So the situation is as follows:
I am studying a gene of which a particular isoform has been described that lacks exon 9. I designed primers around this exon so that after PCR reaction I should get two bands: one of more or less 300 nt (which corresponds to the isoform that has exon 9) and on of more or less 200 nt (the exon 9 lacking isoform). When I run the PCR I indeed get two bands of these sizes, so that seems to ok (see attachment for the gel). Although the lower band is faint, which probably means it is lowly expressed, it is not there because of non-specific amplification.
Because when I isolate the smaller (exon 9 lacking) band from gel, clone it into pGEMT and sequence it, I get sequences that are of different length (200-250 nt), but all of these sequences all correspond to the sequence of my gene (so the exon 9 containing isoform), but not the whole exon 9, only part of it. Moreover, I can't find the sequence of my reverse primer in the sequences, which to me indicates that somehow the PCR only amplifies a part of the sequence, without making use of the reverse primer?
I can't get my head around this, because:
All the sequences are of different length and as they all have different endings (GGCA, ATAT, ATCC, CCAG etc.) I don't think that this is a problem of mispriming.
It's not a problem of sequencing, because the sequencing correctly runs into the pGEMT sequence.
Maybe this smaller band is a result of one primer PCR, but I would not expect this much product? Moreover, why would all the products be 200-250 nt long? As I use Taq and an elongation time of half a minute they should be longer?
It could be a cloning artifact, but when I cut out the larger band (so the exon 9 containing isoform) everything is fine: I get exactly the sequence that I expect. So why would there be cloning artifacts with the smaller band, but not with the larger band?
Input would be much appreciated, because I would like to know what this smaller band is: something interesting or not?
I need to know the sequence, because I would like to design specific qPCR primers for this isoform and maybe do 3'-RACE to find out its UTR (that is, if it is real).
What would you guys do in my case?
Thanks in advance,
Arjen
It sounds like degradation but its unusual that it effects only one end of the fragment. Have you ever tried another primer pair? How do you obtain the cDNA, with random hexamers, oligodT or specific primers?
No did not try another primer pair. That was my next plan actually. I obtained the cDNA with a mix of random hexamers and oligoDT primers.
I would try a new primer pair (and perhaps a crossover old-new primers) because the chance that you find an explanation for these fragments is low.
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