Hi everyone
I sent the PCR products for sequencing (forward and reverse primers separately). The result of the Sanger sequencing was so sharp with high quality for forward sequence but I don't know what happened for reverse sequence. That was completely disappointing. What can be the cause of failed DNA sequencing by reverse primer?
The PCR products were purified by purification kit protocol, the 260/280 and 260/230 ratios were 1.8 and 1.25 respectively and the NA concentration was 50.4 ng/ul. 15 ul cocktails (12ul PCR product +3 ul of each primer) is prepared for sequencing.
The chromatogram of forward and reverse (reverse complement) sequences have been attached.
Thank you in advanced
can you attach the .abi files forward and reverse please as they contain much more information and also what method of primer removal did you use . Ideally also the sequence of the forward primer. Without any evidence at all you can get this effect with an insertion/deletion in the sequence but this can only be tested with the sequencing files
Unfortunately, this problem is not uncommon. There could be a number of reasons this occurs in PCR.
The number one suspect would be that you are generating a second PCR product very close in size to the first product. A 1-2% gel may not provide enough resolution to separate them.
Second would be how are you removing the primers before sequencing. If both primers are still present in the sample, the forward primer could dominate the reaction and the reason it appears clean.
Third would be that the forward primer mismatches the priming site as previously suggested.
How to fix?
Perhaps try testing with a 4 % gel for better resolution. It still may not solve the problem but is a good first step. Be certain you are removing the primers from the template before sequencing. For a mismatch primer, you may try extending the length of the primer or selecting a different primer.
Which is your goal? Understanding the cause of poor sequencing? Or reading the sequence of your PCR product?
I guess the second is your goal.
Then you should design a few more primers for the reverse strand.
The good forward strand read suggest your DNA samples are fine.
Unfortunately, this is uncommon, as mentioned above the one thing that you pay attention to primer dimers only. I don't know you have problem with one primer set or any other too. If you have problem with one primer then I could suggest you to design new primer if necessary otherwise you can go with single sequencing.
It look to me that the sequencing reaction stopped when Polymerase extent to DNA region of single or di-nucleotide repeat (ex. AAAAAAAAAAA, or ATATATATAT). If repeat is short, you can use 5% DMSO in sequencing reaction, optionally increase annealing temp. If repeat longer, you have to rely on forward direction for cover the missing region by designing additional sequencing primers.
Best, ^^
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