I am targeting nuclear genes of vertebrate species. The amplicon size is 300 bp. In my PCRs I get a single band on 2% gel, but in DNA sequencing only the reverse primer generated sequences are clean/good quality. Why this might be happening? How to fix this issue?
Hi Pankaj,
Unfortunately, this problem is not uncommon. Not only do we see this happen with PCR, but also for plasmid sequencing. For plasmid sequencing, it is usually caused when there is a secondary template contaminant containing additional bases on one side of the insert. Therefore, double sequence is only seen in one direction. However, 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 2 % gel may not provide enough resolution to separate them. Can you identify the bases in the unclean direction? Sometimes you can actually see the secondary product matches the first but off by several bases.
Second would be how are you removing the primers before sequencing. If both primers are still present in the sample, the reverse 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 by running a negative control, sequence without adding a primer. For a mismatch primer, you may try extending the length of the primer or selecting a different primer.
Let us know how it goes. Good luck.
May be this helps
http://seqcore.brcf.med.umich.edu/doc/dnaseq/trouble/pcrprimers.html
Hi Pankaj,
if reverse sequencing worked, check if forward primer binding region is correct. Since your amplicon is only 300bp, you should be able to see the sequence where the forward primer is supposed to anneal.
Best Kai
Hi Pankaj,
Unfortunately, this problem is not uncommon. Not only do we see this happen with PCR, but also for plasmid sequencing. For plasmid sequencing, it is usually caused when there is a secondary template contaminant containing additional bases on one side of the insert. Therefore, double sequence is only seen in one direction. However, 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 2 % gel may not provide enough resolution to separate them. Can you identify the bases in the unclean direction? Sometimes you can actually see the secondary product matches the first but off by several bases.
Second would be how are you removing the primers before sequencing. If both primers are still present in the sample, the reverse 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 by running a negative control, sequence without adding a primer. For a mismatch primer, you may try extending the length of the primer or selecting a different primer.
Let us know how it goes. Good luck.
I agree with Kai, and also you can try doing another round of plasmid purification using
NucleoSpin Gel and PCR clea-up kit and give again for sequencing. Let me know if you tried this.
Make a different primer using a slightly different region or adding a few more Ns to the existing primer- the cheapest and quickest solution to your problem.
Good luck!
I had the same problem in my case too. It could be your fwd primer that non specifically binds or may be primer dimer is a huge factor. Try to reduce the primer concentration which is giving trouble.
Make sure that the priming site for reverse primer is present in the template
Is your primer functioning at anneling temperature specified by manufacturer?
Sometimes inefficient primers are ok for PCR but could fail in sequencing.
Also reffer to:
Your PCR primers CAN work very well in sequencing but have some limitiations (http://seqcore.brcf.med.umich.edu/doc/dnaseq/trouble/pcrprimers.html)
Thanks all for various suggestions. As suggested by Doug Binzler, I was getting a faint non-specific band near my expected amplicon. I added DMSO in the PCR and increased the temperature a little bit. This worked well, giving clean sequences generated by fwd and rev primers.
But I have another problem now. I work on animal tissue samples. I wanted to amplify nuclear region. My primers (a pair) for a gene, produced three bands on 2% gel, whereas expected amplicon size was of 380 bp. I did -
1. Increased temperature - problem not solved
2. Added DMSO and BSA in seperate reactions - problem not solved
3. Added DMSO and BSA in seperate reactions and increased temperature - problem not solved.
Any suggestions on this? As these are nuclear primers, they are highly conserved. Average temperature on which the primers work is 56 C.
Maybe your primers are just not specific enough? How did you design the primers? Are you sure they are not in some repetitive sequence?
If the animal you are using already has a complete genome in the databases, you might want to check out Primer-Blast (see link attached). You can check how specific your primers really are....Just add both primers and specify the organism below. You can adjust the stringency criteria as well (how many mismatches etc.). If you add both F and R primers, you don't need to add a target region.
http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome
BSA usually increase unspecificity of the PCR. Try to reduce MgCl2, reduce polymerase amount, and perform gradient PCR for these pair of primer. ( I had recently experience, when with one primer pair lower annealing t works more precisely).
If you haven't done yet, for short fragments use min synthase/ extension time, touch-down pcr can be tried.
of curse, this will not help if the primers are not specific (as Lars-Erik Wehner mentioned). there are cases, when only re-designing of the primers solves problem.
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