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BISULFITE CONVERTION ?? TROUBLE SHOOTING - (Apr/21/2009 )

Dear All,

I need some help working with EZ DNA methylation kit. I wanted to bisulfite convert the FFPE material to study methyaltion status further.
So i have tried the EZ methylation start up kit, which also provided with the universal methylated genomic DNA and control primers hMLH1, hMLH2 to check for the convertion whether it worked or not.
After the bisulfite convertion i have done the control PCR reaction with the above mentioned primers (which should yield a product of 182 bp..if the convertion is complete), but i could only see the product formation in the positve control but not with my FFPE material ??? I have checked the quality of the FFPE DNA using oligo's pairs not related to a mehylated related regions that yileds a 160 bp product and the amplification worked on FFPE material and i could see the product.
Its really an essential step to confirm the bisulfite convertion to proceed further to avoid false positive results. Can someone help me how can i confirm the bisulfite convertion ??? is there any thing which i can change in my PCR conditions etc...i have used 2 l of bisulfite converted DNA for the control PCR reaction and the reaction as follows, 95 for 10 min, 94.5 for 30 sec, 59 for 30 sec, 72 for 60 sec, 72 for 7 min, 35 to 40 cycles...4 degree hold..
FOr the bisulfite convertion...as mentioned in the protocol i used...98 for 8 min, 64 for 3.5 hours, 4 degree hold.

The positive control worked fine??? (this confirms the bisulfite convertion) y not in FFPE DNA ??? ..I have used the unconverted DNA as negative control and ofcourse i could not see any product with that ...Your suggestions are appreciated...Thanks and regards, Raj

-RKA-

How much starting DNA did you use for the bisulfite reaction? Can you post the primer sequences here? What is the expected product size? Are you doing MSP or BSP? You said you have checked the quality of the unmodified DNA by PCR, right?
We need to know more before we can troubleshoot.

-pcrman-

pcrman on Apr 22 2009, 08:09 AM said:

How much starting DNA did you use for the bisulfite reaction? Can you post the primer sequences here? What is the expected product size? Are you doing MSP or BSP? You said you have checked the quality of the unmodified DNA by PCR, right?
We need to know more before we can troubleshoot.


500 ng DNA is used as a starting material and eluted in 10 l after bs convertion (80 % recovery after bs convertion with EZ kit)..
hMLH1 Primer I (sense)
5′-GGAGTGAAGGAGGTTACGGGTAAGT-3′
hMLH1 Primer II (antisense)
5′-AAAAACGATAAAACCCTATACCTAATCTATC-3′
Product size should be 182 bp. I want to use the bs converted DNA for micro array experiments (illumina golden gate methylation assay).

-RKA-

So the primers work with their methylated control DNA (the antisense primer contains a 'G', is that a typo?) but not your modified DNA. You can amplify your unmodified DNA by regular PCR. There are two potential causes for the failure. One is the yield from the conversion kit is too low. You may need another round of PCR using the same primers and the first PCR product as template. Another issue is that the DNA is from FFPE and is already degraded to small pieces. Although you can amplify before conversion, the DNA is further degraded during conversion so that ampifying 182 bp because very difficult. So try 2 rounds of PCRs to see if you can amplify anything.

-pcrman-

pcrman on Apr 23 2009, 05:26 AM said:

So the primers work with their methylated control DNA (the antisense primer contains a 'G', is that a typo?) but not your modified DNA. You can amplify your unmodified DNA by regular PCR. There are two potential causes for the failure. One is the yield from the conversion kit is too low. You may need another round of PCR using the same primers and the first PCR product as template. Another issue is that the DNA is from FFPE and is already degraded to small pieces. Although you can amplify before conversion, the DNA is further degraded during conversion so that ampifying 182 bp because very difficult. So try 2 rounds of PCRs to see if you can amplify anything.


pcrman, thank you very much for your time and suggestions. First thing...the G in antisense primer is true..not a typo..the primers are diesigned to amplify the human invitro methylated and bisulfite converted positive control...may be u should have a look at the explanation as follows.. :rolleyes:

Control Primers
Two hMLH1 Control Primers are supplied at a concentration of 20 μM (each) in 20 μl
TE buffer. The expected PCR amplicon is 182 bp, corresponding to nucleotide
positions 804 - 986 of the hMLH1 DNA sequence that includes the regions spanned
by the primers (see below).
hMLH1 Primer I (sense)
5′-GGAGTGAAGGAGGTTACGGGTAAGT-3′
hMLH1 Primer II (antisense)
5′-AAAAACGATAAAACCCTATACCTAATCTATC-3′

Original sequence of the human MLH1 DNA fragment for bisulfite treatment and
PCR amplification (sense strand 5′ to 3′). All cytosines in CG dinucleotides (bold) are
methylated. Numbers correlate to the nucleotides from the human MLH1 DNA 5 flanking
region (GenBank Accession #U83845). Observed CpG/ Expected CpG is > 1 with a 68%
total GC content.

781 ---------- ---------- ---ggagtga aggaggccaC GggcaagtCG ccctgaCGca
841 gaCGctccac cagggcCGCG CGctCGcCGt cCGccacata cCGctCGtag tattCGtgct
901 cagcctCGta gtggCGcctg aCGtCGCGtt CGCGggtagc taCGatgagg CGgCGacaga
961 ccaggcacag ggccccatCG ccctc

Expected Sequence of PCR amplified product following bisulfite conversion
(sense strand 5′ to 3′).
781 ---------- ---------- ---ggagtga aggaggttaC GggtaagtCG ttttgaCGta
841 gaCGttttat tagggtCGCG CGttCGtCGt tCGttatata tCGttCGtag tattCGtgtt
901 tagtttCGta gtggCGtttg aCGtCGCGtt CGCGggtagt taCGatgagg CGgCGataga
961 ttaggtatag ggttttatCG ttttt

The first question i have asked myself is....if the primers are designed for invitro METHYLATED positive control...how can i see the product in my FFPE as im not sure if the CpGs in that region are methylated or not !!!! but i have asked the company people and they says that..irrespective of methyaltion status, if the DNA is bisulfite converted i should see a product !!! i didnt get them how it is possible...but i thought i will try with unmetylated specific primers (only diffrenec is one nucleotide..between the METHYALTED SPECIFIC PRIMERS PROVIDED BY THE COMPANY...ND UNMETHYLATED SPECIFIC PRIMERS)
For your second query..i have checked the yield of the DNAs after bisulfite convertion and which is approximately 80 to 90 %....and more over i used...50 to 100 ng DNA for the PCR...But i can try second round pcr..

-RKA-