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Bisulfite sequencing problems


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#1 Ming

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Posted 23 February 2005 - 06:16 PM

Dear all:
I've faced a big problem recently...
The PCR product I have sequenced contain about 60 CpG site in 720 bp...
The results I only get is about 100~300 bp...(after cloning)
some people tell me the high GC content problem in it...
Can somebody help me to increase the sequencing performance?

ps. I'm using the Beckman system for sequencing...and I've used high GC reagent, but the results weren't better...

#2 methylnick

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Posted 23 February 2005 - 09:44 PM

The PCR product I have sequenced contain about 60 CpG site in 720 bp...
The results I only get is about 100~300 bp...(after cloning)
some people tell me the high GC content problem in it...
Can somebody help me to increase the sequencing performance?

ps. I'm using the Beckman system for sequencing...and I've used high GC reagent, but the results weren't better...

I am assuming you have checked your clone inserts to ensure you have cloned the full insert. I have experienced instances where I have cloned a PCR amplicon and upon checking the insert, it has shrunk!

If you are looking at a CpG island which I would assume you are with such a high number of CG sites in such a short amplicon you would expect such an island to be unmethylated. This post bisulfite conversion you will have a very AT rich sequence to sequence through.

One thing is to ensure the template DNA you are sequencing from is clean.

I would try lowering the annealing temperature step in your sequencing reaction and increasing the number of cycles.

I would also try sequencing from the other end of your inserts.

Nick

#3 Ming

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Posted 01 March 2005 - 08:29 AM

[quote name='methylnick' date='Feb 23 2005, 10:44 PM'] [quote name='Ming' date='Feb 23 2005, 07:16 PM']
I am assuming you have checked your clone inserts to ensure you have cloned the full insert. I have experienced instances where I have cloned a PCR amplicon and upon checking the insert, it has shrunk!

If you are looking at a CpG island which I would assume you are with such a high number of CG sites in such a short amplicon you would expect such an island to be unmethylated. This post bisulfite conversion you will have a very AT rich sequence to sequence through.

One thing is to ensure the template DNA you are sequencing from is clean.

[quote name='Ming' date='Feb 23 2005, 07:16 PM']
I would try lowering the annealing temperature step in your sequencing reaction and increasing the number of cycles. [/quote]
:unsure:
I'm very appreciate your comment...
That's a good point...

1. I have checked my insert use the vector's primer...and the results are ok...so that will not be the problem...

2. AT rich would be right choice...because the DMSO can't get the better sequence results...conversely...no DMSO will be fine...

3. that's the big issue to adjust the annealing temperature step...It's not a easy way...but it is the best rule...I have gotten the better results to adjusting it...although it is not very satisfying...

Finally, thanks you very much !! :D

#4 pcrman

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Posted 01 March 2005 - 04:09 PM

I don't think you will get all 60 CpG sites methylated so the final sequence won't be GC rich. I have sequenced insert of 300 bp with 29 cpg sites without any problem. I agree with Nick that you make sure your plasmid is clean and the amount of plasmid DNA and primer are optimized. Better send your samples to a sequencing company.

#5 Ming

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Posted 01 March 2005 - 05:38 PM

I don't think you will get all 60 CpG sites methylated so the final sequence won't be GC rich.

you are right...I didn't get all 60 CpG sites methylated...there were less methylated sites in my cell line...uh-huh...that's not my will...the same results I got when I send to sequencing company...thanks for your reply...
Cheers B)




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