Primer Design MSP, BSP, MS-HRM - (Jul/16/2010 )

Hi, I am new in the field of epigenetics and need some help to understand primer design in methylation studies.
I have tried the MSP-Primer and Methprimer programmes but when I want to check the primer-position (especially with MSPPrimer) I never find the antisense primer in the converted sequence. This is probably very basic, but please help me.

Is it possible to use one primer from the above mentioned programmes and design the counterpart (it would have to be the same strand of course)? What has to be minded apart from matching Tm of the primers, control for secondary structures?

I have read a lot about methylation studies with High Resolution Melting. Here, to reduce PCR-Bias, one author (Wojdacz et al.) recomments to include a limited number of CGs at the 5'end of the primer, thus - together with a high annealing temperature - reverse the bias towards the AT-rich sequences. Would you agree?

What are the best primer - programmes to use?

I would be happy to get some help! Thanks in advance, rudi111

I think the programs you cite pick the reverse primers appropriately, they are potentially not found by you because of the nature of bisulphite conversion generating two non-complementary strands of DNA sequence. so you need to mock convert your sequence of interest using find and replace in word or other methods.

Methylprimer express does a pretty good job, I do'nt think there has been a systematic comparison between algorithms.

Another way to increase stringency is to increase the length of your BSP primers. I tend to design 30-mers and a Tm of 60C by primer express.

Good luck

nick

Hi Nick,

thanks for your answer - Methyl Primer Express did the trick, the design is much clearer!
I still have a question regarding methylation analysis using High Resolution Melting. To use HRM you need methylation independent primers, as with BSP. Wojdacz et al. (BMC Research Notes 2008, 1:54 doi:10.1186/1756-0500-1-54) recommend using primers with a limited number of CGs at the 5' end of the primer. Together with high annealing temperatures he was able to avoid PCR-Bias, which is often present in methylation independent primers as they prefer to amplify the CG-poor sequences.
Do you think it would also be possible to use the BSP-Primers with primer-mixes (Y= C and T) and R (A and G) vor methylation analysis with HRM as it should also prevent PCR-Bias?

Do you avoid completely to have CGs in your BSP-Primers, or are 1-2 acceptable?

Another question regards the efficacy of the bisulfite reaction. Unfortunately it is not possible to measure the concentration of the converted DNA after the bisulfite treatment. However I have a bad feeling to assume that the efficacy is always 100%. Do you use a method to control the efficacy of the bisulfite reaction (qualitatively as in 100% convertion and quantitatively as in the concentration of the converted DNA)?

What is the maximum length of an amplicon for nested PCR in your experience? Have you been able to sequence longer amplicons than 200bp with conventional sequencing techniques (capillary electrophoresis)?

Why does the Tm according the Methyl Primer Express programme differs from the Tm of Tm-calculators (which one do you use?). I expect Methyl Primer Express uses salt-free calculation?



Thank you for your help - this forum is really great and I did a lot of reading over the weekend!

Rudi111

methylnick on Sun Jul 18 06:18:39 2010 said: