Differences between MSP and BSP? - (Feb/28/2005 )
Hi everyone,
I might add that for BSP, if it can not be avoided then primers may have CpGs in them, ideally you want them at the 5' end, and to ensure non-bias, make the CG a YG, ie have a degenerate base at the site of potential methylation or non-methylation.
Cheers and good luck!!
nick
There is a bisulfite primer design tool called bisearch which can design such degenerate primers at bisearch.enzim.hu
Hi,methlnick,Thanks for your help.
Sorry I am the newcomer to this Forum ,I could not find where to put my attachment in "my control panel".So I send the attachment to the commen address of the Bioforum, wish it would be transfer to you smoothly.
Of course ,the primers for bisulfit sequencing are different from M and U primers in MSP;In fact in the PCR for Bisulfite sequencing,I follow your protocol and also lower the annealing T ,change the Mg++ concentration,but there is nothing, 3.0/4.3/6.7 mM Mg concentratiin (which works with M and U primer,product <200bp) did not work in this PCR (product should be 505bp)only gived the primer dimer.Even my French boss can not figure out a way.Would you please have a look at the attachment and give me the suggestion?
Biofemme,
here is your file with a new primer seqeunce added to it.
Basically for bisulfite PCR you require two rounds of PCR to obtain the product, you are normally amplifying a larger region than MSP so the larger templates would be more scarce as it is known bisulfite degrades the DNA during the conversion process.
Good luck with it, all the best!
Nick
Is BSP easier than MSP since after bisulfite treatment you can just PCR and sequence the PCR prouct? Did anyone use Methprimer for BSP primers design?
for BSP you are able to assay a greater number of CG sites. It is of the same difficulty as MSP and primer design is a crucial factor!
I have used methprimer before but I would like to caution you when using them, I got artefactual results and sequences that just didn't make sense. I might suggest you try perlprimer I think it does a better job that methprimer in BSP primers.
good luck!
Thanks for your help!
I will take a look at the perlprimer.
I have used methprimer before but I would like to caution you when using them, I got artefactual results and sequences that just didn't make sense. I might suggest you try perlprimer I think it does a better job that methprimer in BSP primers.
good luck!
One more question. I guess I have to use the restriction enzyme to digest DNA first. What is the rule to pick the enzyme except not cut the region you are going to PCR?
Thanks!
It is not a must but an option. You can cut with any enzymes that don't cut within the amplified region.
another way that is simpler is to shear your DNA by running it through a 21g syringe needle about ten times. This will reduce the complexity of gDNA you are using and also help when strands need to be denatured fully for bisulfite treatment.
Nick