primer design for unknown cDNA sequence - (Dec/03/2007 )
I would like to amplify a cDNA for squirrel monkey, however, the sequence is not available at ncbi. There is human sequence as well as mouse, and a "predicted" sequence for macaca old world monkey.
I am new to PCR (primer design) and I am wondering if there is a program that can align human, mouse, and macaca, and help me pick out primers (in regions that are conserved) based on the cdna. I figure that if its found in human and mice then the probability of it being in squirrel monkey is high as well.
Currently what I do is very tedious: I align this myself using SeqMan and find stretches of DNA about (18-25) which are conserved, cross my fingers, and find a subsequent stretch of DNA (<200 mer) from here until I see another 18-25 mer which are conserved. I then paste this onto invitrogen primer finder, and hope that the flanking (18-25 mer) would have compatible primer sets.
There must be an easier more efficient way to do this. Can anyone advice? Thank you in advance.
Muscle is a good DNA alignment program. With a only a single defined primer, you can amplify the cDNA by ligating adapters on the 3' end of the DNA strand (the 5' end of the mRNA) with T4 RNA ligase, and then use the (complement of) the adapter sequence as the other primer. This also has the advantage of getting the entire 5' end of the mRNA strand, and locating the transcription start. The adapters are 5' phosphorylated and usually blocked on the 3' end to avoid self-ligation.