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New to area - (May/15/2007 )

Hi,

a) I have taken over a project in Methylation. I have been using the QIAGEN kit for bisulfite treatment of DNA. Going through the previous lab books, the person looks like they have then directly sequenced from this template using M13 tagged primers. I think that they are for converted sequences. I want to directly sequence my B/s treated DNA but I can' t be certain firstly if the treatment has worked. How do I find this out?

cool.gif I have tried using the sequencing primers to amplify the b/s treated dna but there are no products on the gel. It hasn't worked. Should I be doing a second round of PCR??

Any help would be greatly appreciated.

C

-Casey-

Hi Casey,

There are two potential problems with bisulfite modification: one is incomplete conversion, the other is loss of DNA during purification. To check wheter modification has worked, your PCR should give your some hints because either incomplete conversion or loss of DNA may fail PCR. Usually primers for bisulfite modified DNA are designed on sequences with consecutive non-CpG cytosines. If they have not converted to uracils, the primers will not bind. however, sometimes incompleted converted DNA can still be amplified. sequencing PCR product then becomes necessary to verify the completeness of conversation.

Usually two rounds of PCR are needed to generate product for gel visualization and direct sequencing. You can do that using the same pair of primers or nested primers. The first round of PCR should run 35-40 times, 2nd run 30-35 times.

-pcrman-

Hi,


Thanks for the tips. I did try a second round of PCR with the same conditions as the first PCR (5 cycle touchdown with 35 cycles of amplification). I still did not see any product on the gel. To test whether there was any DNA from the conversion reaction, I tried it with another set of primers for a different gene and there was amplification of the DNA with MSP primers.

My sequencing primers have an M13 tag on the forward and reverse primer and the sequence is as follows

Forward;

TGT AAA ACG ACG GCC AGT/ GTT AGG GGT GGT ATT GTT GG (The first part is the M13 Tag)

Reverse:

CAG GAA ACA GCT ATG ACC/ TCA CAC CAA ACA CAT AAT AAA TAC C (First part is the m13 tag again).

I am using a different kit to the person who had first designed these primers. Would this be influencing their ability to amplify? I don't think it would.

The previous person working on this project had got these primers to amplify. i have ordered new primers and will test whether it is the primer stocks as they have been there two years.

I am using the Qiagen Epitect Kit which people have found to work well. I have SSSi treated DNA as a control.

Any help you can give would be fantastic.

-Casey-

Hi Casey,

You could try to use the M13 tagged primer only in the second round PCR and the untagged version in the first round. With those tags you have a higher probability of unspecific amplification when a lot of different sequences are around. Maybe you don't get enough specific amplification?

-kr├╝melmonster-