Use of W primers to detect efficiency of bisulfite reactions? - (Dec/06/2006 )
Hello there,
Long time reader, first time poster. I've been trying to get MSP to work for MONTHS, without success. Problem is, no one in my lab does MSP. I have read the literature and this forum to try to get some ideas. Whenever I do the PCR, I get NOTHING but primers in my lanes. 
First let me say that I decided long ago to try to get the reaction to work on plain ol' human genomic DNA. I'm using human placental DNA for an unmethylated control, and human placental DNA processed with SssI methylase for a methylated control.
One problem may be is that I am doing the bisulfite reaction without using a kit. I did try the low-melting point agarose bead method as suggested by labtechie, but it didn't work, either. I am using PCR primers described in the literature.
Well, my most recent thought is that perhaps my bisulfite reaction isn't working.... so I decided to try to design so-called "W-primers". In theory, these would amplify the relevant segment of DNA, if no C's had been converted to T's by the bisulfite reaction. I couldn't find any examples of known working W primers in the literature.
In designing these, I simply identified the DNA segment where the M and U primers bind, and made a primer there to anneal to the (non-bisulfite-modified) DNA.
The problem is, when I look at these primer pairs in MACVector, EVERY SINGLE one forms "self 3'dimers, self-duplexes, and hairpins." So I haven't ordered them yet.
ARGH! Does anyone have any suggestions of what to do next? Should I just go ahead and order a MethylEasy kit, and pray for some luck? Or should I go ahead and order these W primers? Also, I've just been using a standard Taq polymerase, but I notice that a lot of you recommend Jumpstart Taq. should I start there?
BTW, I'm not a basic scientist (as if you couldn't tell!). I'm just a hematology/oncology fellow trying to do "research"! and I'm getting really frustrated! 
hi methclueless and welcome to the forum!
The majority of problems occur due to inefficient primer design.
I have told many people to try and not follow primer sequences published in the literature, because I myself have found that they are not the best primers for the application.
ABI have released methylprimer express that, in my hands seems to do a very good job of picking efficient primers.
The bisulfite modification is a robust reaction and you should yield enough DNA for PCR.
Unfortunately the only way to determine you have DNA after conversion is by PCR and you need good primers to do so.
I am happy to have a look at them and see if they are suitable or not.
As for the dimers and hairpins, many form at low temperatures (ie: temperatures well below in a PCR), i simply ignore them and make sure I perform PCR with a hotstart enzyme or perform a manual hotstart. And it seems to work just fine.
Good luck with it
Nick
Unfortunately the only way to determine you have DNA after conversion is by PCR and you need good primers to do so.
Nick
Hi, Nick
What kind of method you think is better for bisulfite modification? Now I am using the QIAGEN Epitect modification kit , (I also tried the Chemicon's CpGenome modification kit) but I could not get my band. Do you think this QIAGEN kit is good for modification? And in your experience, usually I should use how many ug genomic DNA to do modification?
And I use the CpgWare software to design my MSP primers, until now I tried many times and could not get any band of my products, So do you ever try this software to design primer and do you think this one is reliable? Do you think I should redesign my primers using the ABI MethylPrimer Express software?
Thank you for your help
Pink
hi there pink,
I tend to perform homemade bisulfite conversion and this works very well, I have tried methyleasy (http://www.humangeneticsignatures.com) and have nothing but good things to say about it. Zymo's bisulfite kit works as well and my colleagues have now tried the epitect kit from qiagen and say it is easier with the column based methodology.
I think your issue lies within primer design, I have been picking primer's by eye and not required the use of a program although if you need to I would suggest you use ABI's methyl primer express. It seems to do a good job of picking appropriate primers.
good luck!
Nick
I tend to perform homemade bisulfite conversion and this works very well, I have tried methyleasy (http://www.humangeneticsignatures.com) and have nothing but good things to say about it. Zymo's bisulfite kit works as well and my colleagues have now tried the epitect kit from qiagen and say it is easier with the column based methodology.
I think your issue lies within primer design, I have been picking primer's by eye and not required the use of a program although if you need to I would suggest you use ABI's methyl primer express. It seems to do a good job of picking appropriate primers.
good luck!
Nick
Thank you Nick,
I will try again and hope it will work.
Pink
I would also add that certain things like primer concentration, annealing temp, and #pcr cycles are variable when using a previously published primer set. Additionally, the thermocycler, pcr mix, can laso be a variable in reproducing these reactions. In most cases, people give little details in their methods sections, so this usually needs to be played around with.