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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
-Type II = Cut at the restriction site
-The sites are palindromes
-Both strands have same sequence
when read 5’ to 3’
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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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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
Molecular hybridization is the most common way of identifying
a clone in a DNA 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
DNA fingerprinting
-An identification technique used to detect differences in
the DNA of individuals;
-Makes use of a variety of molecular procedures;
-First used in a US criminal trial in 1987
-Tommie Lee Andrews was found guilty of rape
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DNA Analysis
DNA sequencing
-A set of nested fragments is
generated
-End with known base
-Separated by high-resolution gel
electrophoresis, resulting in a
“ladder”
-Sequence is read from the
bottom up
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DNA Analysis
DNA sequencing
-The enzymatic technique develop by Frederick Sanger 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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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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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
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 bcarotene (provitamin A)
-Converted in the body to vitamin A
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Agricultural Applications
Daffodil
phytoene
synthase
gene (psy)
Bacterial
carotene
desaturase
gene (crtI)
Daffodil
lycopene
b-cyclase
gene (lcy)
Genes introduced
into rice genome
Rice
chromosome
psy
crtI
lcy
Phytoene
synthase
Carotene
desaturase
b-Cyclase
Expression
in endosperm
GGPP
Phytoene
Lycopene
b-Carotene
(Provitamin A)
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Agricultural Applications
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