Primer Design..need very basic advice. - Please help, I cant figure this out for the life of me. (Nov/01/2010 )
Hi, after reading through all the posts related on primer design, or even remotely related on primer design, I'm still very confused! I don't know what it is..and my PI thinks I'm dumb or something when I keep telling her I need to design the manually...she thinks its only MSP when you design manually and not for BSP..how do I explain this to her?
Can someone please take me through a "fake" example on how to do this manually? And how do you design the PCR cycling based on what primers you have?
--Indebted to those who reply
so I just ran two gels: One w/ the Bisulfite Modified DNA, and one gel with Unmodified DNA...I ended saw bands for the Unmodified (using Primers from the MethPrimer (designed for Bisulfite Sequencing), but I did not see any bands for the Bisulfite Modified DNA (using the same primers from MethPrimer (designed for Bisulfite Sequencing)....
Why is this happening?
You begin with two strands of DNA, strand A and strand B. After conversion, your DNA strands are no longer complimentary. Therefore, your PCR is only targetting one DNA strand. Lets call this 'strand A'. Your PCR will target strand A and make complimentary copies of this to yield a double stranded DNA product, comprising of strand A and a newly synthesised strand, which we shall call strand C.
Therefore, to quantitate the methylation on strand A, the forward sequence will show C/T peaks at the C position of CG dinucleotides. In the opposite direction, the sequence will show G/A peaks in the G position of CG dinucleotides. This is because you are only looking at the methylation in the C position of the original A strand.I think this is where you are getting confused - the C position in the reverse strand is always going to show C as it corresponds to the original G. It is the G position in the reverse which needs to be measured, as this is the position which corresonds to the C in the orignal A strand.
Confused about the Bolded part? Can someone explain in further details...thank you.