Cloning DNA from protein sequence

Mr. M.J. Lush mlush at hgmp.mrc.ac.uk
Wed Oct 23 09:48:13 EST 2002


In article <3DB64D28.3A87B76F at abdn.ac.uk>,
Bertrand Collet  <b.collet at abdn.ac.uk> wrote:
>Hi There,
>
>Somebody asked me for advice in our department and I was unable to give
>a good reply:
>How easy is it to isolate the gene coding for a protein of known
>sequence ? I have to add that it's in an insect species where the codon
>usage is virtually unknown.

	My advice would be 'degenerate PCR don't spend more than
two years trying to get it to work (it may well be quicker to wait for the
species to be sequenced!)'.  I had a bad experence with the technique 
working from limited highly degenerate sequences of unknown relative 
position.

	Could you get related sequence off out of the databases and use it
to do a library screen?

	The answer really depends on the quality of the known sequence
to make degen PCR work you really need to know the relative position of the
sequences you have to double the number of reactions you need to do
This does not sound too bad untill when you start dealing with 3+ sequence
an therefore 6++ primers,  things get a bit grim.
 
	If you don't know the distance between the primers (4kDa ~100pb)
you have to investiage ever random band you seen on you gel!
I recall the max published range on degenerate PCR is about 1-2kbp 
(probably much less!)

	Any sort of nested amplification is a good thing.

	Tis worth a try but don't peg any grants on getting it to work!


>Second question:
>-If the above is feasable, what is the best way to design degenerate
>primers ? Is the Inosine backtranslation the best method when you don't
>have any idea of the codon-usage ? Does anybody has a good experience
>with degenerate primers with inosine ?
>-If we require specific primers using say the drosophila codon usage,
>where can I find (Web ?) the codon usage information (per species) ?

	Try everything,  preferably at the same time (saves time getting
all your dead ends in one go:-{


I saw this on bionet.molbio.methds-reagnts
/start quote/
From: czhu at changbioscience.com (Chang Zhu)
Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts
Subject: Degenerate PCR primer design software
Date: 19 Oct 2002 18:18:13 -0700

Primo Degenerate designs PCR primers based on a single protein
sequence
or multiple alignments of proteins or DNAs. The melting temperature
may be calculated using the nearest neighbor model, which
automatically corrects for the effective primer concentration for a
degenerate primer. The online version
is available at 

http://www.changbioscience.com/primo/primod.html

/end/

-- 

Michael
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
NPC rights activist  |  Nameless Abominations are people too.



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