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Thursday, March 10, 2005Crappy day (part 1)Well, today was crappy. It wasn't utter shite, which it could have been, but it was definately crappy.First thing, I had a "progress" meeting with SWMNBN. Now it has only been 2 weeks since our last one so expectations were low. Just as well really. None of the experiments that had failed yesterday had started to magically work again, so I was on the back foot slightly. Nevertheless, I didn't make too bad a showing of myself. However, with a conference looming the need for results is only going to get greater. Apart from the implication that I was somehow to blame for a supplier not supplying immediately, most of her wrath and vitriol was aimed at people not actually present at the meeting. Nice. The day got frustratingly worse. I have been trying to create some new probe DNA (see these posts if you don't know what I'm talking about). I use a method called the Polymerase Chain Reaction (PCR). This essentially allows you to copy DNA that you are interested in billions of times. It's the same technique that forensic scientists on shows such as CSI use when they isolate a single hair follicle at a crime scene. The amount of DNA in a single hair follicle is minute, so in order to analyse that DNA they have to amplify it billions of times until they have a useful amount. Think of it as a business meeting, when you run to the photocopier and make 20 extra copies of your report so that everyone present can read one. The technique is deceptively simple. You decide which part of the DNA you are interested in. In my case, I only want about 350bp out of the entire genome. You then design "Primers". These are short stretches of DNA (about 20bp long) that "book end" the DNA you want. If you imagine the genome as a long thin line of ATC and Gs they mark the DNA sequence either side of the region of interest like bookends. The primer designs are then synthesised by a specialist company and posted to you as dry powder which you dissolve in water. The whole thing took 4 days and cost me (the British Tax payer) about £80. To actually amplify your DNA, you take a small amount of your template DNA dissolved in water (in this case genomic DNA, but it could be the DNA found at a crime scene or from a paternity test for example), and add the primers (also in water). You then add some free nucleotides (these are individual As Ts Cs and Gs and are the raw components from which the new DNA will be synthesised - rather like the paper and toner in a photocopier to continue the analogy), mix in some buffer (which keeps the pH constant etc) and then add a special enzyme called Taq polymerase. The reaction is cyclical. The solution is heated to 95C for about 5 minutes. This causes all of the double stranded DNA to break into single strands. The reaction is then cooled to 50C to 60C for about 30 seconds. The primers (which are also single stranded) bind to the single stranded template DNA. The temperature is then raised to 72C for about 30 seconds. At this point, the enzyme Taq polymerase swings into action. It is a so-called "DNA Polymerase". Its job is to synthesise new DNA, using another strand of DNA as a template. Remember that in the world of A always binds to T and G always binds to C? Well, Taq polymerase zips along the single stranded DNA, using the point where the primer has bound as a starting point. Where it finds a T it sticks a free nucleotide (in this case an A), where it finds a G it sticks a C. In this way it turns single stranded DNA into double stranded DNA - thus doubling the amount of DNA that you started with. Now you repeat the cycle - heat it back up to 95c, cool to 50C to 60C then reheat to 72C. This time the newly synthesised DNA can also act as a template - thus you double the amount of DNA again so that you have 4 times the starting amount of DNA. Repeat and you have 8 times etc etc. Typically I do 25 to 30 cycles. By this point, there is a huge amount of my 350bp target DNA, dwarfing the small amount of tempate DNA that I started with. I can then sperate out the DNA on a gel and my DNA will form a single band that I can stain and view under UV light. Well that's the theory. In truth there are many different parameters that need to be tweaked, and we can only predict what those parameters should be to a limited extent. The exact temperature that the primer binds to the template varies between 45C and 70C typically and needs to be pretty close to optimal for the primer to bind. The length of time that the enzyme works at 72C for also has to be calculated (approximately 1 minute for every thousand base-pairs in length - so 30 seconds is plenty for 350bp). A dozen other poorly understood biophysical concepts have to be examined and tweaked. Suffice to say, that after 3 attempts only 3/8 of my probes have been successfuly created - none of which are on my priority list. Bugger. I'm running out of ideas now and I am worried that the primer synthesis company may have sent me a duff batch. (Un)fortunately that is exceptionally rare (although inevitably when you synthesise 10's of thousands of primers a wek, some cock-ups occur), and extremely hard to prove. If it hasn't worked by tomorrow, and I have tried everything that I can think of, I may have to send the order again (and pay again - they won't admit liability) in the hope that that resolves the problem. Stay tuned for tomorrow's post continuing the reasons why today was crap on a personal level also. |
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