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Problems with BSP - loss of signal strength

methylation dideoxy sequencing signal strength cycling conditions BSP

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#1 vesna



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Posted 19 December 2011 - 04:17 AM


I am performing methylation analysis and since this is not the field my lab or myself would have any experience with, I've encountered a problem I can't seem to be able to solve by myself. So I was hoping someone might be able to help me?

I am interested in methylation patterns of promotors of two genes and have been experiencing problems with obtaining a nice sequence reading after methylation-independent PCR of bisulfite-converted DNA. Sequencer is ABI 3730 XL 96 capillary sequencer, BigDye Version 3.1, ABI KB-base caller. So far I have been using regular cycling conditions (50 degrees C annealing).

Problem: signal fades gradually, especially when using differentially methylated templates. When sequencing 100% or 0% methylated control PCR product after bisulfite treatment (therefore there are no mixed methylation sites), I get a reasonably nice reading, signal strenght stays roughly the same or fades in the same manner as, for example, with plasmid DNA. But when I try to sequence 50% methylated PCR product (mixture of 0% and 100%) or some of my samples (which also are a population of different methylation states), the signal strenght fades drastically so it is impossible to distinguish anything after around 120-150 bp.
My PCR products are 250-300 bp and have around 10 CpG sites, mostly towards the end, so it would be really difficult to sequence backwards without losing resolution in some CpG sites (even with a PCR primer with an overhang).Tm of primer for sequencing is 40/50 degrees C (I have tried two different primers for sequencing one PCR product, one a bit longer, but results were the same), primers contain only 3 nucleotides (no C's). I have also tried adjusting DNA concentration (2-10 ng/ul; company, which does sequencing for me, recommends 2-5 ng/ul for PCR fragments of given length), but without any substantial improvement.

What else can I try to get a nice read even for mixed methylation templates? From your own experience, what usually works? Adjusting Ta of sequencing to a lower temperature? 2-step cyle sequencing? Other special cycling conditions? Anything else?

In case you need additional information: I am interested in average methylation level for particular CpG sites. My source of gDNA are human cancerous and non-cancerous cell lines, treated or non-treated with DAC. gDNA was extracted using QIAamp DNA Mini Kit, bisulfite conversion was performed using EpiTect Bisulfite Kit and PCR with HotStar Taq polymerase. My positive (100% methylation) and negative controls (0% methylation) are SssI treated gDNA and PCR-amplified gDNA (before bisulfite treatment). Sequencing of individual clones is too expensive, so I have to optimize sequencing of PCR product.

I would really appreciate any advice.



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