drmax at casbah.acns.nwu.edu
Mon Dec 19 10:49:38 EST 1994
Go for degenerate primers (all possible codon usages for a
given amino acid) and try to pick regions where you think there will be strong
selection for conservation of amino acids. This works best of course if you
have some idea about the function of the protein. Are there related genes
where there are examples from multiple species? If so go for areas that are
conserved between the genes. Once you have even a short streach of the human
gene go to a library screen, you can waste an awful lot of time trying to PCR
a full length gene and even if you get the full length gene by PCR, some
reviewer out there will want to see a library clone anyway.
As an alternative to PCR, why not try a low stringency screen of a human
library (cDNA if you have it or genomic if neccessary) using your bovine
In article <9412182005.AA13786 at siumed.edu>,
Carl Lawyer <clawyer at SIUMED.EDU> wrote:
>Dear PCR netters,
>We have sequenced a bovine gene and are now trying to get the same in
>human. The problem is we found the bovine sequence came out without a
>single nt change when we did the human. This was cross-contamination,
>because we had worked with bovine cells in the same room.
>When redone without cross-contamination, we get nothing.
>Any tips on better cross-species primer design? Are there any good
>references on this?
>Codon degeneracy, cross-species homolgous amino acid substitutions, and
>intra species and intragene codon bias are all problems.
>Any practical solutions? (aside from determining the amino acid sequence of
>the human protein, a big chore)
>Thanks for any help.
> Carl Lawyer M.D., Director of Asthma Center, clawyer at siumed.edu
> Southern Illinois University School of Medicine
> Division of Pulmonary Medicine
> PO Box 19230
> 751 North Rutledge
> Springfield, IL 62794-9230 Fax 217-788-5543
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