BSP question - (Jul/16/2006 )
I've seen it mentioned in multiple posts that the reverse primer seems to work best for direct sequencing reactions, but not the forward. Is there anyone that could explain why? I've tried to search for some information on the topic but am coming up empty. Is this an artifact which is BSP specific? or is it known to occur in normal sequencing too? (i'm just starting with sequencing at the moment so I don't know a whole heck of a lot about it.)
So basically would you recommend that in my primer mix that I send out with my template I should just use the reverse? The reason I ask is because I tested a number of sequencing primer sets on my bisulfite treated template and although the technician told me that my data had a very high signal (saturated) and had to be adjusted, which she attributed to high sequencing efficacy, I ended up with no usable sequencing data using a mix of forward and reverse primers. This was with 33 ng/ul of template and 5 uM primer mix (standard for our sequencing facility). And I know the primers can bind to my template because I tried normal PCR amplification and all the reactions resulted in clean bands. So now I'm not sure if its an issue on my end or something that has to be adjusted on the sequencing end. The worst part about this is that the technician/facility managers have never had any experience with BSP reactions. Any ideas?
in my hands, it seems to be the case that the reverse primer invariably always works if at all. I have not seen anything in the literature about it, but I have heard from others that this is usually the case in most respects. There are some people on this forum who haven't had such a problem, it could be a hemisphere issue
I would say that it maybe due to the base composition of the primer, the forward primers are rich in A,T and G and are very C-poor, conversely the reverse primer is A,T and C rich and very G poor. This would give rise to extension products with similar base compositions.
Now I recall that within the sequencing premix, there are a mixture of dideoxy-nucleotides of which, some are analogues to the conventional bases, I think (I maybe incorrect) that dideoxyinosine is used in place of dideoxyguanosine as it incorporates better during the reaction. this is probably the reason behind why only the reverse primer works as the resulting product doesn't incorporate as many inosine molecules than the forward one does.
i couldnt say why either, it's just what i have witnessed.
for sequencing, i've given up on forward, and just go with reverse.