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Imcomplete bisulfite treatment


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#1 joyphyl

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Posted 22 July 2009 - 07:21 AM

Could you help me with shooting the trouble with bisulfite treatment?

When I performed BSP and direct sequencing, there are still some C signals in non-CpG sites.
I think this reflects the incomplete bisulfite treatment, but with the same template anaylzed in other gene, complete reaction seemed to have occured.
Could bisulfite reaction affect DNA differently according to the region in the genomic DNA?
Then, to do the complete bisulfite reaction in every gDNA region, what kind of factor could I change?

I am purifying genomic DNA by phenol-chloroform extraction and then 1 ug of gDNA is treated with CpGenome modification kit from CHEMICON.

#2 MoB

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Posted 22 July 2009 - 11:25 PM

Could you help me with shooting the trouble with bisulfite treatment?

When I performed BSP and direct sequencing, there are still some C signals in non-CpG sites.
I think this reflects the incomplete bisulfite treatment, but with the same template anaylzed in other gene, complete reaction seemed to have occured.
Could bisulfite reaction affect DNA differently according to the region in the genomic DNA?
Then, to do the complete bisulfite reaction in every gDNA region, what kind of factor could I change?

I am purifying genomic DNA by phenol-chloroform extraction and then 1 ug of gDNA is treated with CpGenome modification kit from CHEMICON.


Incomplete conversion is mainly caused by incomplete denaturation and results in completely unconverted DNA. Small C-signals under a conversion-position are 'quite normal' when using a standard base-caller with your sequencer. This does indicate an imcomplete conversion but is simply an electronical- or software-problem. For the ABI sequencers the manufacturer provides a basecaller, optimized for sequencing of bisulfite-converted DNA.

There are some regions which are extremely difficult to convert by bisulfite-treatment (for instance FRAX). These regions mostly show a high content of cytosine and a high density of CpGs resulting in a high melting temperature. So: yes, there are differences between different regions in the genome. From my point of view the only factor to ensure the highest possible level of conversion is to change the bisulfite-kit. In my opinion the CpGenome kit does not perform very well, and as far as I know, it's very expensive. Try the Qiagen EpiTect Kit or the EZ DNA Methylation Gold Kit from Zymo Research.

Hope that helps...

MoB

#3 joyphyl

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Posted 23 July 2009 - 04:14 AM

Thank you very much for your reply! It helps a lot! :D


Could you help me with shooting the trouble with bisulfite treatment?

When I performed BSP and direct sequencing, there are still some C signals in non-CpG sites.
I think this reflects the incomplete bisulfite treatment, but with the same template anaylzed in other gene, complete reaction seemed to have occured.
Could bisulfite reaction affect DNA differently according to the region in the genomic DNA?
Then, to do the complete bisulfite reaction in every gDNA region, what kind of factor could I change?

I am purifying genomic DNA by phenol-chloroform extraction and then 1 ug of gDNA is treated with CpGenome modification kit from CHEMICON.


Incomplete conversion is mainly caused by incomplete denaturation and results in completely unconverted DNA. Small C-signals under a conversion-position are 'quite normal' when using a standard base-caller with your sequencer. This does indicate an imcomplete conversion but is simply an electronical- or software-problem. For the ABI sequencers the manufacturer provides a basecaller, optimized for sequencing of bisulfite-converted DNA.

There are some regions which are extremely difficult to convert by bisulfite-treatment (for instance FRAX). These regions mostly show a high content of cytosine and a high density of CpGs resulting in a high melting temperature. So: yes, there are differences between different regions in the genome. From my point of view the only factor to ensure the highest possible level of conversion is to change the bisulfite-kit. In my opinion the CpGenome kit does not perform very well, and as far as I know, it's very expensive. Try the Qiagen EpiTect Kit or the EZ DNA Methylation Gold Kit from Zymo Research.

Hope that helps...

MoB


Edited by joyphyl, 23 July 2009 - 04:15 AM.





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