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Biotechnology
Dolly
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Potential Uses for Cloned Genes
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to produce a protein product
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to endow an organism with a metabolic
capability
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plasminogen activator
engineered bacteria that degrade oil spills
create more copies of the gene for further study
Genetic
Engineering
Stages
1. Isolate gene
cleavage of plasmid
2. Recombinant-DNA
3. Clone
* Screen
4. Harvest
Gene
Protein
1. Isolate Gene
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Isolate the desired gene from the genome
the desired gene will be cut with restriction
endonucleases (enzymes)
desired gene to be
isolated
Cleavage
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cut DNA of host with restriction endonucleases
(enzymes)
several hundred RE’s exist discovered 1960s
produce sticky ends or blunt ends
Sticky Ends
Escherichia coli
Eco RI
sticky end
Blunt ends
Haemophilus aegyptius
Hae III
GGCC
CCGG
GG
CC
CC
GG
2. Recombinant DNA
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splice desired gene into host DNA, DNA
ligase seals the strands
Vector, generally
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plasmid of bacteria (prok) or yeast (euk)
viral DNA (not for harvesting protein)
plasmid with recombinant DNA
3. Clone
produce a cell line
in which all members
have identical copies
of a particular gene
Screen
Choose cells that
carry desired gene &
eliminate those cells
that do not carry desired gene
4. Harvest (or Isolate)
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harvest protein
harvest gene
genetic harvesting
protein harvesting
protein molecules
copies of the gene
pest resistant gene
oil eating bacteria
dissolving clot protein
human growth hormone
Cloning
reverse
transcriptase
plus mRNAs
mRNAs
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Directly from an organism
complementary DNA
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made from mRNA template through
reverse transcription (cDNA)
Reverse transcriptase can be used
to make smaller cDNA libraries
These contain only the genes that are
transcribed by a particular type of cell
recognized by the addition of a RE
recognition sequence to it
mRNA is degraded
by an enzyme
DNA polymerase
synthetizes the 2nd
strand
cDNA
Genomic Libraries

“Book,” a clone containing a foreign DNA
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Plasmid library (bacterial, yeast)
Phage library (virus)
Bacterial Artificial Chromosome library
Plasmid Library

Copies of DNA fragments can be stored in a
cloned bacterial plasmid
foreign genome
Each one of these is considered a "book"
recombinant plasmid
bacterial clones
Phage Library
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DNA fragments can be stored in a cloned phage
foreign genome
each phage type is
considered a "book"
recombinant
phage "book"
phage
clones
Bacterial Artificial Chromosome (BAC)
Library
Copies of multiple
DNA fragments can
be stored in a bacterial
artificial chromosome
each clone
occupies one
well
BAC
clone
plasmid with many genes
Gene products
Product
Made in
Use
human insulin
human growth hormone
epidermal growth factor
interleukin-2
bovine growth hormone
cellulase
Taxol
hepatitis B vaccine
erythropoietin
factor VIII
tissue plasminogen activator
E. coli
diabetes
E. coli
growth defects
E. coli
burns, ulcers
E. coli
possibly cancer
E. coli
improving weight gain
E. coli
breakdown of cellulose
E. coli
ovarian cancer
S. cerevisiae
prevents hepatitis
mammalian cells
anemia
mammalian cells
hemophilia
mammalian cells
heart attacks
Other Examples
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"golden rice"
genetically modified
rich in beta-carotene
prevents blindness
news.bbc.co.uk
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papaya's ringspot disease
gene was introduced to
control the plague
http://www2.dpi.qld.gov.au
Human Genome Project
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Collaborative effort to map and sequence
entire human genome
Began 1990
4 goals

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genetic (linkage) mapping
physical mapping
sequencing
analyzing the genomes of other species
Genetic Mapping of the Human Genome

to locate genetic markers spaced evenly
throughout the chromosomes

to make it easier to find other loci
Physical Mapping of the Human Genome

cutting chromosomes into identifiable fragments
then determining their order on the
chromosome
Sequencing the Human Genome
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determining the exact nucleotide pairs
haploid set of human chromosomes contains
approximately 3 billion nucleotide pairs
Genbank
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Database where DNA sequences have been deposited
publically available via the Internet
final draft, 2004 (over 99% of genome was
determined)
remain a few 100 gaps of unknown sequences that
require special methods to figure out
Analyzing Gene Expression
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Analyze genomes of other important species
for genetic engineering
Stem Cells
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unspecialized
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blastula cells
pluripotent
adult stem cells
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gives rise to specific types of cells
bone marrow blood cells
Applications
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Medical
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animal and plant application
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gold rice
salinity resistant gene
Environmental
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Diagnosis
Human Gene therapy
Pharmaceutical products
biofuel
oil cleaning bacteria
Forensic evidence
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The Innocence Project
conviction of guilty
Genomes of other species and H. sapiens
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Bacteria
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1995
1997
S. cerevisiae
6,200
1996
60,000
2002
13,700
22,000
25,000
21,000
2000
2001
2004
2003
Plants
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1,700
4,400
Fungi
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H. influenzae
E. coli
Oryza sativa (rice)
Animals
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D. melanogaster
Mus musculus
Rattus norvegius
H. sapiens
Ethical Issues

Should we engineer new genotypes for
individuals with anomalies?
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diabetes, CF, immune deficiencies, MD, stunted
growth, sickle-cell disease
myopia, altering personalities, increase length of
life
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Should we engineer human germ cells?
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If they are carrying abnormal genes
eugenics - deliberate effort to control the
genetic makeup of human populations
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color of eyes
color of skin
color of hair
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We have technology to test for diseases for
which there is no cure and sometimes no
treatment. (Ex. Huntington’s disease, breast
cancer)
Would you want to be tested?
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Who should have right to examine someone’s
genetic info?
How should that info be used?
Should a person’s genome be a factor in
determining eligibility for a job or insurance
The End