I am doing a little research on the methylation status of human gene NR3C1 . And I found it is difficult to design the BSP primers of its 1C promoter. There are too many CpG sites located in that region.
In paper” Loss of Glucocorticoid Receptor Expression by DNA Methylation Prevents Glucocorticoid Induced Apoptosis in Human Small Cell Lung Cancer Cells”,the authors solved the problem by using degenerate bases in BSP primer sequences.
reverse- 5′- AGGTGAATTCACACRAACTCRCAAAATAAAAAAAA-3′”
But will it cause the ratio of Methylated copies/Unmethylated copies altered?
Yes, of course. Sometimes there is not CpG free region to design BSP primers, you can incorporate one CpG C in the primer (better let the C sit in the 5' end of the primer) by using a degenerate base at the C location.