Hi everyone,
I am having troubles with proofing the results I obtained in Sanger sequencing by other methods.
At the beginning of my PhD I isolated RNA of human cancer cells, transcribed it into cDNA and amplified my gene of interest. I cloned it into the TOPO vector and transformed everything into TG1 cells. the DNA of the different clones was then checked by Sanger and I came up with several different versions of the protein. I repeated this quite often, I sequenced forward and reverse, I sequenced the PCR product directly and I used different polymerases and I always found these versions. I was also able to proof their existence with qRT-PCR and on protein level ( western+MALDI).
I then isolated DNA and came up with WT sequences. So we assumed that alternative splicing must take place.
My problem however is that so far any other sequencing method can' t repeat these results. I tried pyrosequencing, which didin ' t work for somehow the primers couldn't bind or for any other reason. Anyway pyrosequencing didn't give me sequences not even WT. I tried to use LNAs but I couldn't get the LNAs to differ between WT and "mutated" version. I tried digital droplet PCR which didn't work either. In the end we also performed RNA seq. The site of interest was only mapped by 3% however no mutated form was found.
We assume that the site of interest has a quite difficult struture and maybe hard to access but still this is really confusing. We already talked to several splicing experts however so far nobody has explanation for what mechanism might take place.
Is anyone having ideas how to explain these controversing data and how to proof the existence by any other method apart from Sanger? ??????????????
I am having troubles with proofing the results I obtained in Sanger sequencing by other methods.
At the beginning of my PhD I isolated RNA of human cancer cells, transcribed it into cDNA and amplified my gene of interest. I cloned it into the TOPO vector and transformed everything into TG1 cells. the DNA of the different clones was then checked by Sanger and I came up with several different versions of the protein. I repeated this quite often, I sequenced forward and reverse, I sequenced the PCR product directly and I used different polymerases and I always found these versions. I was also able to proof their existence with qRT-PCR and on protein level ( western+MALDI).
I then isolated DNA and came up with WT sequences. So we assumed that alternative splicing must take place.
My problem however is that so far any other sequencing method can' t repeat these results. I tried pyrosequencing, which didin ' t work for somehow the primers couldn't bind or for any other reason. Anyway pyrosequencing didn't give me sequences not even WT. I tried to use LNAs but I couldn't get the LNAs to differ between WT and "mutated" version. I tried digital droplet PCR which didn't work either. In the end we also performed RNA seq. The site of interest was only mapped by 3% however no mutated form was found.
We assume that the site of interest has a quite difficult struture and maybe hard to access but still this is really confusing. We already talked to several splicing experts however so far nobody has explanation for what mechanism might take place.
Is anyone having ideas how to explain these controversing data and how to proof the existence by any other method apart from Sanger? ??????????????