Detection of specific DNA sequence through PCR

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Sep 27 10:05:36 EST 2001


>We will be amplifying a specific DNA sequence from isolated genomic DNA
>through PCR. Our goal is to determine the presence of that gene in another
>frog specie. That specific DNA sequence is  known to be 120 bp long. If in
>our PCR experiment, we were able to amplify a fragment with nearly the same
>value (120 bp), how sure are we that we have really amplified and detected
>the same gene in another specie?? Thanks
>

I did something similar to what you describe, except in plants.  I used
degenerate primers to a region that is conserved in all known versions of
this gene.  I will warn you that I got several bands close to the expected
size, SEVERAL of which were NOT the gene of interest, as determinded by
sequence analysis of cloned PCR fragments.  I did however also amplify a
fragment that I do believe is the gene of interest.  In the end, the
fragment I pulled out shows reasonable homology to the gene I am interested
in.  Actually, I was relieved that the fragment I amplified and cloned had
significant differences from a known homolog I was using as my positive
control.  I was very worried about amplifying a fragment from contaminating
positive control DNA-- something you might need to worry about also-- so
make sure to do appropriate controls (like each primer alone, no template,
etc.).

I am now going to use my PCR fragment for northern blots, which should
futher confirm the identitiy of the fragment-- i.e. it should hybridize to
an appropriately sized transcript.

Hope this helps.  Good luck.

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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