Hello all. I have received great help with BSP from this forum, and I am hoping for help one more time. I am analyzing a promoter that contains a 2.3 kb CpG island by several computational predictors. I want to BSP this promoter, but I'm not if this size of PCR (say 2.5 kb) is even obtainable. I am using a Zymo Research kit, and I am routinely amplifying 0.9-1.3 kb products with great ease.
Has anyone amplified a 2.5-2.7 kb product for BSP and had any success?? I'll take any suggestions right now. I haven't designed any primers because I want to see what the forum has to say. Thanks for the help.
Mike
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4 replies to this topic
#2
methylnick
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Posted 25 September 2009 - 01:22 PM
Hi Mike,
the problem with bisulphite conversion is that it is a destructive process and will chew up your DNA, if this happens, it is usually the unmethylated template that is more susceptible to degradation (because the C is converted to U) compared to methylated template (no conversion).
If this is the case then in theory there are more methylated templates around that are larger in size in your bisulphite converted sample, this would mean you will get bias towards the methylated template because it's there, the larger the fragment you try to analyse. At 2.1kbp it's getting really big and if you do get an amplicon it is most likely to be methylated even though you were expecting no methylation.
I have gone upto 1.2kbp and no larger.
With our sequenon analyses for some samples looking at the H19 DMR we start to see skewing on methylation (increasing to 100%) at fragments greater than 400bp when we have assays designed to the same region of increasing length.
My advice would be to divide the promoter up into smaller amplicons because the current capillary sequencing reactions can still only read out to 800bp, 1000bp if you are really good so it's no real benefit to amplify such a large fragment.
the problem with bisulphite conversion is that it is a destructive process and will chew up your DNA, if this happens, it is usually the unmethylated template that is more susceptible to degradation (because the C is converted to U) compared to methylated template (no conversion).
If this is the case then in theory there are more methylated templates around that are larger in size in your bisulphite converted sample, this would mean you will get bias towards the methylated template because it's there, the larger the fragment you try to analyse. At 2.1kbp it's getting really big and if you do get an amplicon it is most likely to be methylated even though you were expecting no methylation.
I have gone upto 1.2kbp and no larger.
With our sequenon analyses for some samples looking at the H19 DMR we start to see skewing on methylation (increasing to 100%) at fragments greater than 400bp when we have assays designed to the same region of increasing length.
My advice would be to divide the promoter up into smaller amplicons because the current capillary sequencing reactions can still only read out to 800bp, 1000bp if you are really good so it's no real benefit to amplify such a large fragment.
All comments and communication are my own personal ones, and are not tied to any of my affiliations.
#3
PSUMike
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Posted 25 September 2009 - 04:01 PM
How do I divide the promoter up? I had planned to design primers outside of the putative CpG island and then sequence the whole region. Since this isn't possible, do I then design primers inside the CpG island and use them to amplify? Since they are inside the island, how do I design the primers.....as being methylated and unconverted by bisulfite? How would I then interpret a difference between two cell lines or tissues? We have some evidence that there may be a differential pattern between two tissues, and that is why I was thinking of BSP.....before I realized the CpG island so large. Thanks for the help.
Mike
Mike
#4
methylnick
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Posted 28 September 2009 - 03:48 AM
hi mike,
you will have to divide the island up arbitrarily.
Primers are designed to the converted template and you should try an avoid CpG's (if you are looking at mammalian system?) as you don't know the methylation status to them and you could inadvertantly bias your amplification to one or the other state.
try with methyl primer express to design the primers if not you may need to design them by eye.
Nick
you will have to divide the island up arbitrarily.
Primers are designed to the converted template and you should try an avoid CpG's (if you are looking at mammalian system?) as you don't know the methylation status to them and you could inadvertantly bias your amplification to one or the other state.
try with methyl primer express to design the primers if not you may need to design them by eye.
Nick
All comments and communication are my own personal ones, and are not tied to any of my affiliations.
#5
davex
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Posted 09 October 2009 - 07:06 PM
Hello all. I have received great help with BSP from this forum, and I am hoping for help one more time. I am analyzing a promoter that contains a 2.3 kb CpG island by several computational predictors. I want to BSP this promoter, but I'm not if this size of PCR (say 2.5 kb) is even obtainable. I am using a Zymo Research kit, and I am routinely amplifying 0.9-1.3 kb products with great ease.
Has anyone amplified a 2.5-2.7 kb product for BSP and had any success?? I'll take any suggestions right now. I haven't designed any primers because I want to see what the forum has to say. Thanks for the help.
Mike
