Hi. I m having many troubles trying to get amplification from bisulfite modified DNA.
Here is the thing. We want to assess the methylation state of several genes and we design our primers. For almost the totality of genes, the pcr gradient is succesful, but once we try to amplify the samples (tumoral cell lines) the caos is unleashed. Sometimes we get weak strands, sometines smear and other we have nothing. The most shaking thing is that we have this problem with every single gen that we want to study and besides it is perturning as well that we have succeed with pcr gradients but not with subsequent pcr's. I have tried everything: Increase and decrease the concentration of template, Increase and decrease the cycles, pcrs with dmso, pcr with fresly modified DNA, repeated gradients, repeated pcr and on and on and on..... and with have not been able to obtain desired strands (clean and enough).
I will thank any kind of suggestion that could give me back.
best regards.
Dear Javier,
I have been doing bisulfite PCRs followed by direct sequencing since the last two years using DNA from clinical samples. I used to find it very difficult initially to get bands but I got it sorted later.
1. Your DNA quality needs to be good. Run a check gel or if you can afford, use a bioanalyser for both quality and quantity assessment. I quantitate my DNA on Biotek microdrop system at 260/280.
2. Which kit do you use for conversion? I use MECOV-50 from Invitrogen.
3. How much do you convert and in what volume do you elute that much DNA? Warm the elution buffer at 70 deg prior to elution, that helps max recovery of converted DNA. I suggest for 1 microgm, you may elute in 20 microL of elution buffer. Also the product size should not exceed 500 bp oe else you will never get consistent bands unless optimized further.
4. How do you design your primers? I use Methyl Primer Express, which is sadly not freely available anymore. Try designing primers with Tm of upto 55-62 deg C. Increase MgCl2 for your PCR reactions and optimize Ta on higher side of temperature gradient. eg: 3-4mM MgCl2 and Ta of 59-62deg C. Will give you a nice thick band.
6. Use Epitaq from Takara (special Taq for bisulfite DNA). It works wonderfully even for normal genomic DNA PCRs!
5. I am sharing my Bisulfite PCR protocol with you that I have modified for getting amplicons of 500 bp with Epitaq. Hope you find it useful.
Initial denaturation (x1)
95 deg for 5'
Cycles (x 40)
94 deg for 30 sec
(Ta) of reaction for 30 sec
Anneal at 72 for 40 sec
Final Anneal
72 deg for 10 min
Store
4 deg
All the best
Hi, I have some guess for your problem.
1. Is the cancer DNA perfect? No smear or other damage? If DNA is ok, maybe should check bisulfite convert is ok or not. I check bisulfite convert by paper primer, like Col2A1 and ACTB.
2. What program did you use to design primer? Do the primers have CpG site on it? Is it possible that primer have CpG on it?
I hope that I really get your point. Good Luck~
Hi Javier,
I guess there are several things which migh cause your problems. One could be inefficient bisulfite conversion. Partly converted DNA represents such a multitude of different template DNA molecules so that primers might misprime. Which bisulfite method did you use? The innuCONVERT All-in-One Kit is designed to prepare bisulfite DNA from cell line pellets without prior extraction. You might want to test it.
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0093933
http://www.analytik-jena.de/fileadmin/content/import/imported_dam/14-02-28_Fol_innuCONVERT_Bisulfite_EN_WEB_geschuetzt.pdf
Best,
Dimo
Dear Javier,
I have been doing bisulfite PCRs followed by direct sequencing since the last two years using DNA from clinical samples. I used to find it very difficult initially to get bands but I got it sorted later.
1. Your DNA quality needs to be good. Run a check gel or if you can afford, use a bioanalyser for both quality and quantity assessment. I quantitate my DNA on Biotek microdrop system at 260/280.
2. Which kit do you use for conversion? I use MECOV-50 from Invitrogen.
3. How much do you convert and in what volume do you elute that much DNA? Warm the elution buffer at 70 deg prior to elution, that helps max recovery of converted DNA. I suggest for 1 microgm, you may elute in 20 microL of elution buffer. Also the product size should not exceed 500 bp oe else you will never get consistent bands unless optimized further.
4. How do you design your primers? I use Methyl Primer Express, which is sadly not freely available anymore. Try designing primers with Tm of upto 55-62 deg C. Increase MgCl2 for your PCR reactions and optimize Ta on higher side of temperature gradient. eg: 3-4mM MgCl2 and Ta of 59-62deg C. Will give you a nice thick band.
6. Use Epitaq from Takara (special Taq for bisulfite DNA). It works wonderfully even for normal genomic DNA PCRs!
5. I am sharing my Bisulfite PCR protocol with you that I have modified for getting amplicons of 500 bp with Epitaq. Hope you find it useful.
Initial denaturation (x1)
95 deg for 5'
Cycles (x 40)
94 deg for 30 sec
(Ta) of reaction for 30 sec
Anneal at 72 for 40 sec
Final Anneal
72 deg for 10 min
Store
4 deg
All the best
Problems with bisulfite PCR are not uncommon, but are always solvable. A couple of quick points:
Firstly what is the DNA that you use for the gradient PCR? How is it different to your samples DNA? Could you run the gradient PCR using one of your sample DNA preps?
Secondly, is the gradient PCR performed on a different machine from the samples PCR? I have seen variability of PCR amplification in different machines. If it is a different machine then I would try the gradient PCR machine for your sample PCR amplifications.
As for your other replies, I heartily recommend KAPA HIFi polymerase for bisulfite PCRs, as the polymerase is more tolerant of the presence of uracils in DNA, but each group will have their own preferences.
Thank you all for your contributions. i pretty much follow the methodologies and protocols that you use too, unless for the dna modification, because in our lab we do it manually, without kits, that's the way that my partners do with good results. Neither we use softwares to design primers.
Jian Lang, i have not knowledge about this paper primer that you mention. Could you tell me more abou it? .Thank you.
Pooya it's not clear for me the example that you gave about Ta and MgCl2. Could you explain to me please?
Again thank you all for your time.
Dear Javier,
The Ta is the optimum annealing temperature of a set of primers for a PCR reaction. To get maximum specificity of your amplicon and avoid non-specific amplification, your Ta has to be standardized by putting a gradient PCR with 5-6 temperatures at a specific concentration of MgCl2. Thus, if your primers have Tm range of 59-61 deg C, then try bisulfite PCR gradient with 52-57 deg C. with 3mM MgCl2. If you do not get a band, increase MgCl2 and try again. If there are non-specific bands with your band of interest, decrease MgCl2 to say 2.5mM and increase temperature by 1-2 deg. That should give you the band.
Thank you Pooja.
One more thing, My samples are DNA extracted from cancer cell lines and i convert 1 ug elute in 20 ul. Since i have to repeat the pcr several times, do you think that cicles of freezing and thawing could affect the integrity of the DNA thus interfiring the performance of the PCR?
regards.
Hi, in the paper, Promoter hypermethylation of FBXO32, a novel TGF-β/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer, was showed primer for Col2A1 and ACTB. Although they are used for MSP, they also can use for bisulfite conver test.
Good luck
Javier,
Freeze thawing will not affect your DNA unless you are eluting it in water or something other than the commercial buffer. I have not found any difficulty to freeze thaw converted DNA samples so far. Anyway, if it is samples you are worried about, you can convert any other cell/tissue DNA samples to standardize your PCRs first. I will not recommend the use of your precious samples for the same.
Sometimes simply amplifying bisulphite modified DNA is just very difficult, particularly from CpG islands.
It took us over 18 months to get good PCR bands for some of our glucocorticoid receptor bisulphite sequencing protocols. Even now, some protocols work better in the hand of certain lab members, and other protocols in the hand of other lab members.
In the "how to do it", getting bisulphite modified DNA amplified is just the same as normal DNA. You have to play with the same parameters - Tm, MgCl2, and primer sequence. You may have to make multiple primer pairs, and try many different assays with a range of 5 or 6 different enzymes (try the Takara EpiTaq!) until you find the one that works for your particular assays
As you have a very high T content in your template DNA you may have real difficulties designing primers at some of the Tm mentioned here - A CpG island that has been bisulphite modified can be upto 65-70% A/T - and remembering the old-fashioned 2AT+4CG rule for Tm calculation, will always give you a very low Tm.
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