problems with bisulfite PCR - (Sep/02/2005 )
I need your help. I think I am going crazy.
I am trying to analyse the methylation status of a CpG island of a tumor-supressor gene. I'm trying to compare tumor and nomal tissue. Well, I did a conversion of a few tumor samples and performed PCR and after a bit of adjusting it worked perfectly. I got a very nice clean sequence and good results. Then I did a conversion on a few normal samples, performed PCR under the same conditions and got nothing! I can't get a PCR product, everything is blank. I've been playing with this for months now. I tried everything I could think of: lowering the annealing temerature, varying magnesium concentration, diluting the DNA, varying primers concentration, increasing the number of cycles, doing an extra purification step, doing touchdown PCR, even changing the PCR machine, but nothing. I did a new conversion and tried it all all over again. I don't understand. Why does it work on tumor samples but not on normal tissue samples? DNA extraction was performed at the same time, with same buffers, and they have been stored in the same way. I really don't understand what could be the problem.
Do any of you have any idea? Please?
I was wondering, what type of PCR are you performing on your bisulfite treated samples?
Are you performing methylation specific PCR and hence you are using one type of primers on your samples, say the methylated set on your tumour samples and obtained a product? if the same primer sets were to be used on normal samples where you would expect to have no methylation then you will not get a PCR band.....this is a good thing!!!!
I think it is an issue with your primers, if you can find out if they are MSP primers or BSP primers, this will go a long way to solving your problem!!!
I agree completely. You've done everything except the most important thing -- redesign the primers. Likely the primers are designed to bind to a location which is methylated in one set of samples and unmethylated in the second, and you'll need a different set of primers for each state. You could also try changing the location of the primers -- the two sets need not be in exactly the same location, as long as they bracket the region you are interested in.
actually, I designed the primers for bisulfite sequencing, therefore they should work on both methylated and unmethylated DNA. They are situated outside the CpG sites and should sit on the converted DNA. I actually managed to get one normal DNA to work, but only in the beginning, and nothing since then
...oh, yes, my promoter region is rather big, and it requires 4 sets primers to fully cover it. Product sizes are about 400-450 bp long. And I have the same problem with all 4 pairs.
hmmmm that is odd.......
I don't want t o presume anythinig, but i PhD student who has just started bisulfite PCR had a similar problems and we found after a lot of troubleshooting and banging our heads against the wall, that her primer combinations for each PCR were mixed up.
could this be happening with you? It's a really simple mistake to make but one that should be ruled out before anything eles.
I checked and double-checked my primers and it doesn't look like I mixed them up...
What are the chances that I simply don't have enough converted DNA for it to work? I have a protocol for conversion that uses 2 ug of DNA but the bisulfite treatment lasts only 4 hours. Maybe I should try overnight? Maybe that would result in more converted DNA and PCR would work? Also, does the Wizard purification kit work well enough? I mean, maybe there was something left in the DNA after purification that inhibits the reaction?
Anyway, I'll try a new conversion this week, with incubation overnight and see how it works. What do you think? Thnx
I just wanted to let you know I finally succeeded! I ordered a new batch of my primers and all seems to work now. So I guess something went wrong with them, maybe they were thawed and then refrozen too many times. I now made more smaller aliquots so this doesn't happen again. Thank you all for help
Yes primer quality is important for bisulfite PCR success. If you can afford it, use PAGE purified primers which is guaranteed to performed better than desalted.