Download 11-GeneTech

Biotechnology
Chapter 17
You are only responsible for the
material we get through in class
End of Chapter questions:
Understand: 1,2,4,5,7,
Apply: all
Synthesize: 1
Do all mQuiz questions
Gene Technology
1
What are the methods of genetic engineering ?
DNA cleavage
Restriction endonucleases
“sticky ends”
Gene Technology
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Gel electrophoresis
Used to analyze DNA fragments
Agarose – acts like a sieve
DNA has negative charge
—
+
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How can one determine which
bands have a particular
sequence?
Southern blotting
-- involves 3 key steps
1. Transfer to
nitrocellulose
2. “Probe” with 32P labeled
DNA sequence
(e.g., “AATGGCAGGT”)
3. Perform
autoradiography
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DNA fragments are manipulated
(e.g., ‘cloned’) by inserting into a
‘vector’
A ‘vector’ is a carrier
-- Plasmids or viruses
“Cloning” means to copy
Plasmids with
special genes
are used
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How can a “genomic library” be created?
-- Cells with DNA fragments from entire genome
1. Cut up DNA into pieces
2. insert pieces at random into
vector
3. “Transfect” into bacteria
Problems, however:
-- Few bacteria receive a
plasmid
-- Not all plasmids contain a
DNA fragment
Also…
Very few plasmids contain DNA
with any particular gene
Cloning Steps
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How to screen for
recombinant plasmids?
Cells grown on
Medium containing
ampicillin and Xgal
Form blue or white
colonies
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How to screen for cells containing
a specific DNA fragment?
Plate library
Make replica on
Nitrocellulose
Probe with
Radio-labeled
“DNA probe”
Perform
autoradiography
Find original colony
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How can the DNA for a particular gene be
isolated from original organism?
-- one way is a “cDNA”
(complementary DNA)
Starts with mRNA
Reverse transcriptase
DNA polymerase
cDNA lacks intron sequences
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What can cloned genes
be used for?
Transferring to other organisms, e.g.
-- insulin production to bacteria
-- herbicide/ insect resistance to plants
-- crop yield & nutritional value (e.g. ‘golden rice’)
-- biofuel (e.g., rape seed oil)
Gene therapy … some day
Control vs Roundup resistant alfalfa in
field treated with Roundup
-- cystic fibrosis
-- hemophilia
-- various cancers
-- many others
Gene sequencing
-- evolutionary relationships
-- protein structure predications
Sheep engineered to produce the
human protein alpha-1-antitrypsin (AAT)
in milk for treatment of emphysema
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“DNA Fingerprinting”
Used to identify source of DNA
Small genetic differences create
Restriction Fragment Length Polymorphisms
(RFLPs)
Forensic samples
(‘CSI Biology’)
-- blood, semen, saliva
How are RFLPs used to create
a DNA fingerprint?
DNA fingerprint
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PCR is used to analyze very
small amounts of DNA
PCR animation
PCR = Polymerase Chain Reaction
Requires:
Thermocycler
Primers
Taq-polymerase
35 cycles = 235 = 68 billion copies
Gene Technology
PCR
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