Loss of bands at higher Tm in presence of DMSO... - Attempt to optimise DMSO methylation-spec PCR (Dec/14/2010 )
Hi,
I am trying to utilise DMSO in order to perform PCR to detect methylation status (http://jmd.amjpathol.org/cgi/content/abstract/9/5/574). This protocol depends on the fact that methylated and unmethylated DNA show a different sensitivity to the amount of DMSO in the PCR reaction- methylated DNA needs more DMSO in the reaction to show bands.
I have performed a gradient from 56-66oC. (The annealing temp for my primers should be 57-58oC.) At each temperature, I used a master mix with 0%, 2% and 5% DMSO. My hope was to identify a temperature at which the PCR only worked with the addition of DMSO.
However, my experiments are producing very strange results. The addition of DMSO seems to be hindering the PCR reaction. At 56-60oC, the bands with increasing DMSO are fainter. And at 64-66oC, the bands even disappear! I have attached a picture of my results.
I have read numerous posts saying that DMSO really helps annealing and therefore improves specificity and efficiency of PCR. Indeed, the protocol from the paper that I am trying to follow relies on this fact. So why am I getting this result? I really don't understand. Can anyone offer an explanation?
Thanks so much!
beth on Tue Dec 14 11:14:56 2010 said:
Hi,
I am trying to utilise DMSO in order to perform PCR to detect methylation status (http://jmd.amjpathol.org/cgi/content/abstract/9/5/574). This protocol depends on the fact that methylated and unmethylated DNA show a different sensitivity to the amount of DMSO in the PCR reaction- methylated DNA needs more DMSO in the reaction to show bands.
I have performed a gradient from 56-66oC. (The annealing temp for my primers should be 57-58oC.) At each temperature, I used a master mix with 0%, 2% and 5% DMSO. My hope was to identify a temperature at which the PCR only worked with the addition of DMSO.
However, my experiments are producing very strange results. The addition of DMSO seems to be hindering the PCR reaction. At 56-60oC, the bands with increasing DMSO are fainter. And at 64-66oC, the bands even disappear! I have attached a picture of my results.
I have read numerous posts saying that DMSO really helps annealing and therefore improves specificity and efficiency of PCR. Indeed, the protocol from the paper that I am trying to follow relies on this fact. So why am I getting this result? I really don't understand. Can anyone offer an explanation?
Thanks so much!
DMSO will decrease the melting temperature at about 0.6oC per 1% (v/v). It improves PCR specificity by preventing nonspecific primer-template annealing, as it increase the binding strigency. DMSO improves the efficiency of GC-rich PCR as it helps template denaturation.
It likes that the increased DMSO concentration prevent the primer-template annealing at higher temperature in your experiment.
Yes, but if DMSO lowers the melting temperature, AND my PCR works at lower temperatures, then why do my bands disappear?? I.e. According to your calculation, 5% DMSO would lower temperature by 6oC. Therefore, 66oC with 5% DMSO becomes like PCR performed at 60oC and 64 becomes like 58. But I have bands at 60oC and 58 without DMSO, but NOT at 64 and 66oC WITH DMSO.
I hope this is making sense to somebody...?
zhongmindai on Tue Dec 14 13:12:21 2010 said:
beth on Tue Dec 14 11:14:56 2010 said:
Hi,
I am trying to utilise DMSO in order to perform PCR to detect methylation status (http://jmd.amjpathol.org/cgi/content/abstract/9/5/574). This protocol depends on the fact that methylated and unmethylated DNA show a different sensitivity to the amount of DMSO in the PCR reaction- methylated DNA needs more DMSO in the reaction to show bands.
I have performed a gradient from 56-66oC. (The annealing temp for my primers should be 57-58oC.) At each temperature, I used a master mix with 0%, 2% and 5% DMSO. My hope was to identify a temperature at which the PCR only worked with the addition of DMSO.
However, my experiments are producing very strange results. The addition of DMSO seems to be hindering the PCR reaction. At 56-60oC, the bands with increasing DMSO are fainter. And at 64-66oC, the bands even disappear! I have attached a picture of my results.
I have read numerous posts saying that DMSO really helps annealing and therefore improves specificity and efficiency of PCR. Indeed, the protocol from the paper that I am trying to follow relies on this fact. So why am I getting this result? I really don't understand. Can anyone offer an explanation?
Thanks so much!
DMSO will decrease the melting temperature at about 0.6oC per 1% (v/v). It improves PCR specificity by preventing nonspecific primer-template annealing, as it increase the binding strigency. DMSO improves the efficiency of GC-rich PCR as it helps template denaturation.
It likes that the increased DMSO concentration prevent the primer-template annealing at higher temperature in your experiment.
Hi beth,
I use DMSO in my PCR reactions... but I never encounter such incident yet.
Try do a repeat of your PCR and see whether this happens again.
Cheers
Hi, you misunderstand my meaning. If you performed PCR at annealing temperature of 66oC with 5% DMSO, it is likely that you performed PCR at 69oC with normal PCR buffer.
beth on Tue Dec 14 14:08:20 2010 said:
Yes, but if DMSO lowers the melting temperature, AND my PCR works at lower temperatures, then why do my bands disappear?? I.e. According to your calculation, 5% DMSO would lower temperature by 6oC. Therefore, 66oC with 5% DMSO becomes like PCR performed at 60oC and 64 becomes like 58. But I have bands at 60oC and 58 without DMSO, but NOT at 64 and 66oC WITH DMSO.
I hope this is making sense to somebody...?
zhongmindai on Tue Dec 14 13:12:21 2010 said:
beth on Tue Dec 14 11:14:56 2010 said:
Hi,
I am trying to utilise DMSO in order to perform PCR to detect methylation status (http://jmd.amjpathol.org/cgi/content/abstract/9/5/574). This protocol depends on the fact that methylated and unmethylated DNA show a different sensitivity to the amount of DMSO in the PCR reaction- methylated DNA needs more DMSO in the reaction to show bands.
I have performed a gradient from 56-66oC. (The annealing temp for my primers should be 57-58oC.) At each temperature, I used a master mix with 0%, 2% and 5% DMSO. My hope was to identify a temperature at which the PCR only worked with the addition of DMSO.
However, my experiments are producing very strange results. The addition of DMSO seems to be hindering the PCR reaction. At 56-60oC, the bands with increasing DMSO are fainter. And at 64-66oC, the bands even disappear! I have attached a picture of my results.
I have read numerous posts saying that DMSO really helps annealing and therefore improves specificity and efficiency of PCR. Indeed, the protocol from the paper that I am trying to follow relies on this fact. So why am I getting this result? I really don't understand. Can anyone offer an explanation?
Thanks so much!
DMSO will decrease the melting temperature at about 0.6oC per 1% (v/v). It improves PCR specificity by preventing nonspecific primer-template annealing, as it increase the binding strigency. DMSO improves the efficiency of GC-rich PCR as it helps template denaturation.
It likes that the increased DMSO concentration prevent the primer-template annealing at higher temperature in your experiment.