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Biotechnology and Genetic Engineering
- What is it?
- electrophoresis
- PCR
- the Human Genome Project
DNA sequencing
- Cloning (genes),
- Gene therapy
- Cloning (cells, and multicellular organisms)
- Agricultural applications
Refer to chapter 14 in text
What is it?
biotechnology
noun: “the use of living organisms or other biological systems in the
manufacture of drugs or other products or for environmental
management, as in waste recycling: includes the use of bioreactors
in manufacturing, microorganisms to degrade oil slicks or organic waste,
genetically engineered bacteria to produce human hormones,
and monoclonal antibodies to identify antigens.”
Random House Unabridged Dictionary
There is WAY more included than the examples listed here.
1 genetic engineering
. 1. the development and application of scientific methods, procedures, and
technologies that permit direct manipulation of genetic material in
order to alter the hereditary traits of a cell, organism, or population.
2. a technique that produces unlimited amounts of otherwise unavailable
or scarce biological product by introducing DNA isolated from animals or
plants into bacteria and then harvesting the product from a bacterial colony,
as human insulin produced in bacteria by the human insulin gene.
- Mixtures of fragments are
separated by size due to
resistance to passage through
a gel when pulled by an
electric charge.
- Can be used (with different
matrices) to separate proteins
and other molecules
as well as DNA.
→
- May be used for
diagnosis,
paternity testing,
criminal forensics,
to purify a section of
DNA needed for further
study.
a32.lehman.cuny.edu/molbio_course/agarose1.htm
gel electrophoresis of DNA
restriction fragments (in case
you have forgotten….)
→
http://porpax.bio.miami.edu/~cmallery/150/protein/electrophoresis.jpg
electrophoresis
(This is an application from the DNA restriction digest/
electrophoresis lab analysis….)
Fluorescent dyes↑
make bands visible
under UV light.
Southern blot
to identify specific
fragments.
- Gel is pressed onto
a special membrane.
- DNA bands transfer.
- A radio-labeled DNA
fragment base-pairs
with the target fragment.
- This fragment can be
excised for further study.
(Named for Edwin Southern
who pioneered this procedure,
subsequent variations are
called northern and
western blots. Really.)
askabiologist.asu.edu/.../southern/southern.html
application: DNA fingerprinting (Here, paternity testing)
A selected segment of (usually)
“junk” DNA is isolated
←
STR: variable # of short tandem repeats
e.g… ACGGACGGACGGACGG…
between restriction sites results in
RFLP: restriction fragment
length polymorphism (“riflips”).
←The RFLP
pattern
is established for
the persons of
interest….
and compared to
the child’s pattern:
He had to get those
restriction sites
from his parents. →
http://www.thetech.org/genetics/ask.php?id=20
Consequences of DNA fingerprinting (DNA profile):
What are the implications for paternity testing?
There are historical applications, too.
How might it be medically important?
In forensics,
the evidence is much stronger than eye witnesses,
cold cases are often resolved in the alleged prep’s favor…
CODIS (Combined DNA Index System) files:
pros and cons… as it were.
In 1999 180,000 rape kits were unprocessed.1
In 2009 11,000 rape kits in an abandoned Detroit police warehouse.2
1 for
more info, NIJ report 7/2002:http://www.ncjrs.gov/pdffiles1/nij/194197.pdf
2 Alter, Charlotte TIME Magazine, July 28, 2014, p 28ff
PCR:
polymerase chain reaction
-used to amplify DNA samples
for fingerprinting, sequencing,
or further genetic manipulation.
- isolate segment of interest
- warm to denature
- cool to anneal primer
- allow polymerase to proceed
-“Taq” polymerase*
- Each cycle doubles the DNA.
DNA from as few as 20 cells
can be enough to use PCR and
catch a criminal.
This technology was an
essential starting point for ….
* from Thermus aquaticus
(more heat tolerant).
the Human Genome Project
Started in 1990, finished in 2003 (way ahead of schedule,
due to technology and competition issues),
for disease diagnosis, genetic research, evolution studies,
possible drug therapies, identify the 20,000- 30,000 genes…
It was a publicly funded effort of global cooperation between
companies, universities, and nations.
From the start a portion of the funds went to ethical research. Why?
- Began with linkage mapping
to show relative order of genes,
- continued with ordering of restriction fragments
to determine physical distances,
- and ended with sequencing of the DNA.
Since then many other organisms’ genomes have been sequenced.
HGP, under auspices of department of energy:
http://genomics.energy.gov/
Sanger method of
DNA sequencing:
uses ddNTPs to stop
replication of a
target strand. Products
then sorted by size…
The Campbell illustration
adds a very cool touch:
The ddNTPs are each
labeled with a distinctly
colored fluorescent tag,
the gel is run in a tube,
and the colors are read
by a laser detector.
p. 397
genetic engineering
Genes can be transferred from one organism to another,
and the gene products produced
even if the organisms are very different,
because of the universality of the genetic code.
(Prokaryotes have the same genetic language as do you.)
We will discuss some of what can be done,
and what is being pursued.
Whether things should be done is an ethical issue:
Is it OK to change the genome of a corn plant?
Of a human?
What if it alleviates suffering?
What is the risk of genetically engineering cows?
- risk to cows, cows’ neighbors, cows’ consumers? (Science)
Is it justified by the possible benefit? (Ethics)
transgenic technology
- Subject gene isolated
restriction digest,
electrophoresis,
- amplified
PCR,
- inserted into plasmid
restriction enzymes,
DNA ligase,
transformation,
and used to either
generate the subject
gene’s product
(right),
or to alter a new
host’s genome (left).
(E. coli is most common
host, but yeasts or
other cell types work.)
cloning (genes)
(IB doesn’t include this as cloning.)
Recall that prokaryotes
don’t have mRNA processing
like eukaryotes:
In order to get a useful product,
human (e.g.) DNA is transcribed,
pre- mRNA is processed,
mRNA is isolated,
and viral reverse transcriptase
is used to make a cDNA (copy DNA),
and then a complementary
strand is built.
(Sticky ends can be added
for direct insertion)
NOW it is ready for the snipping,
sorting,
and inserting
on the previous slide,
so the bacterium can
generate the protein.
The cloned gene is not of much use
until you can screen for/select which bacteria have picked up the plasmid.
This is part of an AP lab on transformation you will do shortly.
Gene therapy
Somatic cell line therapy: changes made in the DNA of somatic cells.
This can be done in adults,
but has to be redone periodically.
e.g. SCID is the lack of ADA (adenosine deaminase) in white blood cells.
- cells are removed from the patient
- a virus engineered to carry the ADA gene infects the cells
- the gene enters the cell,
allowing for the manufacture of ADA.
- The gene is NOT in the
stem cells,
so the process is
not self propagating.
- These therapies are mostly
experimental still.
- vector here is viral,
liposomes
(phospholipid droplets)
can be used.
Germ line therapy: changes made in the DNA of gametes.
This would be in all cells of the resulting person.
At this point this approach
is theoretical as a
human therapy.
There are practical
and ethical issues.
It is done in lab animals
as a research tool,
and used in agriculture.
cloning (cells)
For single-celled organisms, their simple mitosis or binary fission
is creating identical progeny.
In a lab, they just need to be isolated and cultured.
For cells from multicellular organisms
it is trickier:
- Some isolated cells may grow as sheets
in culture
(such as skin used in burn treatment).
- Therapeutic cloning, (the creation of
an embryo to supply embryonic stem
cells for medical use)
is only allowed to the blastocyst stage in
most places: uses? pros and cons?
- The race is on to reprogram
differentiated somatic cells into stem
cells, for in-vitro tissue and organ
growth.
http://newsimg.bbc.co.uk/media/images/44251000/gif/_44251016_stem_cells_416.gif
cloning (multicellular organisms)
You have a lab for an on-line whole organism cloning
activity. You provide the answers to confirm participation in the
exercise, followed by questions on the ethical considerations.
www.sheep101.info/interestingfacts.html
So how is all of this used?
- β-carotene, (pro-vitamin A, needed
for good vision), made by rice plant,
but not in endosperm…
- PSY gene from daffodils and bacterial
CRTI gene inserted into rice.
- Rice-dependent people experience
significant drop in blindness.
http://www.goldenrice.org/Content2-How/how1_sci.html
Agricultural applications
“Crop production is limited by salinity
on 40 percent of the world's irrigated
land and on 25 percent of irrigated
land in the United States”.
Salt is sequestered
in leaf vacuoles, not
in fruit, and may draw
salt out of soil.
“… Blumwald and Zhang genetically engineered tomato plants that produce higher levels
of …protein known as a "transport protein." The gene that controls increased production
of the transport protein was taken from Arabidopsis, a relative of the cabbage ….
The transport protein uses energy available in the cells to move salt – in the form of
sodium ions -- into compartments within the cells called vacuoles. Once the salt is
stashed inside the vacuoles it is isolated from the rest of the cell and unable to interfere
with the plant's normal biochemical activity”.
http://www.news.ucdavis.edu/search/news_detail.lasso?id=5840
Specific example of GMO battle in ’16 IB syllabus.
Recall…. Potatoes make two starches:
amylose (little branching, less soluble,
slow digesting, poor for gels)
amylopectin (opposite of the above)
http://franziska-brantner.eu/blog/wp-content/uploads/2010/05/basf-500x334.jpg
Industry prefers amylopectin.
Amflora is genetically modified
to not have an enzyme
needed to make amylose.
It was OKed by the EU in 2010,
but the OK was defeated in 2012.
https://thescienceofnutrition.wordpress.com/tag/amylopectin/
Amflora
Pharming
(producing medically useful products from genetically modified plants and animals)
This method is hit or miss
(insertion site is random and often deleterious),
but if desired human protein
is linked to a milk production promoter, and
a gene for antibiotic resistance, eggs or
embryos can be selected before implantation.
Current applications: spider silk in goat milk to make “Biosteel”,
human serum albumin in cow’s milk to expand blood in trauma cases,
factor IX (to treat hemophilia) made in sheep’s milk.
Bt (Bacillus thuringiensis), a highly specific parasitic bacterium
that produces endospores and crystals. The crystals punch holes in the
gut of pests, the spores exit gut, germinate, re-enter gut, and exit the now dead host.
http://www1.umn.edu/news/prod/groups
http://muou.sc.mahidol.ac.th/images/mechanism_bt.jpg
Bt crops: The genes for the crystals are introduced into crop genomes,
so the nibbling pests die,
without needing chemicals or the actual bacterial culture.
Analysis of risks to monarch butterflies of Bt crops.
argument
counter argument
http://www.news.cornell.edu/stories/1999/0
4/toxic-pollen-bt-corn-can-kill-monarchhttp://www.ars.usda.gov/is/br/btcorn/index.html#
bt11
butterflies
Concerns:
- ethics of engineering animals (and plants…)
- unidentified affects of specific gene insertions
- unintended dispersal of gene in broader environment
- ability to choose whether or not to consume such products
- loss of income to previous providers
- lack of understanding/information to public
Advantages:
- cheaper/more accessible protein products
- availability of more nutritious/cheaper foods
- reduced reliance on pesticides/herbicides
- serious money to the developers/new industries
(Discuss pros and cons of any one example of genetic modification.)
You have an unidentified hunk of DNA in a test tube.
Describe a line of research that would investigate this find,
and perhaps put it to some good use.
Employ as many of the techniques explored here as possible.
Pick a technology, such as gene therapy:
How is it done? When should it be applied?
To whom should it be available?
biotechnology
Sanger method
genetic engineering
ddNTP
electrophoresis
transgenic
Southern blot
plasmid
DNA fingerprinting
reverse transcriptase
paternity testing
somatic line therapy
RFLP
vector
STR
liposome
DNA profile
germ line therapy
forensics
Therapeutic cloning
CODIS
binary fission
PCR:
in-vitro
Human Genome Project
pharming
Bt crops