Bisulfite PCR: Smear - Can't amplify a certain sequence (Apr/28/2004 )
I'm having some problems with bisulfite pcr. The template dna was treated with bisulfite, and I've amplified 10 products using it successfully.
One product has been giving me serious problems. I've designed primers of varying lengths (20-25 bp) for amplicons that range in size from 200-800 bp. I've tried modifying primer concentration (200nM - 1000 nM)... Nested PCR reactions, Mg2+ optimization, touchdown, reverse primer alone for 10 cycles, HotStart, etc. However, for the life of me, all I get is a smear.
Are there regions in the genome which are just not amplifiable via bisulfite? Any other tips?
Is your PCR methylation-specific PCR (MSP) or genomic sequencing PCR?
What is your PCR condition?
Knowing that will help me understand your problem.
It is for genomic sequencing.
My primer sequences are about 23 bp and have an annealing temp of 54 deg C.
I've tried annealing temps of 46,50,54 by 40 cycles.
I've also tried touchdown (decr. by 2 deg every 2 cycles, then after 10 deg drop, x 30 cycles) from 62 -> 52, 60 -> 50, 56 -> 46.
I always get a smear or sometimes I get a smear with a couple of sharp bands that do not correspond to my product (low and higher weight bands).
One thought that crossed my mind is that the primers need to be longer. I read that about 30bp is norm for bisulfite. It's odd since I've amplified 10 different products (and sequenced) using primers that were 23 bp. Just this one locus is giving me a hassle!
A few suggestions:
1) Try designing a new set of primers around 25nt
2) Try Sigma JumpStart RedTag, you will see a huge difference
3) 54C Ta should be OK.
4) Increase cycles to 45 (but you already got smear, this may not work).
Thanks! Will give it a go.