1 gene technology chapter 16. 2 outline restriction endonucleases – host / vector systems – dna...
TRANSCRIPT
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Gene Technology
Chapter 16
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Outline
• 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
• 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
• Each restriction endonuclease has a specific recognition sequence and can cut DNA from any source into fragments.
Because of complementarity, single-stranded ends can pair with each other.
sticky ends fragments joined together with DNA
ligase
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Restriction Endonucleases
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Restriction Endonucleases
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Host / Vector Systems
• 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
• 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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Early Genetic Exp.
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DNA Libraries
• 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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cDNA
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Genetic Engineering Experiment
• 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
– Screening clones with DNA fragment of interest
identified from clone librarypreliminary 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
– 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
• Polymerase chain reaction– used to copy specific gene sequences
three basic stepsdenaturationannealing of primersprimer extension
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Polymerase Chain Reaction
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Working With Gene Clones
• Identifying DNA: Southern blotting– sample DNA cleaved into restriction
fragments, and spread apart by gel electrophoresis
gel blotted with sheet of nitrocelluloseprobe 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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Southern Blot
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Working With Gene Clones
• Restriction fragment length polymorphisms (RFLP’s) can be used to identify a particular individual.
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Working With Gene Clones
• DNA fingerprinting– Because two individuals rarely produce
identical RFLP analyses, DNA fingerprints can be used in criminal investigations.
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RFLPs
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Biotechnology
• Medical applications– pharmaceuticals
introduction of protein-encoding genesatrial 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
• 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
• 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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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
• 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
• 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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