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Dec 14, 2012 (4 years and 10 months ago)

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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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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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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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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 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


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 steps


denaturation


annealing of primers


primer 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 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


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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Biotechnology


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


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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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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