methylation specifc primers thermal cycle conditions - (Jul/07/2015 )
I'm new to this technique , I wanted to start with methylation of a specific gene, and decided to use the same primer sets used in a previous study.
But the thermal cycle conditions given in that study are new (and actually confusing) to me :
1- Gradual decrease of annealing temp. after the initial denaturation " annealing temperature decrement of 0.5°C every cycle (from 70°C to 66.5°C)"
2-No denaturation step in each cycle, just annealing and extention.
3- Short annealing and extension times (3 and 5 sec. respectively)
I'd try an ordinary protocol of DNA methylation , but I still want to understand the 3 points above
could any one help?
do you have the reference/paper that stipulates this? sounds like a touch down PCR, but it does require a denaturation step.
Thank you for your response
Hi Iman, that is an interesting strategy, I think it has been done to ensure high specificity of annealing of primers to the right target. As it is MSP there are only a few base differences between the two primer sets you want to be sure your primer is priming the appropriate target and minimise mis-priming. This is done with a touchdown strategy.
Google touchdown PCR for more information and details.
Thanks a lot for your precious advice.
I'll read about touchdown PCR