Polymerase Chain Reaction of Agrobacterium tumefaciens
raum at bio104.alleg.edu
Mon Feb 12 15:38:12 EST 1996
Unlike every other known dicot, the aquatic plant Hippuris
vulgaris does not form a gall (tumor) when it has been inoculated with the
plant bacteria, Agrobacterium tumefaciens. Therefore, to prove that
Hippusis vulgaris has actually been infected (since no visible gall
exits), I am trying to analyze its genomic DNA to see if it contains the
T-DNA region (the transferred DNA region) of Agrobacterium tumefaciens.
[The T-DNA is the portion of the Agrobacterium tumefaciens plasmid which
incorporates itself into the plant's DNA following inoculation.] To detect
this T-DNA region in the infected plant's DNA, I am using the technique
known as Polymerase CHain Reaction (PCR).
I am using PCR to amplify a small region of the infected plants'
DNA using primers that ONLY match up with the T-DNA (transferred) region
of Agrobacterium tumefaciens. Therefore, amplication should occurr ONLY if
the T-DNA region is present (no amplification should occur if it is
absent) So, IN THEORY, this should be an excellent way to determine
whether or not Hippuris vulgaris has actually been infected by
Agrobacterium tumefaciens-- based on the presence or absence of the T-DNA.
HOWEVER, I am having some trouble carrying out this procedure. For
some reason, following PCR (amplification), most of my gel lanes contain
long "fuzzy" streaks instead of nice sharp bands (which would represent
the small region of the T-DNA which was amplified ). I assume this might
be due to non-specific binding of the primers, but I'm not sure.
If anyone has any experience running PCR with plants or has some
knowledge of molecular techniques; please write back with some advice!!!!
p.s. I am writing this from someone else's account so please email your
responses to: cunnink at alleg.edu
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