Primer design for unknown gene - (Mar/10/2005 )
I'm not sure if this is stupid question, but please forgive me because I'm new in this area.
Okey, problem is this:
I'm trying to figure out unknown gene sequence. I have used the DNA sequence of similar species to design my primers as close as I can, to macth the sequence that I'm looking for. The idea have been to determine each exon of the gene separately from genetic DNA and when doing so locate the primers in introns.
My question is,
how far from the begining (or from the end) of the exon primer can locate?I was told that maybe the 20bp distance between primer and exon would be okey, but how much farther off they can be?
So, have anyone or everyone done something similar in the past or now, and what are your experiences?
enthusiastic but frustrated beginner
maybe I didn't understand well.
Why do you want to find primers into introns so far from the exon you want to analyze?
Introns are so different between species that the probability to find righ primers is very low! Why you don't try first to find primers into exons?
Are you sure that there's nothing in the database of the sequence you are looking for? Have you tried first to make a blast with the known sequence of other species? You could find fragment of this sequence that can help you to design your primers.
and thanks for your answer! Yes, I think you understood right and you are right. I could of designed my primer inside the exon, but we just desided to try this intron primer idea. Like that we got the whole exon with one PCR. I have to say, that with all my other exons it has worked, and only the last one has caused sleepless nights.
Anyway, then about my sequence, I'm positive (as sure as I can be) that there isn't any parts of my sequence in any database. I have allso tried blast, but then again I haven't used blast that much that I could say, if there is some feature, that could help me more. Maybe you could help me with that? Untill now I have used sequences of two species that are really similar with mine and played with those to solve my problems and design primers...
Okey, but I have to say if this doesn't work, I should try primers inside exons. Problem with that is that I don't have anyone anymore to explain, how does that work. What I mean is that, how do I get then begining and the end of the exon? I have a feeling, that it's an old and wellknown method/approach, but could you or anyone tell me from where I can find material to study it?
all the best
i want to knoe how does blast help us in this matter
actually I think that you could try the exon-primer research in the same way you approached the intron primer research. Or you can try to use similarity with the other two species to find the beginning and the end of your last exon. and design a primer that could be located between end of intron and beginning of exon .
End of intron= first bases of your primer
Beginning of exon= last bases of your primer
The most important part of your primer is the end because if there's a mismatch there, polymerase can't attach!
p.s. maybe this is not what you mean but is also not easy to understand your problem and give you more information on blast features w/o the knowledge of your sequence.
Hope this can help