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methylation specifc primers thermal cycle conditions


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#1 Iman

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Posted 07 July 2015 - 10:36 AM

Hi everyone

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?
Thanks    



#2 methylnick

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Posted 16 July 2015 - 12:53 AM

do you have the reference/paper that stipulates this? sounds like a touch down PCR, but it does require a denaturation step.

 

Nick


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#3 Iman

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Posted 20 July 2015 - 10:39 AM

Thank you for your response

 

link 
http://www.biomedcen...1471-2407/12/66



#4 methylnick

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Posted 20 July 2015 - 12:35 PM

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. 

 

Nick


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#5 Iman

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Posted 21 July 2015 - 02:28 PM

Thanks a lot for your precious advice.

I'll read about touchdown PCR
 






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