Potentially dumb questions on methylation - Bisulphite sequencing queries (Jun/29/2004 )

My lovely boss has dumped this project on me and as fast as I find things out new questions arise. Here are a few possibly dumb ones!
1. After bisulphite treatment, the strands are no longer complementary and strand specific primers can be designed. This allows amplification, cloning + sequencing on the individual strands to determine their methylation patterns.
Q: Is there any need to study the antisense strand? Can variable methylation of this strand affect expression?
2. Bisulphite sequencing PCRs are described in the literature as “generally shorter than normal PCRs” (ambiguous statement or what?). Programs such as MethPrimer are set to default size of 200bp optimal, 300bp max.
Q: Why short PCRs?
Q: I have 620 bp CpG island to study, could I do it in one big
PCR, or do I have to design 3 sets of primers and do it in 200bp chunks to get the full picture?

3. I’m also trying to PCR across the CpG island of native DNA (not bisulphite treated), using primers either side of it and am having a hell of a job. Is there evidence to suggest difficulty amplifying through CpG islands (I have 100 CpG in 620bp).

Thanks in advance for any help/derision!
Cheers
Chris

Hi Chris,

These are not dumb questions. Everyone got the same questions first starting methylation study.

Q: Is there any need to study the antisense strand? Can variable methylation of this strand affect expression?

A: It's assumed that methylation of both strands is symmetric. Usually there is no such need to map the other strand.

Q: Why short PCRs?

A: The condition of bisulfite modification is hash and may result in strand breaks. Or if you use DNA from paraffin sections, DNA tends to be short. So a size around 200 bp is the easiest to amplify using MSP or BSP. However, 400 bp (DNA from cell line) is also possible (at least in my hand). I heard from one peson in this forum that he was able to amplify over 1kb fragement from bisulfite treated DNA.

Q: I have 620 bp CpG island to study, could I do it in one big
PCR, or do I have to design 3 sets of primers and do it in 200bp chunks to get the full picture?

A: Just try. But if this is the first time you do this, don't be so ambitious.

3. I’m also trying to PCR across the CpG island of native DNA (not bisulphite treated), using primers either side of it and am having a hell of a job. Is there evidence to suggest difficulty amplifying through CpG islands (I have 100 CpG in 620bp).

A: May be difficult, but certainly is possible.

Hope it helps.

I think pcrman is right. I'm trying, too. But I think the critical key is DNA purification step. It's my big barrier !
Hope you to make it!

Thanks for the replies and help.
Ming, what purification methods are you using? I'm currently trialling the Human Genetic Signatures "MethylEasy" kit and I've got the Zymo Research "EZ DNA Methylation" kit on order.
I'll post to let you know how I get on.
Cheers
Chris

I have ever used the Wizard DNA clean-up kit ... Althoough I have good bisulfite conversion, I have poor DNA purification ... so I'm trying new methods....

Hi Ming,
Initial trials with the 2 kits I've been testing:
Both kits gave good yields of DNA.
The DNA from the HGS kit is hard to PCR
The DNA from the ZYMO kit seems to PCR quite well.
This is as far as I've got, I'll be cloning and sequencing next week.
Cheers
Chris

Hi Ming,

I am new to this forum. I just posted a reply somewhere else, but will repeat a little here in case you don't see it. I have found better results using Methyleasy, provided I have clean starting DNA (usually phenol/chloroform extract, even after using a kit).

Love to hear about the results when you get them.

Hi all. Does any of you experts know why I have huge smears as results of my MSPCR, with both M and UM primers (started first with UM). Is the DNA degraded ? Better to store en TE rather than water ? I use dilutions 1:3, in 30 ul, is that too much ?
Cheers

I've had success with long (1Kb) amplification of modified DNA, but in a bacterial context, where the complexity of the DNA sample is very much lower. For your amplification of unmodified DNA, try adding 2-8% betaine, which helps in PCR through high GC regions. I can also reiterate my previous advice to use low extension temperatures in PCR of the modified DNA, which has very low GC content. See PMID: 8628694.