Designing methylation primers on long CpG island - (Mar/21/2005 )

Dear all!

Could anyone give me advice about how to ordering good primers for the attached sequence. I plan to do bisulfitesequencing and need good primers. I tried Bisearch both using the unconverted and the converted sequence as source but I'm not sure if I got any good result. Maybe I should design the primers manually? I would like to sequence the whole region but this may not be either possible or a good idea? Suggestions? Should I divide the sequence into several overlapping regions when sequencing, and if how to plan it, it seems as a difficult task.

Also I've heard that nested PCR give a better result than ordinary PCR, is this true? In that case I also would like to ask anyone in more detail how you plan nested PCR before ordering primers.

Hi Klahar,

Your seqence contains a ver long CpG island with very high density of GC. because of this, it's hard to design BSP primers. However you can try the following:

1) Design primers which amplify the whole CpG island (because there are only ideal priming locations outside the cpg island). Some people have amplified >1 Kb region without problem. I have designed primers with long amplicon on your seqeunce. Here are the primers
Primer picking results for bisulfite sequencing (or restriction) PCR

Thanks a lot pcrman!

I will order one of these primer pairs and check how it works.
Do you have any advice about PCR-conditions or special Taq-polymerase especially for this kind of PCR.

I suppose it will be difficult to plan a nested PCR with internal primers inside the CpG-island, because of high Tm compared to the flanking primers, but if nothing else works I will try to design degenerate primers from Bisearch.

Regarding nested PCR, you don't need to design another pair of primers. You can just run two rounds of PCR using the same primers with the first round run 40 cycles, the 2nd 25-30 cycles. After the first round, you may not see any visible band but that's OK and just go ahead with the 2nd PCR using 0.5-10 ul product as the template.

I strongly recommend Sigma's JumpStart RedTaq polymerase which makes a huge difference.

Use down stream primer for sequencing and also design 3-4 internal antisense primers to seqence the whole region.

Hi Khlar,

here's my two cents worth. Although people have in the past amplified such a large region sucessfully. I have been taught not to be too greedy and maybe design two sets of primers.

Attached is your sequence with the primer locations that I have chosen and the sequences you could order. I have also calculated the Tm using perlprimer, my colleague wrote the program and it has the most accurate Tm algorthim available to date.

You are looking at a CpG island, and it would be safe to say of the CG's assayed with my primer set, you can extend to the whole island, although to be absolutely sure, you will have to select more primer sets.

I am not familiar with all the programs available for such primer design, I have been stung using a particular program only to find out the primers chosen were not optimal.

Good luck with it and let us know how you go!!!

Nick

Hi again pcrman and methylnick!

I have ordered pcrman's 2 primers and methylnicks 3 primers and have made PCR using standard conditions on 30 mkl volume, 57 degrees C annealing temperature, using two different polymerases.
Cycling conditions:

95 degrees for 5 min.

40 cycles with 95 degrees for 30 sec, 57 degrees for 1 min, and 72 degrees for 1 min.

72 degrees for 6 min.

5 mkl of each reaction was run on 0.8% agarose gel.

I got no good bands for any of the pairs (3 pairs totally), not even when running re-PCR using the same conditions except for 25 cycles instead of 40, including 2 mkl from the first PCR (I only got smear on the re-PCR with pcrmans primers). I have ordered the enzyme from Sigma that pcrman recommended, and hopefully it will give better results. Do you have any good advice concerning for example cycling conditions, chemicals to be added to the reactions, or anything else?

Thanks

Sorry to hear that it didn't work out for you the first time.

I didn't see any problem with your PCR. 2nd PCR is necessary but should use less template from the first PCR (no more than 1 ul).

JumpStart RedTaq will certainly make a difference.

If you have known working modified DNA, use it as positive control.

Good luck!

Hi again !

Thanks a lot for recommending the enzyme from Sigma, pcrman, it seems really to make a huge difference, and I really get bands on the first pcr-reaction, using positive control DNA, with the primers designed by you. I hope I will update with further information soon, I didn't check methylnick's primers yet.

Hi again !

Thanks a lot for recommending the enzyme from Sigma, pcrman, it seems really to make a huge difference, and I really get bands on the first pcr-reaction, using positive control DNA, with the primers designed by you. I hope I will update with further information soon, I didn't check methylnick's primers yet.