ready to use primers for BSP - (Jul/20/2007 )
I have to do methylation analysis of hMLH1 promotor. Unfortunately noone from the lab has ever done it before) That’s why I decided to pick primers for direct BSP from an article. I wandered if someone can give me an idea how to see the product of these primers: how many b.p it will be and where it stands in the sequence, how many b.p are there from the R primer to the first CpG and so on…I realy hope there is a site wich presents bisulfite modified sequences of genes rather than the normal ones
Another question – articles use primers for one strand and not for both. Can we be sure that in humans if one strand is methylated, the other one is the same and v.v.???
And finaly, it looks simple to tag modify the primers, but will it realy enhance my sequencing? Has anyone tried it?
As I am enirely new to this field, I find this forum realy helpful and thank you in advance...
Well, to find the product of the primer, you can use the normal sequence of the gene promoter, then change every C to T (e.g.in word) and afterwards search for your primer sequence. To align the second primer, just reverse it's sequence. This should be possible, i guess. There is also the site MethylBlast (sorry; can't find the link), but I never used it successfully.
One strand or both - normally there are some DNA methyltransferases which do nothing but search for hemimethylated DNA and methylate the other strand. So in ,ost cases, one strand should be sufficient.
I use tag modified primers and they really made the sequencing better + it is really convenient to use the same sequencing primer for every gene of interest!
Much fun in methylation land!
Thank you so much monster! (what does krumel mean?)
Word worked fine, great idea! I guess that is the site you mentioned (a little too complicated indeed)
Let me ask something more about tag-modified primers: articles cite primers that are not tag-modified, so I thought I just have to think of a nice GG tag and add it to the 5’ end of my R primer. Then I’ll make two rounds of pcr with the same primer pair (F and the tagged R) and finally I’ll sequence with the same R-tag. Do you think it will work? If so, then 10 to 20 bp of GC should be enough or not ???
Should work. Here is the PMID of an article 16797472 describing the tags.
Krümelmonster is the name of the cookie monster from the sesame street.
OK, i got it
thank you krumelmonster from the sesame street!