is, if there's a change of a single letter in this genetic sequence, they won't switch.

Meera -   So, other methods, say such as fluorescent probes will attach to a sequence even if one base is different because the majority of it is the same, so it will still attach.  Whereas these switches weren't fluoresce unless the sequences match exactly.

Alex -   It may attach, but unless the sequence is right, it won't switch.

Meera -   What's involved with the switch in order to fluoresce and cause the twinkle that we can see here?

Alex -   To measure the switching, we actually use two tags, and depending on whether those tags in the molecule are close together or far apart, we'll get a different signal.

Meera -   So, attaching onto a desired sequence causes the switch to come together essentially and then emit the light.

Alex -   Exactly.

Meera -   What would be the potential applications then of this?

Alex -   It's brought the era where you can contemplate the idea that everybody would have their DNA sequenced.  A lot of people think that the future of medicine is what they called personalised medicine and that's where before you're given a drug treatment, you would have a genetic test to see what is the right drug for you and what is the right dose of that drug.  And the idea is that you don't waste time or money giving people a drug that won't work for them, and of course, you don't give people a drug that will make them ill, that will have a lot of side effects.  So the idea here is that in future, you would have this genetic test and the doctors would then know what is the best drug to give you.

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