Bisulfite Primers- "Top" and "Bottom" Strand
Posted 17 September 2012 - 10:40 AM
As I'm reviewing the literature, I've seen that some papers refer to primer sets designed specificially for either the bottom strand or the top strand. This may have a simple explanation that I am missing, but I am still having a difficult time grasping this... When we design primers for normal PCR, we pick a primer from the top strand and a primer from the reverse strand. How would we pick primers from just one strand if the sequences are the same, but just the reverse complement of each other? I appreciate any insight you can provide!
Posted 17 September 2012 - 08:02 PM
Posted 18 September 2012 - 09:53 AM
Edited by prabhubct, 18 September 2012 - 09:58 AM.
Posted 19 September 2012 - 08:19 AM
Hansen, R. S. "X Inactivation-specific Methylation of LINE-1 Elements by DNMT3B: Implications for the Lyon Repeat Hypothesis." Human Molecular Genetics 12.19 (2003): 2559-567.
Posted 05 November 2012 - 05:11 AM
I mean, I usually use just the sense strand sequence as the design template and use something like Primer3 to pick the primers. If I now use the bisulfate-converted sense sequence, I will obviously get a reverse (antisense) primer that is fully complementary to the sense strand, and a forward (sense) primer that is NOT complementary to the antisense strand because of the bisulfate conversion. So during the first round of the PCR, only the sense strand will be amplified. And after that, the forward primer will bind to the new products and the rest is exponential amplification of the original sense strand. Is this what the strand specificity means?
I just don't see what else could I do. I mean, if I did design the primers fitting to both of the converted strands, they would amplify the original templates all right, but not the products, so the amplification would be only linear.