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Subject: Re: PCR Primers
From: "Thomas M. Grothues" <[log in to unmask]>
Reply-To:Scientific forum on fish and fisheries <[log in to unmask]>
Date:Thu, 8 Feb 2001 10:19:08 -0500

text/plain (70 lines)

There are some genomic primers that one might expect to be nearly
universal, at least in regard to fishes (or "almost" fishes :-)) because
they bracket genomic regions coding for such critical and highly conserved
cellular machinery as ribosomes. Despite that they are themselves
conservative, they may bracket regions that are not. As an example,  the
internal transcribed spacer (ITS) region of the ribosomal gene is not
translated into protein, it merely sits between (spaces) two regions coding
for ribosomal subunits (See Hillis et al. 1996). Primers that contain
translated subunit sequences should work for a wide variety of organisms,
while the amplified spacer region may  be highly variable depending on the
species and it's  natural history. Primers for the ITS regions are
commercially available, and I don't think that this is the only example
from genomic DNA.

As for the tenmer primers: In a RAPD (or AP-PCR) study you want the primer
annealing to be somewhat sloppy so that you can examine more regions. If
you are considering such a marker, don't worry unduly that you don't know
where the primed region is; if it is the same in other specimens, it should
yield similar bands. For reason of achieving this "sloppiness", the primer
will be shorter than that which you might use when targeting a specific
genomic. Of course, even a short primer for targeted region will have high
fidelity under high annealing temperatures, so it could be appropriate
where a known conserved region suitable for priming is short.

I hope this helped. Feel free to contact me off-list for clarification.

Myke Bon wrote:

> Greetings Fish People,
> I'm doing research on Lamprey in Alaska and at the point now that I
> need to perform PCR on the DNA I do have for L. tridentada.  The
> problem I have is that I need to determine a primer to use for the
> process.  I've been to the Gene Bank though all information there is
> mitochondrial; I'm working with genomic DNA.
> We've been told it would be ideal to use a 10mer primer for
> polymorphism research though most literature I've found suggests
> between 18-29 for PCR.  So my question is as follows:
> 1)Can someone suggest how I can determine a primer for a species that I
> dont know the Genomic Map on?  We have access to mitochondrial map of
> L. fluviatis, It has been recommended we use it for a guideline to work
> with L. tridentada, though we can't use it for genomic work I think.
> Is there a universal primer (such as 'alu')that can work?  Any
> suggestions will be recieved with great enthusiasm.
> Thanks in advance!
> Myke Bon
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Thomas M. Grothues
Rutgers University Marine Field Station
C/O 132 Great Bay Blvd.
Tuckerton, NJ 08087
(609) 296-5260 x262
[log in to unmask]

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