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Biotechnology
Chapter 17
DNA Manipulation
The molecular biology revolution started with
the discovery of restriction endonucleases
-Enzymes that cleave DNA at specific sites
These enzymes are significant in two ways
1. Allow a form of physical mapping that was
previously impossible
2. Allow the creation of recombinant DNA
molecules (from two different sources)
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DNA Manipulation
Restriction enzymes recognize DNA
sequences termed restriction sites
There are two types of restriction enzymes:
-Type I = Cut near the restriction site
-Rarely used in DNA manipulation
-Type II = Cut at the restriction site
-The sites are palindromes
-Both strands have same sequence
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when read 5’ to 3’
DNA Manipulation
Type II enzymes produce staggered cuts that
generate “sticky ends”
-Overhanging complementary ends
Therefore, fragments cut by the same enzyme
can be paired
DNA ligase can join the two fragments
forming a stable DNA molecule
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Gel Electrophoresis
A technique used to separate DNA fragments
by size
The gel (agarose or polyacrylamide) is
subjected to an electrical field
The DNA, which is negatively-charged,
migrates towards the positive pole
-The larger the DNA fragment, the slower it
will move through the gel matrix
DNA is visualized using fluorescent dyes
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Transformation
Transformation is the introduction of DNA
from an outside source into a cell
Natural transformation occurs in many species
-However, not in E. coli, which is used
routinely in molecular biology labs
-Artificial transformation techniques have
been developed to introduce foreign
DNA into it
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Molecular Cloning
A clone refers to a genetically identical copy
Molecular cloning is the isolation of a specific
DNA sequence (usually protein-encoding)
-Sometimes called gene cloning
The most flexible and common host for cloning
is E. coli
Propagation of DNA in a host cell requires a
vector
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Vectors
Plasmids are small, circular
extrachromosomal DNA molecules
-Used for cloning small pieces of DNA
-Have three important components
1. Origin of replication
2. Selectable marker
3. Multiple cloning site (MCS)
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Vectors
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Vectors
Phage vectors are modified bacterial viruses
-Most based on phage lambda (l) of E. coli
-Used to clone inserts up to 40 Kbp
-Have two features not shared with plasmid
vectors
-They kill their host cells
-They have linear genomes
-Middle replaced with inserted DNA
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Vectors
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Vectors
Artificial chromosomes
-Used to clone very large DNA fragments
-Bacterial artificial chromosomes (BACs)
-Yeast artificial chromosomes (YACs)
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DNA Libraries
A collection of DNA fragments from a specific
source that has been inserted into host cells
A genomic library represents the entire
genome
A cDNA library represents only the
expressed part of the genome
-Complementary DNA (cDNA) is
synthesized from isolated mRNA using the
enzyme reverse transcriptase
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DNA Libraries
Molecular hybridization is a technique used
to identify specific DNAs in complex mixtures
-A known single-stranded DNA or RNA is
labeled
-It is then used as a probe to identify its
complement via specific base-pairing
-Also termed annealing
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DNA Libraries
Molecular hybridization is the most common
way of identifying a clone in a DNA library
-This process involves three steps:
1. Plating the library
2. Replicating the library
3. Screening the library
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DNA Analysis
Restriction maps
-Molecular biologists need maps to analyze
and compare cloned DNAs
-The first maps were restriction maps
-Initially, they were created by enzyme
digestion & analysis of resulting patterns
-Many are now generated by computer
searches for cleavage sites
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DNA Analysis
Southern blotting
-A sample DNA is digested by restriction
enzymes & separated by gel electrophoresis
-Gel is transferred (“blotted”) onto a
nitrocellulose filter
-Then hybridized with a cloned,
radioactively-labeled DNA probe
-Complementary sequences are
revealed by autoradiography
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DNA Analysis
Northern blotting
-mRNA is electrophoresed and then blotted
onto the filter
Western blotting
-Proteins are electrophoresed and then
blotted onto the filter
-Detection requires an antibody that can
bind to one protein
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DNA Analysis
RFLP analysis
-Restriction fragment length
polymorphisms (RFLPs) are generated by
point mutations or sequence duplications
-These fragments are often not identical in
different individuals
-Can be detected by Southern blotting
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DNA Analysis
DNA fingerprinting
-An identification technique used to detect
differences in the DNA of individuals
-Makes use of a variety of molecular
procedures, including RFLP analysis
-First used in a US criminal trial in 1987
-Tommie Lee Andrews was found guilty
of rape
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DNA Analysis
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DNA Analysis
DNA sequencing
-A set of nested
fragments is generated
-End with known base
-Separated by highresolution gel
electrophoresis,
resulting in a “ladder”
-Sequence is read from
the bottom up
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DNA Analysis
DNA sequencing
-The enzymatic method
was developed by
Frederick Sanger
-Dideoxynucleotides
are used as chain
terminators in DNA
synthesis reactions
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DNA Analysis
DNA sequencing
-The enzymatic technique is powerful but is
labor intensive and time-consuming
-The development of automated techniques
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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DNA Analysis
Polymerase chain reaction (PCR)
-Developed by Kary Mullis
-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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After 20 cycles, a
single fragment
produces over one
million (220) copies!
(Cont.)
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DNA Analysis
Polymerase chain reaction (PCR)
-Has revolutionized science and medicine
because it allows the investigation of minute
samples of DNA
-Forensics
-Detection of genetic defects in embryos
-Analysis of mitochondrial DNA from
early human species
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DNA Analysis
Yeast two-hybrid system
-Used to study protein-protein interactions
-Gal4 is a transcriptional activator with a
modular structure
-The Gal4 gene is split into two vectors
-Bait vector: has DNA-binding domain
-Prey vector: has transcription-activating
domain
-Neither of these alone can activate
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transcription
DNA Analysis
Yeast two-hybrid system
-When other genes are inserted into these
vectors, they produce fusion proteins
-Contain part of Gal4 and the protein of
interest
-If the proteins being tested interact, Gal4
function will be restored
-A reporter gene will be expressed
-Detected by an enzyme assay 43
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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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Genetic Engineering
In vitro mutagenesis
-Ability to create mutations at any site in a
cloned gene
-Has been used to produce knockout mice,
in which 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
Human proteins
-Medically important proteins can be
produced in bacteria
-Human insulin
-Interferon
-Atrial peptides
-Tissue plasminogen activator
-Human growth hormone
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Medical Applications
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Medical Applications
Vaccines
-Subunit vaccines: Genes encoding a part
of the protein coat are spliced into a
fragment of the vaccinia (cowpox) genome
-DNA vaccines: Depend on the cellular
immune response (not antibodies)
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Medical Applications
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Medical Applications
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
-Successful at first, but then patients
developed a rare leukemia
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Agricultural Applications
Ti (tumor-inducing) plasmid is the most
used vector for plant genetic engineering
-Obtained from Agrobacterium tumefaciens,
which normally infects broadleaf plants
-However, bacterium does not infect cereals
such as corn, rice and wheat
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Agricultural Applications
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Agricultural Applications
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Agricultural Applications
Gene guns
-Uses bombardment with tiny gold particles
coated with DNA
-Possible for any species
-However, the copy number of inserted
genes cannot be controlled
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Agricultural Applications
Herbicide resistance
-Broadleaf plants have
been engineered to be
resistant to the
herbicide glyphosate
-This allows for no-till
planting
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Agricultural Applications
Pest resistance
-Insecticidal proteins have been transferred
into crop plants to make them pest-resistant
-Bt toxin from Bacillus thuringiensis
Golden rice
-Rice that has been genetically modified to
produce b-carotene (provitamin A)
-Converted in the body to vitamin A
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Agricultural Applications
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Agricultural Applications
Adoption of genetically modified (GM) crops
has been resisted in some areas because
of questions about:
-Crop safety for human consumption
-Movement of genes into wild relatives
-Loss of biodiversity
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Agricultural Applications
Biopharming
-Transgenic plants are used to produce
pharmaceuticals
-Human serum albumin
-Recombinant subunit vaccines
-Against Norwalk and rabies viruses
-Recombinant monoclonal antibodies
-Against tooth decay-causing bacteria
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Agricultural Applications
Transgenic animal technology has not been
as successful as that in plants
-One interesting example is the EnviroPig
-Engineered to carry the gene for the
enzyme phytase
-Breaks down phosphorus in feed
-Reduces excretion of harmful
phosphates in the environment
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Agricultural Applications
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