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Manipulating DNA
Selective Breeding
• Man has selectively
bred plants and
animals for desired
traits
Mutations of Pointsettias induced
by radiation
New DNA Technology
• Now DNA can be
manipulated to
produce new
combinations not
found in nature
• How far should we
go?
– We need to set
bioethical limits
Restriction Endonucleases
• In 1980, geneticists spliced the human gene
that encodes interferon into a bacterial cell’s
genome.
– Marked beginning of genetic engineering.
• Through natural selection, some bacteria
contain enzymes, restriction endonucleases,
that fragment viral DNA.
– Recognize specific nucleotide sequences, and
cleave the DNA.
– They recognize palindromes
• Methylate their own DNA for protection
Restriction Endonucleases
• Sequences recognized by restriction endonucleases are
typically four to six nucleotides long, and are often
palindromes.
– Restriction enzymes can bind to and cleave both strands
of DNA.
Raven - Johnson - Biology: 6th Ed. - All Rights Reserved -
Restriction Endonucleases
• There are hundreds of bacterial restriction
endonucleases, each with a specific
recognition sequence.
– Given restriction endonuclease can cut DNA from
any source into fragments.
• Complementarity, single-stranded ends can pair with
each other.
– Sticky Ends
» Fragments joined together with DNA ligase.
Using RE’s to Manipulate
Genes
• Chimeras
– One of first recombinant genomes produced
by genetic engineering was a bacterial
plasmid into which an amphibian ribosomal
RNA gene was inserted.
– Viruses can also be used as vectors to insert foreign
DNA into host cells.
Genetic Engineering
Experiment
• Four Stages
– DNA Cleavage
• Restriction endonuclease cleaves source DNA into
fragments.
– Production of Recombinant DNA
• DNA fragments inserted into plasmids or viral
vectors.
– Cloning
– Screening
Genetic Engineering
Experiment
– Screening
• Clones with DNA fragment of interest are identified from
the clone library.
– Preliminary Screening - Eliminate any clones without a vector
and clones with vectors that do not contain DNA.
» Employ vector with gene for antibiotic resistance and lac Z’
gene.
» Expose to growth medium.
Genetic Engineering
Experiment
– Secondary Screening (Gene of Interest)
• Hybridization - Cloned genes form base pairs with
complementary sequences on another nucleic acid
(probe).
– Grow on agar and then transfer to a filter pressed on colonies.
» Treat filter with radioactive probe, and perform
autoradiography.
Working With Gene Clones
• Polymerase Chain
Reaction (PCR)
– Used to copy
specific gene
sequences.
• Three Basic
Steps
– Denaturation
– Annealing of
Primers
– Primer
Extension
Restriction Fragment Length Polymorphism
(RFLP) Analysis
• DNA is cut into small
pieces using restriction
enzymes
• Many copies are made
using PCR
• Radioactive probes
attached to mark ends
• An electric current is used
to separate pieces by
size in a gel
– Larger pieces are slower
and don’t move as far
down the gel
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tions/content/gelelectrophoresis.html
DNA Fingerprinting
• DNA is unique for every
individual
• Comparing the
sequences of small
pieces of DNA can be
used:
– Find relationships
– Narrow crime suspects
– Identify organisms
DNA Sequence Technology
• Genome Sequencing
– Due to technological advances, sequencing of
entire genomes is now practical.
DNA Sequence Technology
• Human Genome Project
– Human genome contains around 3.2 billion
bases and about 30,000 genes.
• Human genes are quite fragmented.
• Fully one-third of genome is composed of
noncoding repeated sequences.
• Genes are not evenly distributed over the genome.
DNA Sequence Technology
• Proteonomics
– Cataloging and analyzing every protein in the
human body.
• Each gene’s nucleotide sequence specifies an
amino acid sequence folded in a certain way.
Biochips
• Gene Microarrays (Biochips) are small
squares of glass covered with millions of
strands of DNA.
– Scanning beam used to build nucleotides.
• Look at Single Nucleotide Polymorphisms (SNP’s).
– Cancer Screening
– Personal Privacy Issues
Medical Applications
• Pharmaceuticals
– Introduction of protein-encoding genes.
• Atrial peptides - High blood pressure and kidney
failure.
• Tissue plasminogen activator - Dissolving blood
clots.
Medical Applications
• Gene Therapy
• These recent clinical
– Add working copies of single
successes have led to a
defective gene.
renewed interest in gene
• Leber's congenital
therapy
amaurosis
• X-linked SCID
• Adrenoleukodystrophy
• chronic lymphocytic
leukemia
• acute lymphocytic
leukemia
• multiple myeloma
• Parkinson's disease
Medical Applications
• Piggyback Vaccines
– Produce subunit vaccines against viruses.
• Herpes
• Hepatitis
– DNA Vaccine
• Cellular immune response.
Agricultural Applications
• Ti plasmid has been early successful
vector.
– Herbicide Resistance
• Insert genes encoding for proteins making crops
resistant to herbicide.
– Widespread herbicide use possible.
– Nitrogen Fixation
• Introduce genes that allow crops to fix nitrogen.
– Reduce need for fertilizer.
Agricultural Applications
– Insect Resistance
• Insert genes encoding proteins harmful to insects.
• Real Promise - Produce genetically modified
plants with traits benefiting consumers.
– Iron Deficiency in developing countries
• Transgenic Rice
– Increasing milk production
• Bovine Somatotrophin
Cloning
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Raven - Johnson - Biology: 6th
Ed. - All Rights Reserved -
Tools needed in Gene Cloning
• Restriction enzymes
– Cuts the desired DNA strands
• Bacterial plasmid
– cloning vector used to move
DNA to another organism
• Host DNA
– Donor gene(s)
Produces a transgenic
organism with recombinant
DNA
Clones
• Copies of one organism
Clones
• Produced by taking cuttings from one plant
– Grafting
– Budding
– Rooted cuttings
Clones
• Produced for many years by dividing an embryo
to form identical quadruplets
– Cows
– Pigs
A New Type of Clone
• The nucleus of a donor cell
• The ova (egg) of another organism
• A surrogate mother
How it’s done…
FDA says cloned animals safe to eat
• Food producers, including
Tyson Foods Inc. and Dean
Foods Co., said they won't
use cloned foods anytime
soon.
• Bloomberg News / January
16, 2008
• WASHINGTON - Cloned
cows, pigs, goats, and their
offspring are safe to enter
the US food supply,
regulators said over protests
from lawmakers, consumer
groups, and worried eaters.
Maryann Mott
for National Geographic News
Champion Horse Cloned by Texas Breeder
April 4, 2006
Meet Royal Blue Boon Too, the first commercially cloned horse in
the United States.
The cost of the frisky foal: U.S. $150,000.
That may be a bargain, considering that the original Royal Blue
Boon, a 26-year-old American Quarter Horse now past
breeding age, has earned more than $380,000 as a competition
and show horse.
.
Some problems to Overcome:
• Many casualties
– Birth defects
– Failures
– Premature aging
• Improve technique
Stem Cells
• Embryonic Stem Cells
– Cells produced very early in developmenteach capable of developing into a healthy
individual.
– Can develop into any tissue.
• May provide ability to restore damaged tissue.
• Tissue Specific Stem Cells
– As development proceeds, tissue-specific
stem cells persist.
Controversial topics…
• Scientists hope to use cloned embryos to harvest precious "stem
cells" — cells that, in theory, can ultimately grow into any kind of cell
in the body.
• These cells could be used to generate new organs or cell clusters to
treat patients with failing organs or degenerative diseases
•
In the case of transplant patients, organs made from exact copies of
an individual's own cells and would not be rejected.
• This research is still in its earliest phase of development. All of the
embryos generated actually died — none grew beyond the six-cell
stage
• Controversial: The thinking is that by allowing any kind of cloning of
human embryos, it is then not such an enormous leap to apply the
same techniques to making copies of people.
Ethics and Regulation
• Questions
– How do we measure the Potential Risks of
Genetically-Modified Crops?
– Is Eating Genetically Modified Food
Dangerous?
– Are Genetically Modified Crops harmful to the
Environment?
– Should We Label Genetically Modified Foods?
Copyright © McGraw-Hill Companies Permission required for reproduction or display