Detection of specific DNA sequence through PCR

Michael L. Sullivan mlsulliv at
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,

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.


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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