I am trying to do Real time PCR for a particular species of earthworm, checking the Genome database of NCBI, I didn't find it there. I am using set of primers from a similar species but I am having difficulties. Could it be that the Primers do not work for each other or they are not the problem
That sounds like it's likely the problem. Many genes have slightly different sequences between close organisms. Here's what I would do:
- Get the sequence of your gene from all the Eartworm's close relatives that are available in the database
- Align them with Clustal and optionally a visualization program like Jalview
- You will see that some regions of the gene are conserved between all the species, and some are variable. That principle will likely carry over to your unknown species: Design your primers against conserved regions of the known species, and it will be more likely to work on the unknown species as well.
- If there isn't enough conservation, you can look at "degenerate primers", which are mixtures covering all the possibilities for each base in any one position.
I don't think it is a problem provided the sequences that the primers bind are identical!
That sounds like it's likely the problem. Many genes have slightly different sequences between close organisms. Here's what I would do:
- Get the sequence of your gene from all the Eartworm's close relatives that are available in the database
- Align them with Clustal and optionally a visualization program like Jalview
- You will see that some regions of the gene are conserved between all the species, and some are variable. That principle will likely carry over to your unknown species: Design your primers against conserved regions of the known species, and it will be more likely to work on the unknown species as well.
- If there isn't enough conservation, you can look at "degenerate primers", which are mixtures covering all the possibilities for each base in any one position.
Dear
Sometimes Universal primers are used but intra-species or genera.
I agree with Mr. Schloendorn that you might try degenerate primers, at least in order to get the sequence of interest. Then you should sequenciate the amplicon and create specific primers against your species' sequence. As far as I know, you should avoid using degenerate primers for real time PCR, since it is likely that you amplificate more than one product, rendering the real time analysis useless.
As Christoph wrote, you could try to sequence your fragment with the primers you have. Then design your species-specific primers using that sequence (e.g. internal to the original primers). Use online-tools to check annealing temperature (should be similar) and primer-dimer formation (must be avoided).
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