Transcript 7/7 - Utexas

Today:
Biotechnology
•Exam #2
W 7/9 in class
Comparative genomics also has been used to
identify recently mobilized transposons in
genetically diverse humans. For example, over 600
recent transposon insertions were identified by
examining DNA resequencing traces from 36
genetically diverse humans.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Tbl 1
Which transposable elements are active in the human genome? (2007) Ryan E. Mills et al. Trends in
Genetics 23: 183-191
DNA fingerprinting using RFLPs
Visualizing differences in DNA sequence
by using restriction enzymes
Sequence 1
Sequence 2
Fig 18.1
Restriction Enzymes cut DNA at specific sequences
tbl 18.3
Examples of some restriction enzymes…
Recognition
Enzyme Sequence
EcoRI 5'GAATTC
3'CTTAAG
BamHI 5'GGATCC
3'CCTAGG
HindIII 5'AAGCTT
3'TTCGAA
TaqI
5'TCGA
3'AGCT
AluI
5'AGCT
3'TCGA
Cut
5'---G AATTC---3'
3'---CTTAA G---5'
5'---G GATCC---3'
3'---CCTAG G---5'
5'---A AGCTT---3'
3'---TTCGA A---5'
5'---T CGA---3'
3'---AGC T---5'
5'---AG CT---3'
3'---TC GA---5'
Fig 20.5+.6
Visualizing differences in DNA sequence
by using restriction enzymes
Sequence 1
Sequence 2
Separating DNA on a gel by size
Fig 20.6
• Gel electrophoresis
Fig 24.21
Visualizing differences in DNA sequence by RFLPs
Fig 22.23
DNA fingerprinting
DNA fingerprinting
DNA fingerprinting
Can DNA be obtained from hair?
How can DNA be obtained from
such a small sample?
The inventor of PCR
Fig 18.6
Polymerase
Chain Reaction:
amplifying DNA
Polymerase Chain
Reaction
Fig 18.6
Fig 18.6
Polymerase
Chain Reaction:
Primers allow
specific regions
to be amplified.
The inventor of PCR
PCR animation http://www.dnalc.org/ddnalc/resources/pcr.html
Areas of DNA from very small samples can be
amplified by PCR, and then cut with
restriction enzymes for RFLP analysis.
Genetic Engineering: Direct manipulation of DNA
Fig 18.2
Bacteria can be modified or serve as intermediates
Fig 18.2
a typical bacteria
Bacterial DNA
plasmid DNA
tbl 18.2
A typical
bacterial plasmid
used for genetic
engineering
Fig 18.2
Moving a gene into bacteria via a plasmid
What problems exist for expressing eukaryotic
gene in bacteria?
Bacterial DNA
plasmid DNA
Fig 18.4
Reverse
transcriptase
can be used to
obtain coding
regions without
introns.
Fig 18.6
After RT, PCR will
amplify the gene or DNA
Fig 18.2
Moving a gene into bacteria via a plasmid
RT and PCR
Fig 18.1
Restriction Enzymes cut DNA at specific sequences
Fig 18.1
Restriction enzymes cut DNA at a specific
sequence
Fig 18.1
Cutting the
plasmid and insert
with the same
restriction enzyme
makes matching
sticky ends
A typical
bacterial plasmid
used for genetic
engineering
Using sticky ends to add DNA to a bacterial plasmid
Fig 18.1
If the same
restriction enzyme
is used for both
sides, the plasmid
is likely to religate
to itself.
Fig 18.1
The plasmid is
treated with
phosphatase to
remove the 5’-P,
preventing selfligation
Fig 18.1
Transformation of bacteria can happen via
several different methods.
tbl 6.1
Bacteria can take up DNA from the environment
Fig 9.2
Transformation of bacteria can happen via
several different methods all involving
perturbing the bacterial membrane:
Tbl 6.1
•Electroporation
•Heat shock
•Osmotic Stress
Fig 18.1
How can you know which bacteria have been
transformed, and whether they have the insert?
Resistance genes allow
bacteria with the
plasmid to be selected.
Bacteria with the resistance
gene will survive when
grown in the presence of
antibiotic
Fig 18.1
Fig 20.5
Is the insert present?
Plasmids with the MCS
in the lacZ gene can be
used for blue/white
screening…
A typical
bacterial plasmid
used for genetic
engineering
Intact lacZ makes a
blue color when
expressed and provided
X-galactose
When the lacZ gene is
disrupted, the bacteria
appear white
Blue/white
screening:
Fig 18.1
Transformed
bacteria plated on
antibiotic and Xgal plates.
Each colony
represents millions
of clones of one
transformed cell.
Fig 18.1
Successful transformation
will grow a colony of
genetically modified
bacteria
RT and/or
PCR
Fig 18.1
Inserting a gene into a
bacterial plasmid
Millions of Hectares
Bacteria can be used to transform plants
Global area planted
with GM crops
http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html
Texas =
70 ha
Agrobacterium infect plants, inserting their
plasmid DNA into the plants genome. Fig 19.15b
Agrobacterium infect plants, inserting their
plasmid DNA into the plants genome.
Fig 19.15
By replacing the gall forming genes with other
DNA when the Agrobacterium infect a plant, it
will insert that DNA into the plant.
Fig 19.16
The generation of a transgenic plant
Grown on herbicide
Fig 19.16
*Not covered in class; will NOT be on the exam; check it out if
you are interested.*
How do you know whether the gene you want
to express has the correct sequence?
DNA sequencing
*Not covered in class; will NOT be on the exam; check it out if
you are interested.*
• The structure of 2’,3’-dideoxynucleotides
Fig 18.14
Fig
18.15
*Not covered in class; will NOT be on the exam; check
it out if you are interested.*
The dideoxy
sequencing method
*Not covered in class; will NOT be on the exam; check it
out if you are interested.*
The dideoxy
sequencing
method
Fig
18.15
*Not covered in class; will NOT
be on the exam; check it out if
you are interested.*
Gel produced by the
dideoxy sequencing
method
*Not covered in class; will NOT be on the exam; check it out if
you are interested.*
Computerized sequencers use a similar method
Fig
18.16
…now enjoy
making
Frakencritters.
What is the largest scale experiment that
is being currently performed?
Genetically
Modified
Organisms
Human resource production and use
Worldwide Grain Production
per Person
http://www.earth-policy.org/Updates/2008/Update69.htm
World Grain Stocks as Days of Consumption
http://www.earth-policy.org/Updates/2008/Update69.htm
~60 percent of the world grain harvest is consumed as food,
~36 percent as feed, and ~3 percent as fuel. While the use of
grain for food and feed grows by roughly 1 percent per year,
that used for fuel is growing by over 20 percent per year.
http://www.earth-policy.org/Updates/2008/Update69.htm
Corn prices late 1999 - Dec 2007
Wheat prices
http://www.earthpolicy.org/Updates/2008/
Update69.htm
http://www.earth-policy.org/Updates/2008/Update69.htm
Increasing oil prices drive up potential profit,
and cost of food crops
Cost of oil----Profitable cost of corn for ethanol
$50 ---- $4/bushel
$100 ---- $7/bushel
$140 ---- $10/bushel
Current corn price: ~$8/bushel
http://www.farmdoc.uiuc.edu/manage/newsletters/fefo07_11/fefo07_11.html
http://www.earth-policy.org/Updates/2008/Update69.htm
Whereas previous dramatic rises in world
grain prices were weather-induced, this one is
policy-induced and can be dealt with by policy
adjustments. If the entire U.S. grain harvest
were converted into ethanol, it would satisfy
scarcely 18 percent of our automotive fuel
needs.
http://www.earth-policy.org/Updates/2008/Update69.htm
Land use in USA, 1997
Artificial
Selection
(breeding)
HunterGatherer
Industrial
Agricultural
Modern cows were
bred from earlier
bovine species.
from Teosinte
Maize
By artificial selection that
began ~10,000 years ago.
Different foods
from one
species of plant.
Created by
artificial
selection.
Wild member of Brassica oleracea:
small side buds
Artificial selection
Number of Individuals
1. Select individuals
that have the largest
side buds and breed
them.
2. Of the offspring,
select individuals that
have the largest side
buds and breed them.
3. Of the offspring,
select individuals that
have the largest side
buds and breed them.
4. After several
generations, bud size
increases dramatically.
0
Brussels sprouts:
extremely large side buds
1
2
Size of buds (cm)
3
Agricultural
HunterGatherer
Industrial
increased
technology and
understanding of
molecular biology
Genetic Engineering: Direct manipulation of DNA
Fig 18.1
Millions of Hectares
Global area planted with GM crops
http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html
Texas =
70 ha
Tbl 19.5
Why Change a Plant’s DNA?
• Can change plant so that it has new
or different characteristics
– Produce needed protein
– Insect resistance (Bt toxin)
– Herbicide resistance (Round-up)
– Drought or other stress resistance
% of Total US Acres
Common GM Crops in the U.S.
http://blog.wired
.com/wiredscien
ce/2007/09/mon
santo-ishap.html
Types of Genetically Modified Crops
Herbicide Tolerance; Insect Resistance; Viral Resistance
% of Total US Acres
Common GM Crops in the U.S.
http://blog.wired
.com/wiredscien
ce/2007/09/mon
santo-ishap.html
Millions of Hectares
Is changing an organsisms DNA a good idea?
http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html
Texas =
70 ha
Is Changing an Organism’s DNA
a Good or Bad Idea?
• Genes flow within species and between
species.
• Are GM foods safe for the
environment?
• Are GM foods profitable for:
– Consumers, Farmers, and Companies?
Pollution in Gulf of
Mexico from run-off in
the Mississippi river
QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this picture.
AAL 25.17
Pollen is easily transferred from plant to plant,
and to related species.
Genetic information moves.
GM crops found
in Oaxaca and
other MX states
GM crops illegal
Genetic information moves,
and is self-replicating.
GM crops found
in Oaxaca and
other MX states
GM crops illegal
Many Questions, but Few
Answers?
• Are herbicide or insect resistant plants
safe to eat?
• Are GM foods safe for the
environment?
• Are GM foods profitable for:
– Consumers, Farmers, and Companies?
Types of Genetically Modified Crops
Herbicide Tolerance; Insect Resistance; Viral Resistance
Patents give 17 year monopoly for
inventor
Examples of current patents:
•Atryn- antithrombin produced in transgenic
goats (in milk) has anti-clotting properties
Examples of current patents:
•Atryn- antithrombin produced in transgenic
goats (in milk) has anti-clotting properties
•Evolutec has patents on proteins in tick saliva
for use as anti-inflammatory
Examples of current patents:
•Atryn- antithrombin produced in transgenic
goats (in milk) has anti-clotting properties
•Evolutec has patents on proteins in tick saliva
for use as anti-inflammator
•GTG in Australia has patents on non-coding
human DNA for detecting risk of various
diseases
Examples of current patents:
•Atryn- antithrombin produced in transgenic
goats (in milk) has anti-clotting properties
•Evolutec has patents on proteins in tick saliva
for use as anti-inflammatory
•GTG in Australia has patents on non-coding
human DNA for detecting risk af various
diseases
•Harvard patented cancer susceptible mouse
Examples of current patents:
•Atryn- antithrombin produced in transgenic
goats (in milk) has anti-clotting properties
•Evolutec has patents on proteins in tick saliva
for use as anti-inflammatory
•GTG in Australia has patents on non-coding
human DNA for detecting risk af various
diseases
•Harvard patented cancer susceptible mouse
•Over 70 U.S. patents for use of Indian neem
tree
Neem, say the women, helps babies sleep, keeps
flies away, is a cosmetic, a disinfectant and a
pesticide. Its leaves make good cattle fodder, its
twigs are good for teeth and gums. It is used, they
say, for snake bites, malaria, hysteria, high blood
pressure, pain relief, skin diseases and a host of
other ailments.
Over 70 U.S. patents for use
of Indian neem tree
Neem, say the women, helps babies sleep, keeps
flies away, is a cosmetic, a disinfectant and a
pesticide. Its leaves make good cattle fodder, its
twigs are good for teeth and gums. It is used, they
say, for snake bites, malaria, hysteria, high blood
pressure, pain relief, skin diseases and a host of
other ailments.
Over 70 U.S. patents for
use of Indian neem tree
One was rejected due to
previous use in indigenous
Indian culture
Concerns of developing nations vs.
industrialized countries
Bioprospecting
Lack of research on third world problems.
(tuberculosis, malaria, cholera, dysentery…
Not profitable.
Where does the information
about genetically modified foods
come from?
Millions of Hectares
Global area planted with GM crops
http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html
Texas =
70 ha
Today:
Biotechnology
•Exam #2
W 7/9 in class