Questions'''agree: green disagree: red

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Questions...agree greendisagree red

• Proteomics is the large-scale identification and
  cataloging of proteins from a particular
  organism.
• MALDI-TOF stands for matrix assisted laser
  desorbtion ionization - time of flight.
• PAGE refers to the separation of proteins through
  agarose gels.
• IEF is the first step of 2D-PAGE and is based on
  a pH gradient
• In situ hybridization gives a better
  representation of gene expression than northern
  blot analyses
• Immunolocalization is to western blotting what in
  situ hybridization is to northern blotting.
• The use of a secondary antibody in western
  analyses increases sensitivity and decreases
  cost.
• GFP is the protein that allows fireflies to glow
  in the dark.
• The expression of reporter genes may be affected
  by position effects.

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• This is the last class!!
• What you should have learned (or need to learn in
  the next week)
• gene structure, transcription, translation,
  global mechanisms controlling gene expression.
• gene cloning strategies.
• targeted vs. random approaches.
• when to use which technique
• uses and limitations of various techniques
  (information, effort, cost).
• Transformation and genetic engineering
• cloning vectors
• transformation techniques
• sense and antisense down-regulation of genes
• ethical concerns
• identifying gene function
• BLAST searches

Final exam Dec. 12, 7-9 pm, BCHM 102 60 pts for
1st half of the class 60 points for 2nd half up
to Exam II 30 pts for recent topics
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This is a short movie to illustrate the use of
reporter genes. This movie and similar movies can
be found at the following web site
http//www.scripps.edu/cb/sullivan/research.htm
(Dr. Sullivans lab at the Scripps Research
Institute in la Jolla, CA). In this movie we are
looking at centromere dynamics during mitosis.
The centromeres are being visualized by a
centromere DNA binding protein that is fused to
the green fluorescent protein (from jelly fish).
The GFP is being visualized through excitation
with UV light. In this case the reporter gene is
used to gain knowledge about cell division.
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Inverse PCR Inverse PCR is used to generate
unknown DNA flanking a known sequence. Because
you dont know the sequence of the unknown DNA it
is impossible to design PCR primers. When we do
inverse PCR we use primers based on the known
sequence, but the primers face opposite
directions. So we have a piece of dsDNA that
contains a known sequence (such as T-DNA, a
transposon or a partially sequenced gene or cDNA)
flanked by unknown sequence. We circularize the
DNA by diluting it in an aqueous solution that
also contains DNA ligase. The dilution is
necessary to maximize the probability of the two
ends of the DNA molecule bumping into each
other. We want intramolecular events as opposed
to intermolecular events!The DNA ligase ligates
the two ends together to form a circular DNA
molecule. With the two primers specific for
the known sequence we can perform PCR which
results in the amplification of the flanking
sequence.
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Metabolic profiling Metabolic profiling, also
called metabolomics, is a high throughput method
to characterize many metabolites in (tissues or
organs of) an organism. The analysis is
typically performed using gas chromatography or
liquid chromatography followed by mass
spectrometry for identification of compounds.
This is abbreviated as GC-MS or LC-MS. You end up
with detailed chemical profiles of your samples
and very large datasets. The datasets are then
subjected to statistical analyses to identify
(sets of) compounds that differ significantly
between samples. So you end up being able to
distinguish your samples based on chemical
differences. What is the rationale for doing
this? The classic genetics paradigm states that
the gene encodes a protein and that the protein
affects a trait. The protein affects the trait
directly or through the chemical conversion of a
metabolite. So by looking at the metabolites we
are as close to the trait as possible. Once we
know which metabolites are important determinants
of the trait, we can (1) specifically monitor
that metabolite while developing new germplasm
optimized for our trait and (2) define genes that
affect that metabolite and use targeted genetic
strategies to affect the trait. DNA/mRNA Genomics
(sequencing projects, EST databases) Protein Pr
oteomics (protein databases) Metabolites Metabol
omics Trait/Phenotype
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We can also use metabolic profiling in
combination with genomics approaches to infer
gene function. For example, metabolic profiling
can be used to evaluate the effects of genetic
modifications as a result of mutations or genetic
engineering. It is typically not very hard to
identify (small) molecules. So when the sequence
of a gene of interest does not enable the
prediction of the gene function (because of lack
of homology with known sequences in the
databases), it may be helpful to at least
identify the chemical changes that occur when the
gene is mutated. Metabolic profiling is a
technique that is still under development, but
likely to become quite important. The
equipment Gas chromatograph this is an oven
that contains a 20-60 meter long capillary column
(wound into a coil). The internal diameter is
0.2-0.5 mm. The column contains a thin film of a
resin inside. The column is connected to a
pressurized tank of an inert gas (usually helium)
that creates a flow of gas through the column.
The sample (containing a mixture of compounds) is
injected at the top of the column when the
temperature is low (typically between 40 and 80
C). The temperature is then raised and the
different compounds are separated based on their
volatility and their affinity for the column. The
retention time is the time it takes a compound to
elute off of the column. Mass spectrometer the
traditional (not TOF) mass spectrometer ionizes
the sample (in this case an individual compound)
with the use of electrons (electron impact
ionization). The resulting ions are then
accelerated in an electric field and then
subjected to a magnetic field that causes the
ions to deviate from a straight course. The
deviation is a function of the mass-to-charge
(m/z) ratio. Many of the ions fragment upon
electron impact. Between the m/z ratio of the
molecular ion (unfragmented) and the
fragmentation pattern that is observed it is
possible to determine the identity of the
compound that eluted off of the GC column.
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A neat web site with an animated overview of
GC-MS can be found at http//www.shsu.edu/7Echm
_tgc/sounds/pushmovies/GC-MS.gif
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The data
chromatogram
Retention time
mass spectrum
m/z
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Data analysis discriminant analysis (as an
example)
Chemical profiles were determined for ten maize
lines with different feed quality (low, medium
and high quality as determined by feeding
trials). The chemical profiles can be used to
classify the lines in the different quality
groups. This helps understand the chemical basis
for feed quality. Discriminant analysis is a
statistical technique that is very useful for the
analysis of large or complex datasets.