TOUCH DOWN PCR
Posted 24 February 2011 - 02:32 PM
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.
Posted 24 February 2011 - 04:21 PM
Posted 24 February 2011 - 08:23 PM
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?