inner primer for BSP PCR? - (Dec/05/2005 )
Hello, I stumbled over that forum here while searching for some informations about methylation testing in DNA - I'm absolutelly new to that topic and nobody in reach here who would know about it ... 
So far I found some very nice protocolls for the PCR conditions and bisulfite treatment here, thanks very much for those sharing their experience. 
But now I'm still lost about the design of the inner primer.
I made a primer pair that includes the whole gene and promotor I need to test (which maybe is already with around 1.3kb way too long?). Those are around 30Bp each and have no CpG included but similar melting temps. So far so good?
Now why do I need a second primer pair inside for a nested PCR?
And since I can't for god's sake find any internal primer inside the gene for a nested version without a CpG how I do that? Is one internal primer enough and the both outer just run towards that one in the second PCR?
I hope my question hasn't already covered, dug through pubmed and the forum for a good while now but with all that new stuff I start to get confused. 
generally speaking, two rounds of PCR are required for BSP to obtain a good yielding amplicon.
As repeating two rounds of PCR with the same primer set has it's problems (if the first round has mis-primed and is amplifying aberant amplicons, this will only get worse in the second round), that is why hemi or fully nested PCR strategy is employed, this also increases specificity.
I have to say 1.3kb is rather large but having said this, it is not impossible, I have managed to amplyify a 1.2kb fragment from BSP with a little optimisation.
Can you design a primer that sits just outside of your current primer set, i can understand why you can not design one within the CpG island as it is full of CpG's but there is no reason for you not to pick one outside of your current set.
good luck
Nick
I strongly discurage you from trying such long PCR. Why cannot you find inner primers on your sequence, too CpG-rich? If CpG sites are unavoidable, you can try degenerate primers.
Hi there and thanks very much for the response so far.
I sat down quite some hours yesterday and finally found one spot a bit in the middle of the sequence with no CpG inside and 27Bp long that would with the outer primer pair give me a 460 Bp fragment and a 860Bp second fragment. So would that be short enough?
So you recommend I run the first PCR with the first primer pair at the outer ends A and B and than have the middle one C with A or in opposite direction with B again, right?
The aberrant amplicons I would get from the changed sequence after the bisulfite treatment?
Thanks again for your efforts.
the smaller fragments you would get from your newly picked primer will be better as pcrman rightly pointed out.
the first PCR reaction will be with your "outer" pair while the second reaction will be with an outer primer (if it is a forward primer) and an inner one (will be reverse).
good luck!! If you would like to post your sequence to the forum, I would be happy to have a look at it for you
Nick
the first PCR reaction will be with your "outer" pair while the second reaction will be with an outer primer (if it is a forward primer) and an inner one (will be reverse).
good luck!! If you would like to post your sequence to the forum, I would be happy to have a look at it for you
Nick
Hi and update on this.
I finally got to this thing back after a pretty busy time with other stuff.
You were right, the Primer for 1.3Kb fragment gave no amplicon at all. I found one other place in the sequence that should give with a nested approach around 500 and around 860Bp long fragments - and yay, I have a band of this size on my gel (plus a few more bands though, but that is to be expected from the conversion, right? I have only the 500 and 860Bp Band with unconverted material).
Running now the final stuff for the gel for extraction, crossing my fingers that it still looks good after sequencing ...
Btw: I used the Genetic signatures Kit MethylEasy for the conversion, as it is not needing a column but works with precepitation of the material. Used amount of DNA was 50ng.
If you are going to do direct sequencing, be sure to recover DNA from a clear and strong band, and send your samples to a service provider that can do a good job sequencing such product. Use both upstream and downstream primer for sequencing. Downstream primer always yields better result.
Good luck!