I have sequences of the Cytochrome c oxidase I (COI) from several populations of Peringia ulvae (Mollusca; Gastropoda; Littorinimorpha; Hydrobiidae). I need additional COI sequences from a specific population. When using the same primers and PCR protocol as for the other populations, I do not get any PCR product for the specific population. My PI suggested me to design internal COI primers for Peringia ulvae. How can I do that? Should I use the COI sequences from the other populations?

  • $\begingroup$ You could try simply ordering a degenerate version of the primers you have. If possible, try aligning the sequences for many known COI alleles from your organism and see which bases in particular are variant. If the sequence is conserved, keep that base, if it is variable, add a degenerate/random nucleotide in to your primer order. The mix of primers you get back will then have every possible variant for a given position, and that might help you to get amplification. $\endgroup$
    – Joe Healey
    Jan 9, 2017 at 13:45
  • $\begingroup$ Try to lower the annealing temperature of your PCR. If there are few mismatches between the DNA sequences of the different populations you may be able to amplify them all with the same primer set by making your annealing less stringent. One question, are the primer annealing inside the coding sequence of COI or on the flanking region? $\endgroup$
    – alec_djinn
    Jan 9, 2017 at 14:06
  • $\begingroup$ @alec_djinn How can I know where the primers are annealing? $\endgroup$
    – jvddorpe
    Jan 12, 2017 at 10:42
  • $\begingroup$ @JustineVandendorpe do you know the sequence of the primers? If so, then just use BLAST to understand where they anneal. $\endgroup$
    – alec_djinn
    Jan 12, 2017 at 15:17
  • $\begingroup$ @alec_djinn I know the sequences and ran a BLAST. Thanks a lot! $\endgroup$
    – jvddorpe
    Jan 13, 2017 at 12:33

1 Answer 1


If you are not getting amplification, there are multiple things to visit before you want to design internal primers. You should try troubleshooting your PCR reactions before designing and ordering internal primers. If you are doing the PCRs wrong, it is likely you will run into the same problems again with internal primers.

There are excellent guides to trouble shooting PCRs - Just a google search "Trouble-shooting PCRs" should give you a lot of material to go through. This is from NEB, but there are several others, each manufacturer having their own, so you could refer to one of whose reagents you are using.

Primers generally give good reads from the 5' end for about 600-700 bp and after that the quality of base calling deteriorates, so if your target amplicon is above 1500bp or so, you would need internal primers because the 'external' primers would give you good sequences for only 1400 bp or so (600-700 bp from both directions, for fwd and rev primers). So internal primers can be used to sequence the regions that cannot be reached by the main (external) forward and reverse primers. The published papers from where you are using the external primers will most likely have the internal primers if CO1 is long enough to require internal primers.

Otherwise, you would need to align the existing sequences from the other populations, and look through regions of the alignment that are conserved which could act as potential priming sites. There are other issues to be taken care of before you can consider a 'conserved region' a 'priming site' and go ahead and order primers. Again there are excellent primer design tutorials available, just a google search away.

Let me know if any parts of the answer are not clear, or sound like jargon and I will do my best to simplify it for you. However, I would strongly suggest troubleshooting the PCR first - for e.g. trying a gradient PCR to find the optimal annealing temperature.

  • $\begingroup$ Thank you very much for your answer. I am also using the reagents from NEB so I had a look at their Tm Calculator. With the parameters I entered, I got a warning message saying: "Tm difference is greater than the recommended limit of 5 °C." Do you know how can I solve that? $\endgroup$
    – jvddorpe
    Jan 12, 2017 at 9:40
  • $\begingroup$ Hi, what is the annealing temperature that you are using in your PCR and what is the Tm for your primers? Also, do you have something like Nanodrop readings of your DNA samples? Do you know their concentration? If the concentrations are too high/too low, PCRs will be inhibited. $\endgroup$ Jan 12, 2017 at 19:59
  • $\begingroup$ I am using an annealing temperature of 52 °C. The Tm suggested for my forward and reverse primers are 49 °C and 59 °C, respectively. The DNA concentration of my samples ranges from 0 to 870 ng/µl. $\endgroup$
    – jvddorpe
    Jan 13, 2017 at 12:39
  • $\begingroup$ 870 is WAY too high. Generally 25-100ng of DNA is recommended in the reaction mixture. $\endgroup$ Jan 13, 2017 at 14:43

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