I amplified my gene of interest via PCR, and had it sequenced. Surprisingly, there is a single base deletion within the sequence. I'm pretty sure it shouldn't be there, at least based on the current bacterial strain's sequence database. So, I'm wondering if PCR has something to do with it. That being said, the chromatogram doesn't show any suspicious peaks or background/noise around that particular region of the sequence. Heck, the peaks are quite defined.
What kind of DNA polymerase did you use in PCR amplification? You should use a high-fidelity DNA polymerase. You can sequence both directions to double-check again.
Normal Taq lacks proof reading activity hence, there are chances of deletion as well as mutation.
Hello Avin,
Although it is rare, but I have seen that too. I even found once a mutation in the primer used for amplification. I agree, it depends on the polymerase too, the high fidelity ones do not give any problem. So, either sequence from both the directions to cross-check or take another clone.
Best,
Simran
Sometimes there are mistakes in the basecalling despite appearing a peak of good quality. I once sent a single sample for sequencing at two different universities and met with surprise that a sequence, in spite of being of good quality, there was an error in a nucleotide. I detected this error thanks to the database. My recommendation is that send to sequence the strands F and R, (x2) and thus you are assured with high probability.
Hi Avin,
As other's have mentioned, this is definitely a possibility, as Taq itself doesn't have a proofreading mechanism. You can repeat the sequencing, or perhaps use a higher fidelity Taq such as Platinum Taq.
If this is in a highly repetitive region, even higher fidelity taqs are going to have a hard time going through it (i.e Poly-T regions).
If it were me, I would try the sequencing again from a couple other clones, and see if you get the same thing.
Hi Avin,
Also you have to be aware that the deposited DNA sequence (in the database) is not always 100% correct. It happened to me on the bar gene. One of the deposited sequence source was not correct for a few DNA bases. I had to search and use two more sequence data deposited in other databases for comparison, and found out that the sequence from the very first one was not correct. Luckily, I could find 3 bar gene sequence data from different databases for comparison. This is just another possibilities for your question.
Best,
Hi Alwin,
To answer your question better, please clarify the following,
1. What kind of DNA polymerase did you use? High fidelity ones?
2. Did you send the PCR product for nucleotide sequencing? Or you performed TA cloning and sequence the plasmid?
After a more careful observation, it would seem that there is an unregistered peak that would otherwise, be registered as my missing DNA base (an adenine). I attached a picture for reference.
The colors are not bright in the picture attached. But if I read the sequencing chromatogram right, then yes, an "A" is not read by the program, but if you see the peak, it is there. That means, your deletion is actually a fault in the reading and not a real deletion. Go ahead with your clone, if rest of the sequence is perfect. Or once again sequence the same clone, from the other direction.
As my supervisor once told me, always check the chromatogram and never believe in the edited sequence, I pass on this piece of wisdom to you.
Cheers :)
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