TOUCH DOWN PCR - (Feb/24/2011 )
WOULD SOME ONE IN A VERY PLAIN LANGUAGE, DESCRIBE TOUCH DOWN PCR.
THOUGH THEOROTICALLY I CAN UNDERSTAND THE CONCEPT, IN REALITY IT SEEMS QUITE DIFFERENT.
AS PER TO THE THEORY, AS THE TEMPERATURE COMES DOWN IT SHOULD BE ABLE TO PICK UP MOST SIGNALS, WHICH I ASSUME IS NON-SPECIFIC, BUT HOW DO THEY CALL TOUCH DOWN PCR INCREASES SPECIFICITY.
ALSO IN SOME OF MY EXPERIMENTS I SEE TOUCH DOWN PCR CAN DETECT EVEN THE LEAST QUANTITY OF DNA PRESENT IN THE SAMPLE, THAN THE REGULAR PCR PROTOCOL.
Touch down PCR, as the name implies, is PCR that starts using higher annealing temperatures and then lowers the annealing temperature for the rest of the PCR reaction. The concept is as follows: you start the first PCR cycles with a significantly higher annealing temperature so that the reaction is much more specific. The theory is that only the right products will be amplified at this higher temperature. Also, the first PCR cycles are critical since the products being amplified at this time will increase in abundance compared to other templates so they will be amplified more efficiently, again in theory, than any other template when the annealing temperature is lowered at later PCR cycles. That's all it is. In short, touch down implies this lowering of the annealing temperature, but in reality the important part is the higher temperature early on that makes the assay work (amplify specific fragments early on, which in theory will make the PCR more specific, even at later lower annealing temperatures).
I agree with your explanation completely. Well, as per to your explanation, and as per to the theory, at higher temperatures, the assay is highly specific and only specific fragments, i.e.,highly complementary fragments amplify. Then why at all do we need to step down in the annealing temperature. Whats the reason for this? This is what I don't understand.
But in actual experiment, touch down assays (scaling down from the highest to the lowest., e.g., 67c - 60c)are able to pick up & amplify samples with very less quantity of genomic copies, when compared with that of regular PCR assays, which has only one annealing temperature, which is high (67c), making it more specific.
This is what is confusing me and I'm not able to explain the results that I've got.
What is making the TOUCH DOWN PCR assay pick up and amplify the lowest genomic copy numbers in the sample?