two bands of one gene in PCR - (Mar/21/2006 )
hi, i'm a beginner in molecular biology and am stuck with the interpretetion of PCR results.. I keep getting two bands (both sequenced and seem to be what I wanted them to be.. just have no idea why they are different size!!??), from the literature I learnt that the gene (exon1(non-coding) - intron -exon2(coding))contains two promoters (in exon1 and intron).. can it be any reason for that?? I can't really get it...
many thanks for any help 
HI
I think you should tell us exactly what you want to amplify (cDNA or genomic DNA) and where your Primers are located. Maybe you could draw a simple scheme.
bye
Morrison
sometimes, the RT-PCR products of the same gene might be difference in length: one of them is shorter than another due to alternative splicing...
one reference
http://www.sciencemag.org/cgi/reprint/302/5653/2141.pdf
Hi,
perhaps two (polymorphic) allels of a diploid organism?
many thanks for any help
Was one band weaker than the other?
It may because the efficiency of the two primers differ. One strain was produced faster than the other, forming triple helix or something else.
I think you should tell us exactly what you want to amplify (cDNA or genomic DNA) and where your Primers are located. Maybe you could draw a simple scheme.
bye
Morrison
good point! thanks
I want to amplify cDNA. The gene consists of:
noncoding exon 1 - intron - coding exon 2.
my primers are within exon 2..
I don't think those two bands are due to different splicing considering the short length of the gene (unless process similar to splicing can occur within one exon (?)) also the strength of the bands between each other doesn't differ that much (rarely I get only one random band respective to the gene but this is not regular)
many thanks for help!
Jaga
Anything can be the case, including alternative splicing in a small gene.
But before you mentioned that you had sequenced the fragments and that they are the same. Surely you did not sequence the entire fragments for they would have a difference at some point.
You could opt for cloning both in a vector, check -by restriction digest- that you have a clone of each and then sequence both entirely.
Who knows it might turnout to be interesting...
Maybe you found a new splice variant or a pseudogene. I also would suggest that you clone your PCR Products and sequence several clones. Sequencing the PCR product directly I would not recommend, because gel separation sometimes isn´t good enough. You could sequence artefacts.
Morrison
Thanks a lot for the suggestions! I might do the clonign then (I havent' done it yet so also nice new thg to learn).. seems a very good idea!
an interesting thg that came up in the results of sequencing both bands is that the sequences part of the bigger band actually consists within the sequence of the smaller band.. (gene has only one coding exon..) have you come across such thg? is it possible at all that a process similar to splicing happens within one exon?? thanks in advance!
and again many thanks fro your help