Transcript Chapter 16 - drtracey.net

Gene Technology
Chapter 16
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Outline
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Restriction Endonucleases
– Host / Vector Systems
– DNA Libraries
Genetic Engineering Experiment
Working With Gene Clones
Biotechnology
– Medical Applications
– Agricultural Applications
– Risk and Regulation
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Restriction Endonucleases
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Restriction endonucleases recognize specific
nucleotide sequences, and cleave DNA
creating DNA fragments.
– Type I - simple cuts
– Type II - dyad symmetry
 allows physical mapping
 allows recombinant molecules
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Restriction Endonucleases
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Each restriction endonuclease has a specific
recognition sequence and can cut DNA from
any source into fragments.
 Because of complementarity, singlestranded ends can pair with each other.
 sticky ends
 fragments joined together with DNA
ligase
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Restriction Endonucleases
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Host / Vector Systems
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DNA propagation in a host cell requires a
vector that can enter the host and replicate.
– most flexible and common host is E. coli
– two most commonly used vectors are
plasmids and phages
 viruses and artificial chromosomes also
being probed for use
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Plasmid and Phage Vectors
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Using Vectors to Transfer Genes
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Chimeras
– One of first recombinant genomes was a
bacterial plasmid into which an amphibian
ribosomal RNA gene was inserted.
 Viruses can also be used as vectors to
insert foreign DNA into host cells.
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Early Genetic Engineering
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DNA Libraries
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A collection of DNA from a specific source in
a form that can be propagated in a host
– genomic library - representation of the
entire genome in a vector
– cDNA library is limited to expressed genes
isolated by reverse transcriptase
 isolated from retroviruses
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DNA Libraries
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Genetic Engineering Experiment
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Four stages
– DNA cleavage
 restriction endonuclease cleaves source
DNA into fragments
– production of recombinant DNA
 DNA fragments inserted into plasmids
or viral vectors
– cloning
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Genetic Engineering Experiment
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Screening
 clones with DNA fragment of interest
identified from clone library
 preliminary screening - eliminate any
clones without a vector and clones with
vectors that do not contain DNA
 employ vector with gene for
antibiotic resistance and lac Z’ gene
 expose to growth medium
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Genetic Engineering Experiment
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Secondary screening (gene of interest)
 hybridization - cloned genes form base
pairs with complementary sequences on
another nucleic acid (probe)
 grow on agar then transfer to filter
pressed on colonies
 treat filter with radioactive probe, and
perform autoradiography
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Genetic Engineering - Stages
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Genetic Engineering - Stages
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Working With Gene Clones
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Polymerase chain reaction
– used to copy specific gene sequences
 three basic steps
 denaturation
 annealing of primers
 primer extension
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Polymerase Chain Reaction
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Working With Gene Clones
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Identifying DNA: Southern blotting
– sample DNA cleaved into restriction
fragments, and spread apart by gel
electrophoresis
 gel blotted with sheet of nitrocellulose
 probe of purified, single-stranded DNA
poured over sheet
 if radioactive probe used, band of
radioactivity appears where probe
hybridized with complementary
fragment
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Working With Gene Clones
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Restriction fragment length polymorphisms
(RFLP’s) can be used to identify a particular
individual.
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Working With Gene Clones
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DNA fingerprinting
– Because two individuals rarely produce
identical RFLP analyses, DNA fingerprints
can be used in criminal investigations.
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Biotechnology
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Medical applications
– pharmaceuticals
 introduction of protein-encoding genes
 atrial peptides - high blood pressure
and kidney failure
 tissue plasminogen activator dissolving blood clots
– gene therapy
 add working copies of single defective
gene
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Medical Applications
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Piggyback vaccines
– produce subunit vaccines against viruses
 herpes
 hepatitis
– DNA vaccine
 cellular immune response
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Subunit Herpes Vaccine
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Agricultural Applications
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Ti plasmid has been early successful vector.
– nitrogen fixation
 introduce genes that allow crops to fix
nitrogen
 reduce need for fertilizer
– herbicide resistance
 insert genes encoding for proteins
making crops resistant to herbicide
 widespread herbicide use possible
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Ti Plasmid
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Agricultural Applications
insect resistance
 insert genes encoding proteins harmful to
insects
Real promise - produce genetically modified
plants with traits benefiting consumers
– iron deficiency in developing countries
 transgenic rice
– increasing milk production
 bovine somatotropin
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Transgenic Rice
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Bovine Somatotropin
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Risk and Regulation
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Questions
– How do we measure the potential risks of
genetically modified crops ?
– Is eating genetically modified food
dangerous ?
– Are genetically modified crops harmful to
the environment ?
– Should we label genetically modified
foods ?
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Summary
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Restriction Endonucleases
– Host / Vector Systems
– DNA Libraries
Genetic Engineering Experiment
Working With Gene Clones
Biotechnology
– Medical Applications
– Agricultural Applications
– Risk and Regulation
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