I was wondering if anyone out there can answer these questions for me.
I am currently analysing silencing in my transgene promoter in rice. Methylation restriciton digests suggest that my promoter (553bp) is methylated and so is the UBI enchancer gene (1050bp) that follows the promoter (the construct is - Promoter-Enhancer-Transgene-Terminator). We would like to carry out some Bisulfite sequencing to confrim that they are methylated and I am currently designing BS Primers. According to MethPrimer I may have a CpG Island (411bp) that spans the end of my promoter and into the beginning of my enchancer gene. So my questions are:
1) Should I be interested in seqencing only the overlapping CpG Island? As I understand it only methylation of the Islands has biological significance, or should I be trying to amplify the entire promoter.
2) If I bisulfite sequence the CpG Island should I amplify only a region that is within the promoter? Most of the programs that generate BSP only give me primers for the region of CpG Island within the enchancer gene.
Apologies for the very basic questions but I've very new to epigenetics and no-one in my lab studies methylation.
Thanks in advance