Transcript Reverse Transcription PCR (RT-PCR)

Gene Regulation: What it is,
and how to detect it
By Jordan, Jennifer, and Brian
What is the Promoter Region
of a Gene?
•Upstream region of a gene,
which regulates when/where
the gene is to be transcribed.
• Essentially the promoter
region acts as the “on
switch” for the gene.
How Can the Gene Promoter
Region be Located and Isolated?
Location:
Isolation
• The vital region of the
promoter is somewhere
upstream from the gene of
interest, and downstream from
the “stop” codon directly before
the gene of interest.
• Knowing that the region of
interest is somewhere between
these two points is all that is
necessary!
• If the entire genome of the
organisms has been
sequenced (as is the case with
arabidopsis), then the exact
nucleotide sequences of these
areas is known.
• From the sequence
information primers can be
designed and the gene
promoter fragment can be
amplified by the polymerase
chain reaction (PCR).
How can an isolated promoter region
show where a gene is active?
G.F.P. (Green Fluorescent Protein)
• The GFP is a protein derived from the jellyfish •
Aequorea victoria (actual protein name is
aequiron), which fluoresces green when exposed
to blue light.
• Because other factors are not required for
fluorescence, GFP expression can be used to
monitor gene expression in living organisms.
•
•
A previously amplified promoter
region (as described in the slide
prior) can be fused to a GFP reporter
gene.
The fused region can then be
transformed back into the original
organism’s genomic DNA.
Observing where the green
fluorescence is present will visualize
where the promoter region is active
in the original organisms.
Isolate RNA from Plant
Silique
Inflorescence
Leaf
mRNA
mRNA
mRNA
Synthesize cDNA Using RT
mRNA
Reverse Transcriptase
cDNA
Amplify cDNA Using Primers
FW
RV
Polymerase Chain
Reaction
Visualize with Gel Electrophoresis
mRNA
Present
mRNA
Absent
Positive
Control
Negative
Control
Presence of mRNA
• Starting point for determining expression
• Expression of gene determined by
observing proteins
• Can only determine transcription
GeneChip Analysis
• Using cDNA created from the
mRNA isolated from various
organs, we can analyze the
mRNA accumulation levels for
all genes.
• This is done by creating the
complementary strands of the
known gene sequences and
assembling them on a chip.
• The sequences are tagged
with flourescent tags that glow
a certain color when in contact
with the complementary
strand.
This is what you get as a resulting
image:
Red means active
Green means not
as active
No color means no
activity
We can then translate the data into
graphs like
This is an
example of
GeneChip data
accumulated for
one gene across
many organs of
the plant