We assembled the genome of a new rodent species. We also did gene annotation. There is one gene showing expansion in our rodent genome (7 copies in our genome, 2 copies in mouse and 1 copy in human). Below show our evidence that we consider the gene are very likely under expansion.
Now we want to design primers to experimentally verify those copies and show those gene do express by RT-PCR and translate by western-blat. Since our assembly is new and those copies are highly similar in the coding sequence (>90% identity), I just wonder what is the accurate pipeline to design copy-specific primers?
Evidence for expansion
(1) The coding sequencing of these copies show high identity (>90%) and also show differences among copies.
(2) Different copies, also show different gene structure (e.g., different length of introns between exons).
(3) RNA-seq data support there are different transctipts: the sites showing differences among copies are covered by RNA-seq for each copy.
Thanks in advance!