I did a PCR experiment and sequenced the product. The result is very surprising.
I had it sequenced in both directions with forward and reverse primer respectively and aligned them together and the structure of the product looks like this. The bold parts indicate sequence in accord to the primers. I aligned the two parts hanging outward from each other and they don’t match.
R --------------------------
F -------------------------------
I have also attached the seq information here so take a look if you are interested.
How could this happen? Thanks!
Dear Eve, if you read Sanger sequencing by dideoxy chemistry then you will realize that only a single strand DNA synthesis occurs that too of various length but after PCR when the ssDNA of different length are resolved in the capillary the you get the sequence due to dyes attached with each nucleotide added at last (A, T, C & G). The smaller fragments accumulate more (from the reactions terminated very early) so due to crowding they are not resolved and you won't get the sequence near the primer that you used for amplification/sequencing PCR. In your case also you haven't got it but you got that particular stretch from the opposite primer or vice-verse. For better understanding plz see the slide attached. I hope it will help. Get it sequenced three times from each primer and make consensus sequence or eliminate errors by reading peaks in chromatogram.
Thank you so much Kamal! You are real expert! I understand what you are saying but I still have a little doubt.. We have been working with this sequencing company for quite a while and usually the first 10 to 20 nt are off the mark but this time there's a ~45 nt missing from the beginning...and the template for this PCR reaction is RTed from miRNA with stem-loop primer, this is the first time I'm dealing with it...does this have anything to do with the stem-loop partial structure of the template? Thanks again, just need one more confirmation!
1. The sequence you posted-- is the size (225 bp) a correct size?
2. If you got 'noisy' reading results (such contains NNNNNNN.....), at the end of both sequences, then when you assemble these two sequences with a program, the noisy ends will be cut off, so you would get the sequencing pattern you posted above. Is the '(outward) not-matched part' good reading or noisy reading?
3. Another thing is-- are those primers unique? Have you BLASTed your primers to see whether or not it/they may bind to other places (which may contains a short identical sequence)?
The missing of several bases at the beginning near a primer is common. This should not cause a big problem if you sequence both directions of your DNA. Using your diagram above as an example: generally speaking, the 'not-readable' bases (at the beginning) near the primer F can be revealed by the sequencing result from using the primer R, especially when your fragment is cloned in a plasmid.
However, your problem is that the ends of your sequences are missing/or not matched from both sequencing (if the 'red'-color sequences are the 2 primers in your attached file).
Hi, Dr. Yau, thanks for your attention!
1. i didn't count but 225 is overlapping+hanging-out together.
2. i read the chromatogram myself nt by nt and already removed the NNNNN at the end and disordered part at the beginning (~20nt) So all these nt shown are good reading, no noise reading.
3. I recovered this product from agrose gel and the size on gel looks like 150 or so. I blasted the primers and found no match in human rna database. the primers are designed to bind with the adapter part of RT primer (stem-loop primer for specific miRNA).
Thanks!
Dear Liu,
Few points i want to make clear:
1). You have fragment size which is very small and in this case you do not have to do sequencing from both side. Either Forward or reverse primer based sequencing can give you upto 600-800bp length nt sequence accurately.
2). If you are going to do sequencing of a PCR amplified product then PCR must be carried out using high fidelity DNA polymerase. If you have used normal Taq-polymerase then there are chances of wrong bases but rarely.
3). In every sequencing product from start to a few nt length the nt bases does not come accurately because of overlapping peaks. If you wand to avoid of this then first you should clone the pcr amplified into a cloning vector and then do sequencing using primers which are designed on flanking region of insert. Such primers are always 100-300bp (upto your choice) away from insert and during sequencing the overlapping peaks will come in this region and your nt sequence of actual fragment will be safe from such noise.
good luck
The number of missing nucleotides will depend upon the template amount used for PCR before capillary resolution. May be this time they used a bit more template. Some sequences near the primer you can check by chromatogam. I don't think the structure has anything to do with it.
Put out chromatograms of Sanger sequencing for everybody to see! The data you are sharing are secondary, one needs primary data to be able to help you without guessing games. I suspect it will resolve all the problems. Most likely, as some other researcher already noticed, you have bad sequencing results, which means, literally, you CAN NOT compare sequences and make any conclusions based on these data.
Hi, I think Olga´s suggestion are crucial. We need the rawdata to help you Eve.
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