Multiple bands after bisulfite PCR - (Jul/07/2010 )
I have recently started studying DNA methylation. I have used the Qiagen EpiTech kit for bisulfite conversion.
I start with 500ng to 1ug Genomic DNA. For bisulfite PCR analysis I have used primers designed by the Methyl Express
software amplfying around 400 bp of my gene of interest. I do two rounds of PCR and have longer annealing and extension times.
While primer optimisation I found out that 51C is the best Tm that I could use for my primers. I get two bands one at 400bp (desired)
and one at 150bp (undesired) with the smaller one being stronger. Due to this after gel purification of my fragment (400bp) my yield is very
low, just 4ng/ul!!! I cannot use it for direct sequencing noe cloning due to this!!! Please help!!
I have tried changing the primers but I get multiple bands repeatedly. Following is my PCR protocol that I got from this forum.
I use the promega Go taq enzyme mix for amplification.
95°C 4min (denaturation)
95°C 30 sec
52°C 90 sec (annealing. Temperature is set according to calculated Tm from primer express. 2°C lower than the lowest Tm primer, round one PCR the primer was 54°C, round 2 48°C).
72° 120 sec (extension time to ensure no PCR recombination)
× 5 cycles.
95°C 30 sec
52°C 90 sec
72°C 90 sec (ex tension time is still longer than usual as the product is about 300 bases.)
× 25 cycles.
72°C 4 minutes
Welcome to this forum pcrgirl!
You did not mention what PCR you are doing--MSP or BSP.
your cycling time for all steps is unnecessarily long because your amplicon size is only 400 bp.
Try the following cycling conditions:
94C - 10''
55C - 20"
72C - 20"
x 35 cycles
If you are doing BSP, reamplify the first round PCR product for another 25-30 cycles if you do not get anything from the first round.
Thank you for your prompt reply!!
I am doing BSP.. tried amplifying with the following conditions but it did not work-
no of cycles=35
if the tm is raised then the non specific band increases and my specific band ie at 400 bp decreases, almost vanishes..
I already do two rounds of pcr..
pcrgirl, are you trying to amplify a repetitive region or a gene who has many family members (ie: pseudogenes or orthologues with similar sequences)? this is probably why you are getting non-specific bands.
Another trick to try is a nested pcr approach where you design internal primers to your 400bp amplicon of interest and PCR a 2nd time with nested primers, this increases specificity.
I dont think the primers are amplfying anything other than my own gene, because when I gel purify the
spc fragment and reamplify it I get multiple bands again!!! It seems my primers might be binding
elsewhere in the same fragment and amplfying a part of it!!!
Btw will reamplification of the pcr product erase the methylation signature?
Nested PCR may help.
By bisulfite conversion the methylation pattern is sort of transformed into a sequence change. So firts round PCR and also reamplifiying does not erase the methylation pattern. Maybe you can sequence your unspecific product to get an idea of what it is.