Nanog Methylation and sequencing - (Apr/27/2007 )
Greetings to everyone,
currently I am analysing the methylation pattern of Nanog promotor and CpG island in human germ cell tumours. I am using DNA from tissues and cell lines. I am converting the DNA by Qiagen EpiTect Kit. My PCR amplicons are 306 bp and 225 bp in length. I am using Invitrogen TA Cloning Kit pCR2.1 for ligation and competent E.coli TOP10 for transformation. I am screening for positiv clones by ampicillin resistence and blue/white test. Plasmids are isolated by alkaline lysis method (Birnboim&Doly). Plasmids are controlled for right inserts by a restriction digest. Sequencing is performed after Sanger by a company. Now to my problems. I am achieving no reproducible results. All samples are sequenced in triplicate but unfortunately the methylation status of the CpG's is different from each other. In case of the analysed CpG island even the whole sequence is not complementary to the original sequence (after imitation of bisulfite treatment). Can anymone give me some hints/tips or can find any bugs I made.
Thanx a lot, Daniel
Hi Captian meth,
a couple of things come to mind. Your tumor samples could be a mixed population of cell types and you are seeing methylation on clones from a range of difference cell types.
Your primers are not strand specific because you are not seeing the complementarity with your original sequence, if designed incorrectly, you will get PCR recombination and funny results.
As for reproducibility, how many clones are you sequencing? per sample 6 is a good start, 12 is better and ideally the more the better.
good luck!
Nick
a couple of things come to mind. Your tumor samples could be a mixed population of cell types and you are seeing methylation on clones from a range of difference cell types.
Your primers are not strand specific because you are not seeing the complementarity with your original sequence, if designed incorrectly, you will get PCR recombination and funny results.
As for reproducibility, how many clones are you sequencing? per sample 6 is a good start, 12 is better and ideally the more the better.
good luck!
Nick
Thank you for yor quick answer and your tips!
If I compare the bisulfite treated sequence of the promotor region to the original sequence I see up to 95% complementarity (differences only because of methylation). When comparing the sequences of the CpG Island I see 85% complementarity, leading to the conclusion that the amplified region should be correct but something must have happend that alters parts of my sequence. Can you imagine which errors could lead to this result?
Thank You, Daniel
CaptianMeth
are you seeing non-CpG methylation in your CpG islands and is it this that is leading to your lower complementarity? If so it means you have not converted your DNA efficiently.
IF this is the case, add a 100C step in your denaturation prior to addition of bisulfite, I am not too familiar with epitect but that is what I do with the home brew method.
Nick