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puzzled with PCR outcome after BS treatment


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#1 Illinu

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Posted 10 July 2014 - 04:26 AM

Hi,

 

I am new to the forum and to epigenetics. I have a simple question. I am reading that after BS the PCR should be either BSP or MSP. If I understood well (reading from other post), BSP PCR uses 1 pair of primers designed to not contain any CpG Cs, whereas MSP PCR uses 2 pairs of primers one of which is designed for the methylated template and the other for the unmethylated template. I only found out this now and up to now I was using 1 set of primers where all Cs where transformed into Ts, something like a BS treated primer. Here is the deal:

 

I am aiming at a 500bp fragment which is supposed to have methylated Cs. After the PCR I obtained fragments <100bp. I clonned and sequenced them. They seem to be primer dimer, they hybridise in 4 bases but the funny thing is that the sequence belongs to untreated DNA. So they are exactly the sequence of the primers but there are Cs where there should be Ts. I can't figure out what happeded. How could 'treated' primers hybridise with untreated DNA?

 

Also I wanted to know if it is absolutely necessary to do either BSP or MSP or if using one set of 'treated' primers as I did is ok.

 

Thank you

Linu



#2 Illinu

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Posted 10 July 2014 - 05:36 AM

I just realised that by definition I have BSP primer, because the forward one contains no CpG C's (Ts) and the second one contains only one. So ignore the second question. I am still puzzled as how a set of BSP primers produces a product of untreated DNA

 

Any thoughts on this?



#3 rakun

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Posted 24 July 2014 - 12:54 PM

I just realised that by definition I have BSP primer, because the forward one contains no CpG C's (Ts) and the second one contains only one. So ignore the second question. I am still puzzled as how a set of BSP primers produces a product of untreated DNA

 

Any thoughts on this?

Hi IIIinu,

 

I am not sure that I fully understand, but it seems that the primer pair was poorly designed.






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