I found a pair of primers of my target gene in references, which is Staphylococcus-specific, as
F 5’-GGCCGTGTTGAACGTGGTCAAATCA-3’
R 5’-TIACCATTTCAGTACCTTCTGGTAA-3’
I want to verify its specificity, but there is an "I" in the reverse primer, and the NCBI blast will directly ignore the "I" to find the matched sequence.
I am confused how can I blast such a primer sequence?
(The target gene of this primer varied in some bases in the target Staphylococcus genus, that maybe why there should be an I in this primer.)
Thank you so much!
If you are familiar with Linux, I highly recommend using the multiPrime script available on GitHub (https://github.com/joybio/multiPrime). In particular, the primer_specificity.py and primer_coverage_validation_by_BWT.py tools can be used to evaluate the specificity of a primer pair. Good luck!
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