methylation analysis with MS-HRM (primer design)
Posted 14 October 2011 - 02:03 AM
I am studying promotor methylation. Some primers I have ordered do not work (MS-HRM with Rotor-GeneQ).
As I am reading Wojdacz and his recommendations for primer design I don't understand one thing -
he is recommending that in primer sequence there should be at least on C (the one from CpG, potentially methylated) included at 5´ end of the primer, to ensure amplification of both methylated and un-methylated sequence. Still - does this have to be a wobble base C/T (when ordering this should be Y) or not?
Example from his article: Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay, BioTechniques, 2006: Forward primer sequence: TCGGAGGGGTGTAGATAGAGT - so no wobble base. Wouldn't this primer extend only methylated sequence?
Bit confusing for me, I would appreciate help,
Posted 26 October 2011 - 07:41 PM
As for the C at the 5' end, I think it works two ways, in some ways it stabilises the primer during hybridisation in the PCR step and another is that you have four bases incorporated into the sequence during primer synthesis.
All comments and communication are my own personal ones, and are not tied to any of my affiliations.