Direct BSP using tag-modified primers - (Jul/10/2006 )
I was hoping someone out there might be able to help me clear up a point of confusion. I'm trying out a new published method for direct bisulfite sequencing published by:
Spivak SD, DNA methylation mapping by tag-modified bisulfite genomic sequencing, Analytical Biochemistry June 2 2006
But I'm running into a point of confusion and I was hoping someone here could clear it up. Unless I'm completely missing something, in the paper they show the sequencing primers that they used (both forward and reverse) but the reverse primer is not reverse complementary to the 3' G-tag. Is that a typo? I tried contacting the corresponding author, but haven't gotten a result yet.
This new method of BSP primer design is based on the observation that in a given sample, if it is methylated, direct sequencing yields better result than for samples less methylated. By adding C/G tag to PCR primers is a clever idea and will certainly improve the readability of sequencing chromatograph.
I don't quite understand your question here. Why the reverse primer has to be complementary to the 3' G-tag? The G-tag is just an artificial sequence added to the 5' end of the reverse primer.
Can you elaborate more on your question?
Thanks for the response PCRman, but I just had a misunderstanding where I thought the tags were both placed on the same strand.