Plant gene isolation - query

Alan Smith alansmith at students.wisc.edu
Fri Jun 8 09:47:00 EST 2001


In my opinion the best way to clone genes is using a race type procedure
when you are trying for a gene that is already cloned in plants.  Degenerate
PCR can make you go crazy because of double priming and false positives.  It
is almost like magic when degenerate PCR works IMO.  If you are good with a
race procedure you might not even need to screen a library unless you are
after the genomic sequence.  It is easy to "walk" using race, just don't be
scared to spend money on primers with race or dPCR.  A good library is a
blessing, but many times libraries wont give the full clone if they are home
made. RACE=fast cloning, libraries=slow going. I would try an RT-PCR
approach first and resort to libraries latter if it cant be cloned by race.
The only problem with a race procedure is that the gene needs to be
expressed in the tissue you extracting from (this can be complicated for
weird genes with isoforms).  Happy hunting.

Alan Smith


-----Original Message-----
From: owner-methods at hgmp.mrc.ac.uk
[mailto:owner-methods at hgmp.mrc.ac.uk]On Behalf Of juamber
Sent: Friday, June 08, 2001 8:41 AM
To: methods at hgmp.mrc.ac.uk
Subject: Plant gene isolation - query


I would appreciate to have your opinion
in the following approach for plant gene isolation.
What are the possibilities of success?
Where are the "dark points" explaining why this estrategy is never used?

Assuming there are no introns in the gene (and perhaps this is the biggest
"dark point") the proposed method would include:

1. Use degenerate primers from conserved domains of known protein sequences
to
amplify DNA sequence/s from genomic DNA.

2. Use the PCR product/s as probe/s in Southern analysis to estimate copy
numbers and restrict future work

3. Use the PCR product/s as probe/s in Northern analysis of RNA extracted
from a certain tissue to confirm the expression of the amplified sequences

4. Sequence the products and compare them in databases to confirm homology
with
heterologous proteins

5. Use these amplified sequences to design new primers for genome walking to
retrieve the 3´- and 5´-ends.

Why the standard procedure is to start from RNA and then by RT-PCR generate
a
cDNA sequence that is later on used for screening a library?

Thank you very much for your comments.
Juamber

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