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CHAPTER 17
LECTURE
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Biotechnology
Chapter 17
DNA Manipulation
• Restriction endonucleases revolutionized
molecular biology
• Enzymes that cleave DNA at specific sites
– Used by bacteria against viruses
• Restriction enzymes significant
– Allow a form of physical mapping that was
previously impossible
– Allow the creation of recombinant DNA
molecules (from two different sources)
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• 3 types of restriction enzymes
• Type I and III cleave with less precision
and are not used in manipulating DNA
• Type II
– Recognize specific DNA sequences
– Cleave at specific site within sequence
– Can lead to “sticky ends” that can be joined
• Blunt ends can also be joined
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• DNA ligase
– Joins the two fragments forming a stable DNA
molecule
– Catalyzes formation of a phosphodiester bond
between adjacent phosphate and hydroxyl
groups of DNA nucleotides
– Same enzyme joins Okazaki fragments on
lagging strand in replication
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Gel Electrophoresis
•
•
•
•
•
Separate DNA fragments by size
Gel made of agarose or polyacrylamide
Submersed in buffer that can carry current
Subjected to an electrical field
Negatively-charged DNA migrates towards the
positive pole
• Larger fragments move slower, smaller move
faster
• DNA is visualized using fluorescent dyes
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Transformation
• Introduction of DNA from an outside
source into a cell
• Natural process in many species
– E. coli does not
• Temperature shifts can induce artificial
transformation in E. coli
• Transgenic organisms are all or part
transformed cells
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Molecular Cloning
• Clone – genetically identical copy
• Molecular cloning – isolation of a specific
DNA sequence (usually protein-encoding)
– Sometimes called gene cloning
• The most flexible and common host for
cloning is E. coli
– Vector – carries DNA in host and can
replicate in the host
– Each host–vector system has particular uses
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Vectors
• Plasmids
– Small, circular chromosomes
– Used for cloning small pieces of DNA
– 3 components
• Origin of replication – allows independent
replication
• Selectable marker – allows presence of plasmid to
be easily identified
• Multiple cloning site (MCS)
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• Artificial chromosomes
– Plasmids have limited insert size
– Yeast artificial chromosomes (YACs)
– Bacterial artificial chromosomes (BACs)
– Allow for larger insert for large-scale analysis
of genomes
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DNA Libraries
• A collection of DNAs in a vector that taken
together represent the complex mixture of
DNA
• Genomic library – representation of the
entire genome in a vector
– Genome is randomly fragmented
– Inserted into a vector
– Introduced into host cells
– Usually constructed in BACs
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Plasmid Library
DNA fragments
from source DNA
DN A inserted
into plasmid vector
Transformation
Each cell contains a
single fragment. All cells
together are the library.
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• Complementary DNA (cDNA)
– DNA copies of mRNA
– mRNA isolated
• Represents only actively used genes
• No introns
– Use reverse transcriptase to make cDNA
– cDNA used to make library
– All genomic libraries from a cell will be the
same but cDNA libraries can be different
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• Molecular hybridization
– Technique used to identify specific DNAs in
complex mixtures such as libraries
– Also termed annealing
– Known single-stranded DNA or RNA is
labeled
– Used as a probe to identify its
complement via specific base-pairing
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• Molecular hybridization is the most
common way of identifying a clone in a
DNA library
• This process involves three steps:
1. Plating the library
•
Physically the library is a collection of bacteria or
viruses in bacteria
2. Replicating the library
3. Screening the library
•
Probe is specific sequence of interest
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DNA Analysis
• Restriction maps
– Molecular biologists need maps to analyze
and compare cloned DNAs
– Initially, created by enzyme digestion,
separation by electrophoresis, and analysis of
resulting patterns
– Many are now generated by computer
searches for cleavage sites
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• Southern blotting
– Sample DNA is digested by restriction
enzymes and separated by gel
electrophoresis
– Double-stranded DNA denatured into singlestrands
– Gel “blotted” with filter paper to transfer DNA
– Filter is incubated with a labeled probe
consisting of purified, single-stranded DNA
corresponding to a specific gene
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• Northern blotting
– mRNA is separated by electrophoresis and
then blotted onto the filter
• Western blotting
– Proteins are separated by electrophoresis and
then blotted onto the filter
– Detection requires an antibody that can bind
to one protein
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• RFLP analysis
– Restriction fragment length polymorphisms
– Generated by point mutations or sequence
duplications
– Restriction enzyme fragments are often not
identical in different individuals
– Can be detected by Southern blotting
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• DNA fingerprinting
– Identification technique used to detect
differences in the DNA of individuals
– Population is polymorphic for these markers
– Using several probes, probability of identity
can be calculated or identity can be ruled out
– First used in a U.S. criminal trial in 1987
• Tommie Lee Andrews was found guilty of rape
– Also used to identify remains
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DNA Analysis
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• DNA sequencing
– Determination of actual
base sequence of DNA
– Basic idea is nested
fragments
– Each begin with the same
sequence and end in a
specific base
– By starting with the
shortest fragment, one
can then read the
sequence by moving up
the ladder
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• Enzymatic DNA
sequencing
• Developed by Frederick
Sanger
• Dideoxynucleotides are
used as chain terminators
in DNA synthesis reactions
• 4 separate reactions, each
with a single
dideoxynucleotide, to
generate a set of
fragments that terminate in
specific bases
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• Automated DNA sequencing
– Enzymatic technique is powerful but is laborintensive and time-consuming
– Automation made sequencing faster and
more practical
– Fluorescent dyes are used instead of
radioactive labels
– Reaction is done in one tube
– Data are assembled by a computer
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• Fundamentally new method for DNA sequencing
– DNA is cleaved into smaller pieces
– Both ends are ligated to adapters that are
complementary to specific primers
– DNA fragments are injected into a flow cell
– Each of 7 channels contains a solid substrate with
primers that complement the ligated ends of the DNA
fragments
– Like Sanger sequencing, uses fluorescent tag on
dNTPs
• However, blocking group removed after each round
– Camera records colors after each round of extension
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA
Adapters
a.
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Adapter
DNA fragment
Dense primer lawn
in flow cell
Adapter
DNA
Adapters
Flow cell
a.
1 cm
b.
b: © 2007, Illumina Inc. All rights reserved
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Bridge
amplification
with unlabeled
dNTPs
Free end
binds to
primer
c.
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Bridge
amplification
with unlabeled
dNTPs
c.
Free end
binds to
primer
Fragments
become
doublestranded
Attached
Free
terminus
d.
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Bridge
amplification
with unlabeled
dNTPs
c.
Free end
binds to
primer
Fragments
become
doublestranded
d.
Attached
Free
terminus
Denature
doublestranded
molecules
Attached
e.
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Bridge
amplification
with unlabeled
dNTPs
c.
Free end
binds to
primer
Fragments
become
doublestranded
d.
Attached
Free
terminus
Denature
doublestranded
molecules
e.
Attached
35 cycles
of bridge
amplification
Clusters
f.
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
G
C
T
A
T
G
C
A
First round of
synthesis with
labeled dNTPs
g.
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
G
C
T
A
T
N
G
C
O
A
A
–O
T
O
O–
4´
1´
3´
G
Image capture for each
round of synthesis
g.
N
P O CH2
5´
C
First round of
synthesis with
labeled dNTPs
N
NH2
2´
OH
A
Reversible terminator
h.
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Adapter
DNA fragment
Dense primer lawn
in flow cell
Adapter
DNA
Adapters
Flow cell
1 cm
a.
Bridge
amplification
with unlabeled
dNTPs
c.
b.
Free end
binds to
primer
Fragments
become
doublestranded
d.
Attached
Free
terminus
Denature
doublestranded
molecules
e.
Attached
35 cycles
of bridge
amplification
Clusters
f.
b: © 2007, Illumina Inc. All rights reserved
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• Polymerase chain reaction (PCR)
– Developed by Kary Mullis
• Awarded Nobel Prize
– Allows the amplification of a small DNA
fragment using primers that flank the region
– Each PCR cycle involves three steps:
1.Denaturation (high temperature)
2.Annealing of primers (low temperature)
3.DNA synthesis (intermediate temperature)
– Taq polymerase
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After 20 cycles, a
single fragment
produces over one
million (220) copies!
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• Applications of PCR
– Allows the investigation of minute samples of
DNA
– Forensics – drop of blood, cells at base of a
hair
– Detection of genetic defects in embryos by
analyzing a single cell
– Analysis of mitochondrial DNA from early
human species
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• Yeast two-hybrid system
– Used to study protein–protein interactions
– Gal4 is a transcriptional activator with a
modular structure
• DNA-binding domain that binds sequences in
Gal4-responsive promoters
• Activation domain that interacts with the
transcription apparatus to turn on transcription
– System uses 2 vectors – each with one of the
above – neither vector alone can activate
transcription
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• When cDNAs are inserted in each of these
vectors, they produce fusion proteins
• Contain part of Gal4 and the protein of
interest
– DNA-binding hybrid is called the bait
– Activating domain hybrid is called the prey
• If the proteins being tested interact, Gal4
function will be restored
• A reporter gene will be expressed
• Detected by an enzyme assay
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Genetic Engineering
• Has generated excitement and
controversy
• Expression vectors contain the sequences
necessary to express inserted DNA in a
specific cell type
• Transgenic animals contain genes that
have been inserted without the use of
conventional breeding
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• In vitro mutagenesis
– Ability to create mutations at any site in a
cloned gene
– Has been used to produce knockout mice
• A known gene is inactivated
– The effect of loss of this function is then
assessed on the entire organism
– An example of reverse genetics
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Medical Applications
• Medically important proteins can be
produced in bacteria
– Human insulin
– Interferon
– Atrial peptides
– Tissue plasminogen activator
– Human growth hormone
– Problem has been purification of desired
proteins from other bacterial proteins
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Genetically engineered mouse
with human growth hormone
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• Vaccines
– Subunit vaccines
• Genes encoding a part of the protein coat are
spliced into a fragment of the vaccinia (cowpox)
genome
• Injection of harmless recombinant virus leads to
immunity
– DNA vaccines
• Depend on the cellular immune response (not
antibodies)
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• Gene therapy
– Adding a functional copy of a gene to correct
a hereditary disorder
– Severe combined immunodeficiency disease
(SCID) illustrates both the potential and the
problems
• On the positive side, 15 children treated
successfully are still alive
• On the negative side, three other children treated
have developed leukemia (due to therapy)
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Agricultural Applications
• Ti (tumor-inducing) plasmid
– Most used vector for plant genetic
engineering
– Obtained from Agrobacterium tumefaciens,
which normally infects broadleaf plants
– Part of the Ti plasmid integrates into the plant
DNA and other genes can be attached to it
– However, bacterium does not infect cereals
such as corn, rice, and wheat
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• Other methods of gene insertion
– Gene guns
• Uses bombardment with tiny gold particles coated
with DNA
• Possible for any species
• Copy number of inserted genes cannot be
controlled
– Modification of Agrobacterium system
– Use of other bacteria like Agrobacterium
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• Herbicide resistance
– Broadleaf plants have been engineered to be
resistant to the herbicide glyphosate
– Benefits
• Crop resistant to glyphosate would not have to be
weeded
• Single herbicide instead of many types
• Glyphosate breaks down in environment
– In the United States, 90% of soy currently
grown is GM soy
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• Bt crops
– Insecticidal proteins have been transferred
into crop plants to make them pest-resistant
– Bt toxin from Bacillus thuringiensis
– Use of Bt maize is the second most common
GM crop globally
• Stacked crops
– Both glyphosate-resistant and Bt-producing
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• Golden rice
– Rice that has been genetically modified to
produce b-carotene (provitamin A)
– Converted in the body to vitamin A
– Interesting for 2 reasons
• Introduces a new biochemical pathway in tissue of
the transgenic plants
• Could not have been done by conventional
breeding as no rice cultivar known produces these
enzymes in endosperm
– Available free with no commercial
entanglements
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• Adoption of genetically modified (GM)
crops has been resisted in some areas
because of questions
– Crop safety for human consumption
– Movement of genes into wild relatives
• No evidence so far but it is not impossible
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• Biopharming
– Transgenic plants are used to produce
pharmaceuticals
– 1990 – Human serum albumin produced in
genetically engineered tobacco and potato
plants
– In development
• Recombinant subunit vaccines against Norwalk
and rabies viruses
• Recombinant monoclonal antibodies against tooth
decay-causing bacteria
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• Transgenic animal technology has not
been as successful as that in plants
• Molecular techniques combined with the
ability to clone domestic animals could
produce improved animals for
economically desirable traits
• Main use thus far has been engineering
animals to produce pharmaceuticals in
milk (also biopharming)
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