Problems with methylation analysis!!! - Primers crossreaction etc. (Aug/11/2005 )

Hi,
I have been working with methylation analysis searching for methylation status of two gene promoters (hMLH1 and MGMT, both been analyzed many times before). I have problems with the reliability of the method and I don’t know how to move on from this point… So I briefly explain what I have been done, could you please help me; any suggestions .
I have extracted DNA from paraffin blocks using phenol-cloroform extraction method (proteinase K treatment about two days at 55 °C), after extraction I have bisulfite treated DNA using Chemicon kit. I have designed primers for MLH1 at two different CpG islands (I call those far and near islands) and below is the primers I use. For MGMT I have designed two different set of primers MGMT1 and MGMT2, both also been presented below. For CpG island prediction and primer design I have used MethPrimer. Primers I have analyzed with many kinds of primer design programs (e.g. NetPrimer) and these analyses have shown a few primer-dimer etc. formations, which have not been disturbingly high. The size of the PCR products got from these analyses has been between 110-130 bp. I have optimized PCR conditions using gradient PCR to find proper annealing temperature. I have used Sigma’s JumpStart hot start polymerase. Now I have tested all primers with control DNA (Chemicon’s universal methylated and unmethylated control DNA) and also my own sample DNA. My problem is that only MGMT1 primers are not crossreacting with methylated/unmethylated control DNA, all other do that. If only MGMT1 is not crossreacting it gives positive result with water blank. I have tried to get rid of it with almost all possible ways (change clean water, new jumpstart, new primer dilution, new pipet etc.) but it always shows positive result, only smaller size than sample DNA should give. Maybe it is primer DNA that copies itself or I don’t know! The other not so nice problem is that some sets of sample DNA doesn’t give any result at all, there are not any lanes seen in the gel and the next set gives result! Where the DNA disappears or why it does not work. Am I losing it during bisulfite treatment? I would be very grateful if somebody could help me because now I have huge amounts of bisulfite treated DNA (which is useless soon), primers that won't work and hopeless feeling that this will not ever work!

Best wishes
Karoliina

MLH1, FAR:

Methylated forward-primer:
FmfarMLH1 5’- TTT TTT AGG AGT GAA GGA GGT TAC G-3’

Methylated reverse primer:
RmfarMLH1 5’- GCC ACT ACG AAA CTA AAC ACG AA-3’

Unmethylated forward-primer:
fumfarMLH1 5’- TTT TTA GGA GTG AAG GAG GTT ATG G-3’

Unmethylated reverse-primer:
rumfarMLH1 5’- AAA CAC CAC TAC AAA ACT AAA CAC AAA-3’


MLH1, NEAR:

Methylated forward-primer:
FmnearMLH1 5’- TGT TTT TAT TGG TTG GAT ATT TCG T-3’

Methylated reverse primer:
RmnearMLH1 5’- TCC CTA AAA CGA CTA CTA CCC G-3’

Unmethylated forward-primer:
fumnearMLH1 5’- TTG TTT TTA TTG GTT GGA TAT TTT G-3’

Unmethylated reverse-primer:
rumnearMLH1 5’- CTC CCT AAA ACA ACT ACT ACC CAC T-3’




MGMT1 primers:
Methylated forward-primer
fmMGMT1 5’-TTT CGA CGT TCG TAG GTT TTC GC-3’

Methylated reverse primer
rmMGMT1 5’-GCA CTC TTC CGA AAA CGA AAC G -3’

Unmethylated forward-primer
fumMGMT1 5’-TTT GTG TTT TGA TGT TTG TAG GTT TTT GT-3’

Unmethylated reverse-primer
rumMGMT1 5’-AAC TCC ACA CTC TTC CAA AAA CAA AAC A-3’



MGMT2 primers:
Unmethylated forward-primer
fumMGMT2 GGTTTGTATTGGTTGAAGGGTTATTT
Unmethylated reverse-primer
rumMGMT2 CCTAAAACAATCTACACATCCTCACT
Product size: 111

Methylated forward-primer
fmMGMT2 TTGTATCGGTCGAAGGGTTATTC
Methylated reverse primer
rmMGMT2 CTAAAACAATCTACGCATCCTCG
Product size: 107, Tm: 67.7

Hi Karolina !
If you just start methylation research and don`t have any expirience on this field, then welcome in club! I had similar problems ( it was a very sadomasochistic time) till I tried the higher magnesium concentrations.
Please. just play with diff. magnesium concentrations, eg from 3mM to 7mM. Dont`t use old dNTP`s, which were rethawed many times.
In my opinion, your "positive result" are primer dimers.
I don`t have any expierence with JumpStart. But I guess, it requieres Magnesium optimization too.

I also use Chemicon Kit. It usually works very well. Hold your starting DNA amount <=1 µg. I also use MethPrimer for MSP. It`s OK too. But not for BSP-Primer. See disscussion on this forum http://www.protocol-online.org/forums/inde...?showtopic=8281.

Good Luck!

Boris.

Hi.
thanks for you advices. But I was wondering, how does the changed magnesium concentration can make a difference with the fact that U (unmethylated) primers creates a result when I'm using methylated control DNA and M primers the same way with unmethylated control DNA. I thought that problem might be the polymerase.... or the primers that pairs with "wrong" DNA and aren't spesific.
Thanks again

-Karoliina

Altering magnesium concentration will alter the Tm and binding properties of the primer to the DNA template and may help in your situation.

Is it possible that MGMT is imprinted? My knowledge of this gene is rather limited, if it were imprinted then you will get a positive result using both methylated and unmethylated primers.

I think it is possible that the primer design may be suboptimal and you could try another primer set.

Nick