Quantifying Bisulfite treated DNA - (Oct/03/2008 )

Hi,

I've been using Qiagen's EpiTect Bisulfite kit with reasonable success. I've tried to quantify by NanoDrop (in ssDNA mode) but I've found this to be very inaccurate. Does anyone have a method of how to quantify modified DNA?

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Hi,
The Nanodrop is usually a very accurate method of measuring all forms of Nucleic Acid. Have you contacted your distributor to make sure the unit is functioning properly? Our dealer is very helpful with such matters.
Cheers
JB

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HI I'm also using the Qiagen Epitect kit but my recovery was low and not sure if the conversion is complete because I couldn't detect any bands in my MSP. I started with 1ug gDNA (200-700bp) without addition of carrier RNA and eluted the DNA in 30ul EB, the most I can get is 300ng. Anyway to increase the recovery rate?

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I think most of the DNA is not bound to the membrane of the spin column. The binding-efficiency of DNA to a silica membrane strongly depends on the fragment-length. The longer the DNA, the stronger the binding! Just keep in mind that your DNA is additionally degraded and fragmented during the bisulfite conversion due to the harsh reaction conditions.

To overcome that problem I add an equal amount (560µl) to the binding buffer BL of ethanol to the binding mixture and allow to incubate at RT for 10 minutes. Loading to the column has to be done in two seperate centrifugation steps. I obtained recovery rates of >80 percent even for highly fragmented DNA.

Hope that helps...


MoB

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So after conversion you added 560ul of the buffer BL to your DNA mixture and allow for 10min incbuation before centrifuge. How much DNA mixture did you add to each column?

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You have to add 560 ul binding buffer BL and 560 ul abs. ethanol to the bisulfite reaction mixture. Mix well and allow the mixture to incubate for 10 minutes at room temperature. As the final volume is larger than the maximum capacity of the spin column you have to add the mixture in two seperate centrifugation steps onto the same (!) spin column. After loading continue as described in the manual.

Best

MoB

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you will always have problems quantifying BiS treated DNA. the mutagenesis is harsh and creates many nucleotide fragments.
Even with traditional clean up methods you will have carry over of mutagenized products.

qPCR is by far the most accurate way to quantitate converted DNA.

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I think small fragments will be washed away during purification. Abasic sites will be cleaved during desulfonation. Therefore quantification by UV should be similar to quantification by PCR. Accurate qPCR depends on the length of your qPCR. For short assays (<100 bp) I found that the amplification is identical to the quantification by UV.

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