No Product from MethylEasy Kit Bisulfite Treatment! - (Nov/21/2008 )
I have been relentlessly reading your postings for the past 4 months while trying to get my Bisulfite reactions to work, and I must now summon your help!
I am looking to do BSP and sequence clones on a ~1kb region of CpG dense DNA. I have optimized genomic/ nontreated primers for the whole fragment and get robust product. At each step I have run my starting DNA on a gel and it is of high quality.
Moving on to the Bisulfite Treatment...I started out treating my (cell line) DNA with the conventional protocol (MethylNick's version). Result was no PCR band with many different primer sets.
-Used 2ug Starting DNA for Conversion
-Predicted fragment lengths = ranging from 1kb - 100bp
-Conversion Time Length = 4-16hrs
-PCR Cycling = 1 Round + 2 Round attempts + Nested attempts
-PCR Conditions = Annealing Temp 1 degree below Tm, 30s Denature, 30s Anneal, 1 min Extension (at 72 degrees)
-I have tried [DNA] and [MgCl] gradients
-PCR Cycles = 35
-We use regular Taq Polymerase
-I will also mention that I actually used MSP primers (with the intention of cloning and sequencing) because the region is so CG rich it is nearly impossible to find BSP primers.
We then decided to purchase the MethylEasy Kit to ensure my conversion protocol isn't tainted. From their protocol, I am able to pcr their control fragment from both their DNA and my DNA. However, my primers do not work- and have tried varying the options as above. I will also mention that my PI had performed a pilot study about 6 months ago on this same gene. He had 2 MSP primer sets (with a 150bp band) that gave him product reliably. These primers are not working in the MethylEasy converted DNA.
In the MethylEasy manual, they show a strong high molecular weight DNA band after treatment. We ran some of our treated DNA and their control treated DNA and we see no band (but I am still able to get product from their primers). My boss has speculated that their last step in the protocol "Add Reagent 3 and incubate at 72 degrees for 60 mins" contains the NaOH and is possibly degrading the DNA because it is not re-precipitated at this point and we also put a sample of our treated DNA on a pH strip and it is very high, around pH10, which could affect the enzymes.
So some of my questions are:
Has anyone come across the same issue with the MethylEasy Kit?
Should I be lengthening my PCR cycling times or Dropping my Extension to 68?
Do I need to plan on doing nested or is it possible to get non-nested products?
Should I change my polymerase? Perhaps Taq can't handle the long stretches of TA's
Thanks everyone in advance!
I am having very similar problems, with DNA extracted with Methyleasy Xceed. The protocol is very easy and I get good strong bands with their primers, but none with mine. I too have tried varying cycles and temperatures and DNA concentrations, Mg concentrations and different primer sets with no luck. I have used both ABgene Reddymix 2X and Solis Biodyne HotFire polymerase. I get beautiful primer-dimer bands but very little else!
I had the same problem with the MethylEasy Kit. Even though I measured a significant amount of DNA after bisulfite treatment by UV, I obtained no PCR products with BSP or MSP primers for PCR products of 100 bp up to 1kb. In my opinion it's a conversion problem. With unspecific primers (no conversion positions in primer binding sites) I got strong bands but found after sequencing a significant C-signal under every converted cytosine position.
A pH of 10 is definately too high and will have a negative impact on your PCR performance. Increasing the number of cycles to 45 in your PCR might have a positive effect. But it seems questionable if the results reflects the methylation-status of your sample DNA...???
The high pH of the Reagent 3 is required for an effective desulfonation of your DNA. But it also degrades your DNA. At first abasic sites will be cleaved yielding a highly fragmented DNA. But it seems also possible that caused by the long incubation time- and temperature the DNA is more degraded. Desulfonation is typically done either at roomtemperature or at the maximum of 37°C for 10 to 15 minutes.
Try another bisulfite conversion kit. I obtained excellent results with the Qiagen EpiTect Kit or the Zymo EZ DNA Methylation Gold Kit even for CpG rich promotor regions.
strange to be hearing these problems,
if the controls work on your DNA samples it would suggest that your primers to your region of interest were not designed properly. Fragment length too big, not targeting converted DNA etc because the controls work. The methyleasy kit has some good controls that can control for conversion process and PCR amplification.
if the same primers work on the same dna converted with another kit, well that would be a combination of issues, one that would stem from the quality of your starting DNA and amount I would say.
We have found that for very limited amounts of gDNA it would seem the current Zymo Kit does well here however when you have high amount of good quality DNA both kits perform just the same.
I would not sequence MSP amplified products because you are already biasing the reaction towards either methylated or unmethylated templates depending on the primer you use, it would be pointless to sequence them.