I ordered two Predesigned assays for quantification of CpG methylation by Pyrosequencing from Qiagen and I do not have any product after PCR. I have dissolved them in TE ph8, tried different annealing times and temperatures. I used bisulfite treated DNA from 100ng up to 500μg and still cannot see any product. If this only happened to one primer set, I would think it is a faulty primer, but it is the same for both.
What is the od260/280 ratio of your dna?
It is possible that the dna contains pcr inhibitors so in this case using less dna and more cycles may produce a product as the inhibitor is diluted too much to interfere. To test this you could run an ordinary pcr with other primers on a known goos dna sample and also run a sample with 100ng of your dna as well as the good dna.If the pcr fails when you have added your dna then it has inhibitors and need further purification. Some inhibitors remove divalent ions so you could try adding 2x as much Mg as usual in your pcr and use a low annealing temperature. Is your template particularly Gc rich. 500ug is a huge amount of dna and will definately stop pcr reactions
Paul Rutland, thank you for the answer.
After the bisulfite conversion of my DNA I started to run some tests with primers designed by our lab, and they never had any problems (clear bands, unregarding DNA concentration, and good peak heights in Pyrosequencing). I then proceeded to using the PyroMark CpG Assay PCR primers and they just didn't work.
I seem to have difficulties only when using these primers for some reason I cannot explain. I used the same bisulfite converted DNA and I also ran a Pyrosequencing test for the chance that maybe there was just too little DNA for me to see in the gel, but it was an absolute flatline.
I also used different concentrations of Mg but I will try using 2x the next time.
The od ratio is generally from 1.8 to 1.9. I have used other DNA which was way worse (again, with primers I had designed, not a kit) and I never had a problem.
It does seem that your dna quality is fine which leaves the sequence and the oligos. Is there any chance that your dna is deleted or has a polymorphism near the 3' end of the primer spoiling the chance of extension ?. As your in house designs work it would be reasonable and cheap to design your own primers. Qiagen is a good company but if your cohort is strongly bottlenecked for a rare polymorphism that could spoil pcr or pyrosequencing extension. If the problem is the pcr step could you redesign primers further out just to get a product then use the internal sequencing primer which is now inside the amplimer to get round the poor pcr step. If the template is high GC then the use of 1M betaine and/or 5-10% DMSO may help to produce an amplimer. I always found tech help at Qiagen very helpful.It is worth giving them a call to check tech problems and also any known production issues with exactly that batch number of the kit.
Paul Rutland , it turns out that the TE buffer is problematic for primers. Until now we dissolved our primers in RNA-free distilled H2O and had no problem, this time the manufacturer insisted on TE buffer. After adding TE -in various concentrations- in a PCR with unproblematic primers it seemed that they were silenced.
So, keep in mind, if you ever buy ready or designed primers that it is better to dissolve them in water even if the manufacturer insists on TE.
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