Problems with PCR following bisulfite treatment - (Aug/30/2006 )
I have been having alot of problems amplifying a promoter region of interest to analyze it's methylation status following bisulfite treatment of the DNA. I keep getting a smear following the PCR reaction. I practically did everything that I could think of that might be a possible hinderance, re-did the bisulfite treatment using another PH meter to make sure the PH is accurate, ran the PCR in a different machine, ordered new primers, played with MgCl concentration, altered annealing temperature. Nothing seems to help. All I get is this big blotty smear when I run my samples on a gel. Any help on what else I can do or how I can get this reaction to work would be greatly appreciated.
I know how depressed you are now,because I was in the same dilemma as you during last 4 months.Then I went here for help,nick gave me lots of useful advices,and I got my bands,though they were not very perfect.Now I use primers designed by ABI MethyPrimer Express and the agarose bead protocol on the forum.Can you tell me how you treat you sample and what primers you use ?I wish I can help you.
what kind of bisulfite treatment?
I usually dilute my mutagenized DNA if i am doin PCR. I find there is always a lil contaminant in my recovered DNA from the desulphonization and so on. It depends on your setup, but i convert 2.0 ug of DNA with the ZYMO EZ kit, recovery elute of about 15-20ul. Then i dilute the DNA by 1:5 into water.
try setting up a dilution gradient 1, 1:1, 1:5, 1:10 ,if you have enough DNA. I really notice a difference when running this on qPCR.
How do you set your primers? Just 2 pair? If so,you can find another one pair primers and do a hemi-nested MSP /nested MSP,and you will get your bands.
the bisulfite reaction is robust and a lot of the problems usually stem from poor PCR primer design, how did you pick your primers?
Thanksl everyone for their replies. I actually carry out a nested PCR reaction. the PCR primers have been used by other members of the lab successfully. The starting genomic DNA is from human tissues, could that be a factor?
I really don't know what else to do. What is the optimal MgCL concnetration which people usually use?
I think two major factors attribute to frequent failure of bisulfite PCR. one is the modified template DNA which is highly degraded, very low in quantity; 2nd is the primers which have long strings of T's or A's. There is no much room for improvement in primer design because there are very limited places where you can design primers. To improve your template quality and PCR, I definitely want to recommend two things:
1. Qiagen modification kit - We just tried this kit. The DNA modified by this kit has much higher success rate for amplification than any other kits.
2. Sigma JumpStart Taq - the JumpStart can produce bands where regular taq cannot, eliminate smear or primer dimers.
did you try Qiagen HotStarTaq also? I am just trying to get some products also and was wondering if my taq was not the best for the purpose?
I have also seen some papers with high concentrations of MgCL (>60mM) - do you normally need concentrations that high?
Does it matter if instead of using 10uM primer concentration, a higher concentration is used for the PCR reaction?
do you mean final primer concentration in your reaction mix? Then, 10µM would be a much to high concentration. Try 0.2 - 0.4µM instead. I had the same problem, using 1µM primer concentration and did only get primer dimer.
Hope that helps.