Trends in Biotechnology
110512 3d Reporter Genes
Reporter Genes.
A. Connected to the gene of interest
and used to indicate if the desired DNA
is expressed.
B. Allows researchers to measure how
well a gene is expressed.
Reporter Genes
A reporter gene is connected to the gene
of interest and used to indicate if the
desired DNA is expressed.
It allows researchers to measure how well
a gene is expressed.
Here are some examples:
Reporter Genes
β-glucuronidase gene (GUS) - an
enzyme that breaks down chemicals
called β-D-glucuronides. Can produce
a blue or fluorescent color.
Reporter Genes
Luciferase gene—found in firefly and the
bacteria Vibrio harveyi, the gene
produces light in response to the
molecules luciferin and ATP.
Fig. 3.19 A
tobacco plant
transformed
with the firefly
luciferase
gene.
Reporter Gene
Green fluorescent protein (GFP) produced by the jellyfish Aequorea
victoria and interacts with the protein
aequorin to produce fluorescence.
The GFP gene can be fused with another
gene, allowing GFP to indicate the
production of the desired protein.
The jellyfish
Aequorea victoria
showing the
fluorescence, and
a model of the
protein.
The green fluorescent protein (GFP) can
be expressed in mammals.
Baby monkeys which have been engineered to
express GFP.
GFP has
been
mutated
to
produce
different
colors.
GFP has been engineered (amino acid
substitutions etc) to produce many
colors.
It has also been used to engineer brain
cells to show different expressions.
In this Harvard University photograph released October 8th, 2008, brain cells of a laboratory mouse are shown
glowing with multicolor fluorescent proteins at Harvard University in Cambridge, Mass. The Nobel prize in
chemistry was awarded to two Americans and a U.S.-based Japanese scientist for research on a glowing
jellyfish protein that revolutionized the ability to study disease and normal development in living organisms.
(AP Photo/Harvard University, Livett-Weissman-Sanes-Lichtman)
Video: Brainbow
http://www.youtube.com/watch?v=y51la5
gS5Ws
Southern Blot Hybridization.
Another way to see whether a particular
DNA is present was invented by
Edward Southern in the mid-1970s.
How to do it (Figures 3.20):
1. DNA fragments in a gel are denatured
by alkaline buffer.
2. DNA fragments are transferred to a
nylon or nitrocellulose membrane.
3. DNA probes are hybridized to the
membrane, and the membrane will be
exposed to show a band representing
where the probe successfully hybridized.
Fig. 3.20 The steps involved in conduction a Southern blot hybridization.
Southern
blotting and
hybridization
with probes
can be used
to locate a few
specific
fragments in a
large pool of
DNA.
Sensitivity of Southern blots have been
increased by:
1.Increased activity of labeled probes.
2.Development of nonradioactive (color,
fluorescent, or light-producing) probes.
3.Treatments to prevent nonspecific binding of
probes to membranes.
4.Use of detectors (phosphoimagers) to obtain
images of hybridization signals.
5.Use of vacuum blotting or electroblotting to
reduce the DNA transfer time.
6.New methods of DNA transfer, such as
downward capillary transfer.
Fig. 3.21 Southern blot capillary DNA transfer. (a) Downward capillary transfer.
Fig. 3.21 (b) Bidirectional capillary transfer.
Southern blot hybridization can be used to
detect banding pattern produced by
restriction enzymes, called “restriction
fragment length polymorphisms”
(RFLPs).
Restriction Fragment Length
Polymorphism
Video:
http://highered.mcgrawhill.com/sites/0072437316/student_view
0/chapter16/animations.html#
Southern blot hybridization is different
from colony hybridization in the following
ways:
1. Blotted DNA can be cloned or not
cloned, and does not have to be in
vectors.
2. DNA is separated on an agarose gel
and transferred to the membrane, and
bacterial colonies do not need to be
lysed to release DNA.
Northern Blot Hybridization.
A. RNA is transferred from a gel to a
membrane, and DNA probes are used.
B. Used to measure the quantity and
determine the size of transcribed RNAs.