Transcript Ch20 Biotechnologyx

Ch.20
Biotechnology
Overview: The DNA Toolbox
History

1970’s Recombinant DNA

2001 Human Genome
Project
 10

years, $3 billion
2010 1200 Genomes
Sequenced
DNA microarray
20.1 DNA Cloning
Bacteria E. coli

Plasmids

Gene Cloning

Pesticide/Herbicide
Resistance

Oil Spill Clean-up
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Dissolve Blood Clots
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Human Growth Hormone
Plasmid
20.1 DNA Cloning
Restriction Enzymes

Discovered in 1960’s
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Protect Bacteria from
Viruses
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Very Specific-Restriction
Site

Restriction Fragments
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Sticky End-single sided
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DNA ligase-rejoins
20.1 DNA Cloning
Eukaryotic Genes
Figure 20.4 pg.399

Cloning Vector-original plasmid
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Genetic Marker (ampicillin)

Same restriction enzymes
remove and insert gene of
interest

Plate to sort
20.1 DNA Cloning
DNA Libraries

Shotgun approach


Genomic Library
Bacterial Artificial
Chromosome (BAC)

Larger Segments of DNA
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Complementary DNA
(cDNA) library
20.1 DNA Cloning
Screening Libraries



Gene of Interest

Nucleic Acid
Hybridization

Nucleic Acid Probe
Expression Vector-inserts
gene of interest into
correct reading frame
Electroporation-electrical
pulse opens membrane
20.1 DNA Cloning
PCR

Polymerase Chain
Reaction-thermocycler
copies DNA fragments

Use bacteria from hot
springs

Millions of copies in 24
hours
20.2 Sequence, Expression &
Function of Genes
Gel Electrophoresis
Southern Blotting
20.2 Sequence, Expression &
Function of Genes
Dideoxy Chain Termination
Northern Blotting

Fig.20.12 pg. 408

4th Generation

3rd Generation


1980 Nobel Prize-Sanger

“Sequencing by
Synthesis”
Used to study expression
of single genes at a
particular time in
development.
20.2 Sequence, Expression &
Function of Genes
5th Generation

Reverse Transcriptasepolymerase reaction (RTPCR)


cDNA is used as a
template
in situ Hybridization

Shows where in body
genes are being
expressed w/ fluorescent
dyes
20.2 Sequence, Expression &
Function of Genes
Interacting Genes

DNA microarray assays
(DNA chip)


Shows how groups of
genes are expressed as
an organism develops
in vitro mutagenesis

Specific mutations
introduced, used to
produce organisms for
scientific studies
20.2 Sequence, Expression &
Function of Genes
RNA interference (RNAi)

Blocks translation

Turns specific genes off so
that you determine its
function. “knocking out”

Prohibited in humans
20.2 Sequence, Expression &
Function of Genes
Genome-wide Association Studies

Large-scale analyses

Used to study: heart
disease, diabetes, autism,
etc.

Test for genetic markers

Single nucleotide
polymorphism (SNP)
20.3 Stem Cell Research
Plants
Animals

1950’s Steward at Cornell
University

1950’s Briggs & King w/
frogs
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Totipotent-cell that can
differentiate into a
specialized cell.

1970’s Gurdon-inversely
related to age
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1997 Scotland-Dolly the
sheep
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2007 Monkeys
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Many crop and
decorative species:
grapes, apples, orchids,
etc.
20.3 Stem Cell Research
Problems w/ Animal Cloning

Low success rate
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Defects

Obesity, pneumonia &
liver failure

Caused by epigenetic
changes in chromatin
during development
(methylation)
20.3 Stem Cell Research
Embryonic Stem Cells

Blastula stage of
development
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Reproduce indefinitely

Pluripotent-can produce
any type of cell

Could be used to replace
cell that don’t normally
divide: nerve & heart
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Controversial-fetal tissue
Adult Stem Cells

Ex. Bone marrow
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Can only differentiate into
limited cell types

2007 reprogramminginduced pluripotent stem
cells (iPS)
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Still in development,
limited success
20.4 Practical Applications
Medical




Identification of faulty
genes
Identification of
pathogens
“personalized medicine”
Gene therapy-replacing
defective genes, limited
success. Ex. SCID bone
marrow replacement
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Pharmaceuticals

Cancer treatments
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Proteins

HGH, Insulin, etc.
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Transgenic animals used
to make medicines
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Forensic Evidence
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Genetic Profile, STRs
20.4 Practical Applications

Environmental Cleanup
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Ethics/Safety/Regulation
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Agriculture
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GMO debate

Insecticides/herbicide
resistance
Disease resistance
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Wild varieties

Ti plasmid
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Allergies

Environmental
adaptation

Personal information

Designer babies
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Selective breeding

Increased production
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