Download biotechnology: tools and applications

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Biotechnology - use of living organisms to create products
or help processes
Ex. HGH, insulin
Recombinant DNA - segment of DNA containing
sequences from different organisms
How is DNA manipulated?
Restriction enzymes cut DNA at specific sites
and create sticky ends
G T G G
T
T
A A
C A C C
A
A
T T
A
T
T A
C C T
C C A
G A
G G A
G G T C
T
G A
T C
G
C T C
T G T
T
C A G
A A
A
C
A
C
T
G
G
C A
A
A
G
G T C
T T A
T
GA
GA
T
TC C
C
G
G
G T
T
C T
T
T
C G A
T
C C A G AA C G G A
A A
T TC
T
C
C TAG
AG
C
A C
G A
G
A
C
A
A
G
C TA A
G G T C T T G G C T
T TA
G
Complementary ends will fuse to produce
a long strand of DNA
• The DNA is then integrated into the recipient
cell’s chromosome
Donated DNA
Degraded DNA
Crossovers
Recipient cell’s
chromosome
Figure 12.1D
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Recombinant
chromosome
Plasmids are extra rings of DNA that replicate in bacteria.
DNA can be inserted into plasmids.
bacterium
bacterial
chromosome
plasmid
Cloning Vectors
Bacterium
Human cell
Plasmid
DNA
Human protein
Bacterial
chromosome
1. Use restriction enzymes.
2. Insert gene into plasmid.
recombinant DNA
transformation
3. Transfer the plasmid back
into bacterial cell.
replication
4. Let bacterial cells replicate.
bacterial
clones
Recombinant DNA products
• “seed” protein for artificial snow
• Insulin for diabetes treatment
• Enzymes that clean up toxic waste spills
• Growth Hormones (Human, Bovine)
• TPA: Tissue Plasminogen Activator for
treatment of heart attacks
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• The polymerase
chain reaction
(PCR) can
quickly clone a
small sample of
DNA in a test
tube
Initial
DNA
segment
• Selection of
specific sequence
1
2
4
Number of DNA molecules
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8
Figure 12.12
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Gel electrophoresis sorts DNA molecules by size
• Restriction fragments of DNA are compared by
size
Mixture of DNA
molecules of
different sizes
Longer
molecules
Power
source
Gel
Shorter
molecules
Glass
plates
Completed gel
Figure 12.10
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
DNA
forensics
Egg microinjection to produce
transgenic animal
Credit: © Science VU/Visuals Unlimited
Egg manipulation via microinjection.
Grow bigger fish faster.
Salmon with gene from another fish species
• Uses of
transformed
animals:
• Produce
medicines more
easily
Ex. sheep and gene to
treat cystic fibrosis
Goats and AT3 gene to
prevent blood clots
Figure 12.16
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Fig. 11-14, p.173
Genetic engineering of plants
Methods to insert DNA:
1. Ballistics
2. Protoplasts
3. Agrobacterium as vector
- Ti plasmid
Credit: © Brad Mogen
An extremely large Agrobacterium tumefaciens tumor (crown gall disease) and secondary tumors
on Kalanchoe stem.
a A bacterial
cell contains
a Ti plasmid
(purple) that
has a foreign
gene (blue).
b The bacterium
infects a plant
and transfers the
Ti plasmid into it.
The plasmid DNA
becomes
integrated into
one of the plant’s
chromosomes.
c The plant cell
divides. Its
descendant
cells form an
embryo, which
may develop
into a mature
plant that can
express the
foreign gene.
A young
plant
expressing
a fluorescent
gene product
Fig. 11-12, p.171
Genetically modified crops
• Golden rice with Vitamin A
• Cotton resistant to boll weevil
• Soybeans resistant to herbicide (Roundup)
• Corn resistant to European corn borer
• Rapeseed with healthier vegetable oil
Benefits and risks
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
How Dolly was cloned
DNA
udder cells
white sheep
embryo
surrogate
mother
egg cell
black sheep
Dolly
Cloning of human cells
• Regenerative medicine
– Bone, pancreas cells, skin
Stem cells - the $6 billion promise?
Gene therapy may someday help treat a
variety of diseases
• treat disease by altering an
afflicted individual’s genes
Cloned gene (normal allele)
1 Insert
normal gene
into virus
Viral nucleic
acid
– Ex vivo
Retrovirus
– In vivo
2 Infect bone
marrow cell
with virus
– Stem cells
3 Viral DNA
inserts into
chromosome
Bone marrow
cell from patient
Bone
marrow
4 Inject cells
Figure 12.19
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into patient
Human Genome Project
• 3.2 billion bases in 22 autosomes + X, Y
• Draft sequence completed in 2003
• Available at
www.ornl.gov/sci/techresources/Human_Genome/
home.shtml
• www.ucsc.genome.edu
What does the human genome
sequence tell us?
• 20 K to 25 K genes
• 99.9% alike, across all races
• 97% of DNA is not transcribed
- Spacers between genes
- Structural (centromeres, telomeres)
- Regulatory (enhancers, promoters)
- Leftovers of evolution?
How are specific genes identified?
1. Isolate it from a genomic library by
homology with a gene from another
organism.
2. Find mRNA for the gene, make cDNA
from it.
3. Make DNA sequence based on protein
sequence.
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1. Nucleic acid probes identify clones
carrying specific genes
• A nucleic acid probe can tag a desired gene in a
library
Radioactive
probe (DNA)
Mix with singlestranded DNA from
various bacterial
(or phage) clones
Single-stranded
DNA
Base pairing
indicates the
gene of interest
Figure 12.8A
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
cDNA
mRNA
• Complementary
DNA
mRNA
• Using reverse
cDNA
transcriptase
• Assembles DNA
on mRNA
template
reverse
transcriptase
DNA
polymerase
DNA
DNA
Fig. 11-4, p.164
DNA microarrays test for the expression of
many genes at once
• A labeled probe can
reveal patterns of
gene expression in
different kinds of
cells
cDNA
DNA of gene
DNA
microarray,
actual size
(6,400 genes)
Figure 12.9
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Gene Therapy
• What is it?
• How is it done?
• Does it work?
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Gene therapy
• Goal - Treat diseases caused by mutated genes
• Method - Add a normal gene or block an
abnormal gene in enough cells to restore
normal function
• Target - somatic cells
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Which disorders are candidates for gene therapy treatment?
•
Disorders due to mutations in one or more genes
•
The responsible gene is known
•
The affected tissues are known and accessible
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Knockout gene therapy
• Goal: turn off a gene that is causing a
disorder
• Strategies:
– Antisense
– Triple helix oligos
– Spliceosome
– Ribozyme
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
How is gene therapy done?
1.
Identify the gene(s) responsible for the disorder
2.
Make copies of the normal gene
3.
Insert the copies into vectors (i.e., viruses)
4.
Infect the affected cells with the vectors
5.
Activate the gene
–
Transcription and translation take place
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Critical factors in choosing a vector
Gene size
– Limited room in vector genome
•
Target tissue
– What cells can the vector infect?
•
Integration into the genome
– Without integration, only short-term
effect
– Random integration may disrupt other
genes
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Photo courtesy of Van de Silva
Gene Therapy Successes
Ashanti de Silva
successfully treated for
ADA deficiency - 1990
Ryes Evans successfully
treated for SCID - 2001
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Gene Therapy Problems
Jesse Gelsinger died of
complications due to an
immune system response while
participating in a clinical trial
Three children treated for SCID developed
leukemia due to disruption of a gene that
regulates cell division
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Ethical and Social Issues
• Patient safety while participating in
clinical trials
• Which applications are therapies and
which are enhancements?
– “Designer” babies
• Access to gene therapies
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings