How do you ensure that the region you select on the mRNA transcript does not include introns; as this would not allow correct annealing of the primer?
Have mRNA for a gene of a closely related species. But need to design primer for DNA for the same gene in different species.
If you don't have information about the exon-exon juntion in your mRNA it hard to determine were intron could be in the genome. It is a unknow gene in other species??
On the other side, if you can't find information on pubmed about your gene in other species its gonna be hard even to know the sequence to use for the primers design.
yes you can - but - but if you suspect splicing look at specific structures
Hello Asif
Based on the percentage homology of the gene, you can design you oligos based on the gene sequence from the most closely related species. or you can align the gene sequences from all the close relatives and design degenerate primers. in both the cases you can try optimizing the annealing temperature by setting up gradient PCR. In the close relatives try to map exon-exon junctions, that can throw some hint on intron locations
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