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Biotechnology and Genomics
Chapter 17 & 18
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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Recombinant DNA
• Recombinant DNA (rDNA) contains DNA
from two or more different sources.
• Restriction enzymes cleave the vector
DNA and the source DNA at a specific
sequence.
• Restriction enzymes
• Used in manipulating DNA
– Recognize specific DNA sequences
– Cleave at specific site within sequence
– Can lead to “sticky ends” that can be joined
to allow a portion of the source DNA to be
inserted into the vector DNA.
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• DNA ligase - Joins the two fragments
forming a stable recombinant DNA
molecule.
• After recombinant DNA enters the host cell,
it may be copied.
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Molecular Cloning
• Clone – genetically identical copy
• Occurs naturally in new plant shoots, bacterial
colonies and identical human twins.
• Gene cloning – isolation of a specific DNA
sequence, producing identical copies of a gene.
• 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
• A vector plasmid (a small accessory ring
of DNA in bacteria) or virus is necessary to
insert foreign DNA into a host cell.
• Plasmids
– Small, circular chromosomes
– Used for cloning small pieces of DNA
Bacterial cells take up recombinant plasmids
and clone the new DNA.
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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
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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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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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Gel Electrophoresis
• Separate DNA fragments by size (restriction
enzymes)
• Gel usually made of agarose
• 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
• Results in a pattern unique to the individual (Genetic
fingerprint)
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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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• 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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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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• Polymerase chain reaction (PCR)
– Developed by Kary Mullis
• Awarded Nobel Prize
– Produces many copies of a single gene or
piece of DNA. Requires DNA polymerase
and a supply of nucleotides for the new DNA
strand.
– Each PCR cycle involves three steps:
1.Denaturation (high temperature)
2.Annealing of primers (low temperature)
3.DNA synthesis (intermediate temperature)
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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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Genetic Engineering
• Has generated excitement and
controversy
• Expression vectors contain the sequences
necessary to express inserted DNA in a
specific cell type
• Transgenic organisms have had a foreign
gene inserted in them.
• Transgenic animals contain genes that
have been inserted without the use of
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conventional breeding
Medical Applications
• Transgenic bacteria - Medically important
proteins can be produced in bacteria
– Human insulin
– Interferon
– Hepatitis B vaccine
– Human growth hormone
– Problem has been purification of desired
proteins from other bacterial proteins
– Bacteria has been added to degrade waste
and help mine metals.
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Genetically engineered mouse
with human growth hormone
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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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Ex Vivo Gene Therapy
• The ex vivo method withdraws tissues from the
patient. Bone marrow stem cells are withdrawn
from the body, a retrovirus is used to insert a
normal gene into them, and the stem cells are
returned to the body.
• This method works for (SCIDs) and may work for
familial hypercholesterlemia where liver cells
lack a receptor for removing blood cholesterol.
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In Vivo Gene Therapy
• Cystic fibrosis patients lack a gene that codes
for a membrane carrier of chloride ions;
researchers try to deliver the gene by nose
sprays.
• Many researchers are trying to cure cancer by
inserting genes to make healthy cells tolerant of
chemotherapy or use gene p53 to bring about
apoptosis of cancer cells.
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Agricultural Applications
• Transgenic plants - Foreign genes are
added to protoplasts (lack a cell wall)
using electric current.
• Foreign genes in cotton, corn, and
potatoes have given them pest resistance;
soybeans are made resistence for no till
farming.
• Transgenic plants produce human
hormones, clotting factors and antibodies
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in their seeds.
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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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• Golden rice
– Rice that has been genetically modified to
produce b-carotene (provitamin A)
– Converted in the body to vitamin A
– Could not have been done by conventional
breeding as no rice known produces these
enzymes.
– 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
• Main use thus far has been engineering animals
to produce pharmaceuticals in milk (also
biopharming)
• Can be inserted into eggs by hand or vortex
mixing.
• Gene farming uses transgenic farm animals to
produce pharmaceuticals in milk. Plans to use
animals to produce drugs for treatment of cystic
fibrosis, cancer, etc.
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Human Genome
The Human Genome Project has two
goals:
• to know the sequence of bases on all
the human chromosomes
• to construct a map of genes on all the
human chromosomes.
• This task has been completed and researchers know
the sequence of three billion base pairs after 15
years of research.
• The two agencies that completed the task are The
International Human Genome Sequencing
Consortium and Celera Genomics, a private
company.
• However, knowledge of the sequence merely leads to
additional work to determine how it acts. DNA
sequences are similar among organisms and the
differences may be due to regulation of genes.
The Human Genome Project found fewer
genes than expected
-Initial estimate was 100,000 genes
-Number now appears to be about 25,000
-However, the complexity of an organism is
not necessarily related to its gene number
Genomics
Bioinformatics
- Use of computer programs to search for
genes, and to assemble and compare
genomes
Each cell in our bodies has about 6 feet of
DNA stuffed into it
• However, less than one inch is devoted to
genes!
Genome Organization
Genomes consist of two main regions
• Coding DNA
- Contains genes than encode proteins
• Noncoding DNA
- Regions that do not encode proteins
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Comparative Genomics
• Comparative genomics, the study of whole
genome maps of organisms, has revealed
similarities among them
• Comparing genomes (entire DNA
sequences) of different species provides a
powerful new tool for exploring the
evolutionary divergence among organisms
• Genomes are instructions and a history of
life
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Applications of Genomics
• The genomics revolution will have a lasting
effect on how we think about living systems
• The immediate impact of genomics is being
seen in diagnostics
- Identifying genetic abnormalities
- Identifying victims by their remains
- Distinguishing between naturally occurring and
intentional outbreaks of infections
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Applications of Genomics
• Genomics has also helped in agriculture
– Improvement in the yield and nutritional
quality of rice
– Corn resistant to drought stress
• - Doubling of world grain production in last
50 years, with only a 1% cropland
increase
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• Genome science is also a source of
ethical challenges and dilemmas
- Gene patents
- Should the sequence/use of genes be
freely available or can it be patented?
- Privacy concerns
- Could one be discriminated against
because their SNP profile indicates
susceptibility to a disease?
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• Stem cells
– Cells that are capable of continued division,
but can also give rise to differentiated cells
– Degree of determination
• Totipotent – cell that can give rise to any tissue in
an organism (embryo and extraembryonic
membranes)
• Pluripotent – give rise to all cells in the adult
organism’s body
• Multipotent – give rise to limited number of cells
• Unipotent – give rise to only a single cell type
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• Embryonic stem cells (ES cells)
– Form of pluripotent stem cells
– Made from mammalian blastocysts
– ES cells isolated from inner cell mass and
grown in culture
– In mice, have been shown to develop into any
type of cell in the tissues of the adult
• Cannot develop into extraembryonic membranes
– 1998 – first human ES cells
• Great promise and controversy
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Nuclear reprogramming
• Early experiments showed nuclei could be
transplanted between cells
– Cells do not appear to undergo any truly irreversible
changes, such as loss of genes
– More differentiated the cell type, the less successful
the nucleus in directing development when
transplanted
• Nuclear reprogramming – nucleus from a
differentiated cell undergoes epigenetic changes
that must be reversed to allow the nucleus to
direct development
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• 1984 – sheep was cloned using the
nucleus from a cell of an early embryo
• 1996 – Dolly, the first clone generated
from a fully differentiated animal cell
– Used somatic cell nuclear transfer (SCNT)
– Dolly matured into fertile adult
– Established beyond all dispute that
determination in animals is reversible
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Preparation
Cell Fusion
Mammary cell is extracted and grown in
nutrient-deficient solution that arrests the cell cycle.
Nucleus containing
source DNA
Egg cell is extracted.
Development
Embryo begins to
develop in vitro.
Mammary cell is
inserted inside
covering of egg cell.
Cell Division
Electric shock fuses
cell membranes and
triggers cell division.
Nucleus is removed fro
egg cell with a micropipette.
Implantation
Birth of Clone
Growth to Adulthood
Embryo is
After a five-month pregnancy, a
implanted into lamb genetically identical to the
surrogate
sheep from which the mammary
mother .
cell was extracted is born.
Embryo
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© APTV/AP Photo
• Reproductive cloning
– Uses SCNT to create animal genetically
identical to another
– Efficiency is quite low and other problems
• Only 3–5% of adult nuclei transferred to donor
eggs result in live births
– Due to lack of genomic imprinting
• Normal mammalian development depends on
precise genomic imprinting
• Organization of chromatin in adult and embryo
very different
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• Much work has been put into trying to find ways
to reprogram adult cells to become pluripotent
cells without the use of embryos
• Different lines of inquiry showed that
reprogramming of somatic nuclei was possible
• 2006 – genes for 4 different transcription factors
introduced into fibroblast cells in culture
– Named induced pluripotent stem cells (iPS cells)
– Appear to be similar to ES cells in terms of
developmental potential, as well as gene expression
pattern
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Oocyte
Nuclear
Transfer
Somatic cells
Somatic cells
Fusion
Blastocyst
ES cells
Defined
factors
Culture
Pluripotent
stem cells
Somatic cells
Germ cells
Some adult stem cells
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• Therapeutic cloning
– Produce patient-specific lines of embryonic
stem cells
– Artificial embryo created using same process
as Dolly (SCNT)
– Its cells are used as embryonic stem cells for
transfer to injured tissue
– Body readily accepts these cells with no
immune rejection
– May be obsolete with development of iPS
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cells
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
The nucleus from a skin cell of a diabetic patient is
removed.
The skin cell
nucleus is inserted
into the enucleated
human egg cell.
Cell cleavage
occurs as the
embryo begins to
develop in vitro.
The embryo
reaches the
blastocyst stage.
Inner cell
mass
Diabetic
patient
Early embryo
ES
cells
Blastocyst
Therapeutic cloning
Embryonic stem cells
(ES cells) are extracted
and grown in culture.
The stem cells are developed
into healthy pancreatic islet cells
needed by the patient.
Healthy pancreatic islet cells
The healthy tissue is
injected or transplanted
into the diabetic patient.
Diabetic
patient
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Morphogenesis
• Generation of ordered form and structure
• Product of changes in cell structure and cell
behavior
• Animals regulate
–
–
–
–
–
The number, timing, and orientation of cell divisions
Cell growth and expansion
Changes in cell shape
Cell migration (not used in plants)
Cell death
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• Apoptosis
– Programmed cell death a part of development
• Human embryos begin with webbed fingers, but
webbing “dies” as part of development.
– Necrosis – cells that die due to injury
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