Fw: Plant gene isolation - query

Deanne Bell dbell at qnis.net
Tue Jun 12 10:51:25 EST 2001


Hi all

I am trying to follow this cloning stuff, but I am afraid I am missing some
basic vocabulary, can someone refer me to a basic, descriptive article
where I could learn such things as "race procedures", "walk", and the pros
and cons of degenerative PCR?

Thanks ahead of time
D.Bell

----------
: From: "Alan Smith" <alansmith at students.wisc.edu>
: To: methods at hgmp.mrc.ac.uk
: Subject: RE: Plant gene isolation - query
: Date: Friday, June 08, 2001 7:47 AM
: 
: 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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