[Zbrafish] Re:Sequence homology for protein expression - help!

[Joanna Wilson]

Hi there,
I am totally unsurprised that you are finding sequence differences  
with the gene you cloned and what is in NCBI.  We see this in the  
genes we clone in the lab - one to 10 nucleotide sequence differences  
(our genes are 1500 bp) with aa changes.  You can see this if you pull  
out sequence information from NCBI for which there are multiple  
entries - they will not be completely identical.  Some of this is  
strain differences.  It doesn't mean it won't be expressed.  You need  
to be sure that the differences are real (sequence several clones so  
that you can show it isn't a PCR or sequencing error) and that it will  
translate in frame without stops.
Joanna Wilson

Re:
Message: 1
Date: Fri, 18 Apr 2008 11:34:55 -0700 (PDT)
From: dykvar from yahoo.com
Subject: [Zbrafish] Sequence homology for protein expression - help!
To: bionet-organisms-zebrafish from moderators.isc.org
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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!!


Dr. Joanna Wilson
Assistant Professor
Department of Biology
McMaster University

1280 Main Street West
Hamilton ON
L8S 4K1

Tel: 905-525-9140 ext 20075
Fax: 905-522-6066
joanna.wilson from mcmaster.ca



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