[Zbrafish] Sequence homology for protein expression - help!

dykvar from yahoo.com via zbrafish%40net.bio.net (by dykvar from yahoo.com)
Fri Apr 18 13:34:55 EST 2008


Hi,
I have a general question regarding rescue and protein expression via
construct in ZF. To do this one must first clone the protein mRNA
sequence from cDNA, then insert into an expression vector (I’m using
PCS2+). However, whenever I clone and then sequence I never get 100%
homology when blasting on NCBI. Although I use high-fidelity (Roche)
enzyme, I get about 4-5 nucleotides different (out of ~900) and 2-3 aa
different from what is in the database.
Is this normal? Is it a result of polymorphism? Or is it a problem
with my methodology? And what degree of homology is needed (assuming
no gaps exist) for the protein being expressed properly?
Your help is appreciated!!



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