Transcript Genetic Engineering and Recombinant DNA

Genetic Engineering and
Recombinant DNA
Chapter 13
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Biotechnology
• The use of living organisms for practical
purposes.
• How long has biotechnology been around?
• Since man began planting crops, breeding
livestock and brewing beer – about 10,000
years! It is even mentioned in the Bible.
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• Today we can alter an animal or plant one
gene at a time, more rapidly and precisely
producing altered organisms.
• Even the boundaries between species are
becoming blurred as we move genes from
bacteria into plants and animals.
– Cotton-polyester blends grown in cotton plants
– Alter biochemical pathways :
• Damage genes for ethylene production
• Damage genes for polygalacturonase
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Recombinant DNA
• A DNA molecule consisting of two or more
DNA segments that are not found together
in nature.
• We can insert a gene into a plasmid, and
infect a cell with the plasmid.
• “designer genes”
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DNAase and RNAase cut up genetic
material at random.
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Restriction enzymes cut only at certain
sequences of bases, called restriction sites.
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Restriction Enzymes
• Bacteria produce many different restriction
enzymes that cut genetic material at different
sites.
Some make “blunt” ends: ATTC GGATC
TAAG CCTAG
Some make “sticky” ends: ATTCGG
ATC
TAA
GCCTAG
These pieces are restriction fragments.
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For recombinant DNA to be useful, we
needed to be able to produce large
quantities of a gene.
One way is to use plasmids.
A plasmid can be cut down to an origin of
replication and one or more genes for
antibiotic resistance.
Now any other gene can be inserted into the
plasmid and the bacterium will copy it.
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PCR – polymerase chain reaction
•
Now we can easily copy or amplify DNA
in the lab.
1. Produce two oligonucleotides that match
up with the DNA before and after the
DNA you want to copy.
2. Heat DNA to break the hydrogen bonds
and open the DNA.
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3. Add oligonucleotides (primers)
4. Cool, allowing primers to bind to DNA
5. Add nucleotides and DNA polymerase.
6. Repeat
Can now do the whole thing at higher
temperatures using the more stable
enzymes from Thermos aquaticus
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• PCR can be applied to :
– Amplifying DNA in a drop of blood
– Find DNA from the HIV virus
– Find early signs of cancer
– Find genetic defects in human embryos
– Examine the DNA of ancient organisms
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If we want to put eukaryotic DNA into
prokaryotic DNA we have one more
problem:
Introns! Bacteria do not have a means for
dealing with introns, so we must give them
a copy of a gene without introns in it.
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Start with mature mRNA.
Use reverse transcriptase to copy it into
DNA.
This DNA is called complimentary or cDNA.
Unfortunately, some proteins need to be
modified after they are translated, which
can only be done in eukaryotic cells.
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To get our gene into another cell we need a
vector – a means to carry it there.
This vector can be a plasmid or virus or
another means of carrying the DNA into
cells.
Now we can have bacterial or eukaryotic
cells making useful proteins – purer
vaccines, enzymes, drugs and human
proteins.
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• We could also make new proteins:
– “Abzymes” antibodies that act as enzymes
that would attach to and break up synthetic
chemicals polluting the environment.
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• Recombinant DNA can be inserted into the cells
of whole plants and animals – these animals
are called transgenic organisms.
• Transgenes
• Insulin in the milk of cows, polyester blends
from cotton plants.
• To produce an organism that has the transgene
in all the appropriate cells of the organism the
DNA must be added to:
The zygote – the original single celled
organism – this is called germ line gene
therapy
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So far, only one transgenic organism is born
for every hundred zygotes.
Few eggs incorporate the DNA
DNA is inserted into the chromosome at
random –
can disrupt key genes and kill organism
Replacing one gene with another is easy in
prokaryotes but difficult to do in animals.
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• It is easier to create a genetic “knockout”
–in which a particular gene has been
removed or disabled.
• This can be used to remove a harmful
gene, for producing research animals and
for studying the effects of a specific gene.
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• Genetically engineering plants is common
and lucrative
• >2/3 of all genetically engineered plants on
earth are grown in the U.S.A.
• Mostly corn, soybeans and cotton
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It is relatively easy to insert genes into plants:
We can use the Ti plasmid in the bacterium
Agrobacterium tumefaciens
We can also use a gene gun – particles of
gold or tungsten are coated with
recombinant DNA and shot into young plant
tissue which is chopped up and grown in
culture
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Examples:
• Corn which has an natural insecticide
“built in”
• Soybeans that resist a root-rot fungus
• “Roundup Ready” seeds resist the
herbicide Roundup
• “high-oil” corn makes twice the normal
amount of oil
• Tobacco plants that manufacture
antibodies and vaccines
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What are the risks?
• These new genes can infect wild strains
• A gene for herbicide resistance could find
its way into weeds
• Danger to animals that interact with the
plants
• Dispersing genes of viruses and bacteria
that cause disease in plants
• Dispersing genes for antibiotic resistance
• Tendency of these genes to jump to other
organisms
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• Problems with patents:
– Farmers that save seeds for next year’s crop
– Pollen that blows or is carried into neighboring
fields
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Gene Therapy
• Ideally, we could use these techniques to
treat genetic diseases such as cystic
fibrosis and hemophilia
• Engineer rice to contain vitamin A and
prevent blindness
• Vaccines in bananas
• Tomatoes with cancer fighting substances
• Production of fibrinogen for bandages
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• Right now we can test for genetic diseases
without being able to do anything about
them:
– What if your insurance company finds out?
– Breast cancer genes
• Peanut genes in other plants?
• Jesse Gelsinger of Tuscon, AZ was one of
the first deaths recognized to be caused
by attempted gene therapy.
– Ornithinetranscarbamylase deficiency
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Cloning
• Risk death of clones and pregnant mother
• Abnormalities in pregnancy
• Half of all clones of large mammals have
abnormalities of the heart, lungs and other
organs
• Do clones age faster than normal?
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Other questions
• Where do “undesirable” traits stop?
• Can we patent human genes?
• ?????
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