Why primers for bisulfite converted DNA amplify unconverted DNA? - (Jan/26/2006 )

Hello All! I'm looking at DNA methylation in brain tissue. To evaluate the sodium bisulfite treatment efficiency, I designed unconverted and converted primers for the sequence of interest. If the treatment converts unmethylated Cs to Ts ~100% of the time, then the unconverted primers (without Cs converted to Ts) shoul not amplify treated DNA. For the most part this is true. The bizarre thing is that the converted primers amplified the UNTREATED DNA. Ruling out contamination as a cause, does anyone know why converted primers would anneal to DNA that was not even treated with SB?? I am quite puzzled by this and welcome any suggestions. Many thanks!

mispriming of these primers could cause the amplification, are you sure the primers are amplifying the region of interest or somewhere else in the genome?

N

Hi Cristi,
I had the same problem when I started with my bisulfite sequencing experiments. I solved it by redesigning my primers so that the 3' ends of the primers were one or two T which in original (not converted DNA) corresponded to non-CpG cytosins. I thought that even if the primer would anneal to not converted sequences, the polymerase will not start the elongation because it needs a perfect match on the 3' end of the primer. It worked very well - in about 100 clones I sequenced in those experiments I never found an unconverted one.
Good luck
Gosia