1. After bisulphite treatment, the strands are no longer complementary and strand specific primers can be designed. This allows amplification, cloning + sequencing on the individual strands to determine their methylation patterns.
Q: Is there any need to study the antisense strand? Can variable methylation of this strand affect expression?
2. Bisulphite sequencing PCRs are described in the literature as “generally shorter than normal PCRs” (ambiguous statement or what?). Programs such as MethPrimer are set to default size of 200bp optimal, 300bp max.
Q: Why short PCRs?
Q: I have 620 bp CpG island to study, could I do it in one big
PCR, or do I have to design 3 sets of primers and do it in 200bp chunks to get the full picture?
3. I’m also trying to PCR across the CpG island of native DNA (not bisulphite treated), using primers either side of it and am having a hell of a job. Is there evidence to suggest difficulty amplifying through CpG islands (I have 100 CpG in 620bp).
Thanks in advance for any help/derision!
Edited by Chris Harris, 29 June 2004 - 07:09 AM.