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Genetic Engineering
4.4.7 – State that when genes are transferred between
species, the amino acid sequence of polypeptdies
translated from them is unchanged because the genetic
code is universal.
 Genetic engineering –
•
DNA technology has resulted in biotechnology,
•
DNA technology is now applied in areas ranging
from agriculture to criminal law
4.4.7 – State that when genes are transferred between
species, the amino acid sequence of polypeptdies
translated from them is unchanged because the genetic
code is universal.
 Recombinant DNA –
•
•
This works because the genetic code is
Each codon codes for
Human DNA (ex.gene
coding for human
growth hormone,
insulin, etc.)
Plasmid
(bacterial
DNA)
Recombinant DNA
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
Genetic engineering is possible because of
restriction enzymes (restriction endonucleases):
• Very specific –
• These are often a symmetrical series of four to eight
bases on both strands running in opposite directions.
• If the restriction site on one strand is 3’-CTTAAG-5’, the
complementary strand is 5’-GAATTC-3’.
• In nature,
– each enzyme has a different restriction site
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
• Restriction enzymes
oSticky ends will form
o
bonds the
complementary sticky ends together
oRestriction enzymes and DNA ligase are used to
DNA pieces
together
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
Bacterial Transformation –
– uses include:
• Bacteria that can produce hormones such as human
growth hormone and insulin
• Bacteria that eat oil slicks
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
Escherichia coli
• Often used for genetic engineering
• Common inhabitant of human colon –
• Can be easily grown in suspension culture
• Has a simple circular chromosome with about 1/600th
the haploid amount of DNA in a human cell
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
• E. coli often contain small
circular DNA molecules called
(extrachromosomal)
oPlasmids usually confer a
particular trait
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
• A vector is a device for
• Plasmids are easily isolated and re-introduced to bacteria
so they are
.
• We can easily introduce our own genes to plasmids to
produce desired products.
• Vector plasmids are produced by:
oCutting DNA out of a chromosome using
oCutting the plasmid open with
.
oInserting the desired gene into a plasmid to act as a carrier.
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
oThe recombinant plasmid is then inserted into a
bacterial cell.
oThe bacteria that have the recombinant plasmid
inside of them are said to have been
These bacteria can now
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
oThe desired gene is often inserted into a plasmid with
genes for
so that the
transformed bacteria can be easily selected from other
cells that did not pick up the plasmid.
Plasmid
(bacterial
DNA)
Human DNA (ex.gene
coding for human
growth hormone,
insulin, etc.)
Genetic marker (tells us if the
bacteria cell has the plasmid)
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
In nature, genes can be transferred between
bacteria in three ways:
• Conjugation –
• Transduction –
• Bacterial Transformation – involves the transfer of
genetic information into a cell by direct uptake of the
DNA (
)
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
Transformation in the Laboratory:
• Transformation was first performed in the laboratory by
Griffith and later by Avery, MacLeod, and McCarty
(experiment using mice and pneumococcus bacteria)
• Bacteria can take up DNA only during the period at the
– cells are said to be
(can accept DNA that is introduced
from another source)
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
• E. coli competence can be
• Plasmids can transfer genes and act as carriers
for introducing DNA from other bacteria or from
eukaryotic cells
• E. coli cell membrane is weakened using
• E. coli cells are then “
• Sterile technique must be used
” to
4.4.8 – Outline a basic technique used for gene
transfer involving plasmids, a host cell (bacterium,
yeast, or other), restriction enzymes
(endonucleases) and DNA ligase.
• Transformation Lab –
o Ampicillin kills bacteria by
Plasmid
Gene coding
for ampicillin
resistance
DNA Profiling
• Restriction Enzymes are also used for DNA
profiling
oDNA profiling creates
• DNA fingerprints are based on parts of an individual’s
DNA that can by used for identification.
obased on
ononcoding regions have
o
oWe inherit these noncoding regions from our mom and
dad
• We can use
to cut
near these noncoding sequences.
• Because the restriction site (recognition sequence)
usually occurs (by chance) many times on a long
DNA molecule,
• Result:
– Restriction Fragment Length
Polymorphs (RFLPs)
• Since all individuals have unique sequences of DNA,
restriction enzymes cut each individual’s DNA into
4.4.2 – State that, in gel electrophoresis, fragments
of DNA move in an electric field and are separated
according to their size.
4.4.3 State that gel electrophoresis of DNA is used
in DNA profiling.
•The RFLPs are then separated by gel
electrophoresis resulting in a bar-like
pattern
oElectrophoresis means “
”
oDifferent sized RFLPs will be carried
• Electricity is run through the gel box creating a
4.4.2 – State that, in gel electrophoresis, fragments
of DNA move in an electric field and are separated
according to their size.
o
charged DNA migrates
from the
of the gel box
through the pores in the gel to the
of the gel box
o
o
visible
is used to make the DNA bands
SEM photo of a 1% LE Agarose gel at 22kX magnification
4.4.4 – Describe the application of DNA profiling to
determine paternity and also in forensic
investigations.
Uses for DNA profiling:
• Allows scientists to compare DNA from various
organisms and identify a particular individual (DNA
can be extracted from blood, saliva, hair roots, and
skin)
• Crimework: rape and murder cases (forensics)
• Paternity suits
• Missing persons and unidentified bodies
• Immigration disputes
• Animal work - breeding
4.4.4 – Describe the application of DNA profiling to
determine paternity and also in forensic
investigations.
4.4.9 – State two examples of the
current uses of genetically modified
crops or animals
Agricultural uses of DNA technology
• Animal Husbandry – many farm animals are treated
with products made by recombinant DNA methods
(examples include vaccines, antibodies, and growth
hormones)
oSome milk cows are injected with
oBGH also improves
4.4.9 – State two examples of the current uses of
genetically modified crops or animals
• Transgenic animals –
oExamples: beef and dairy cattle, hogs, sheep and several
species of commercially raised fishes
4.4.9 – State two examples of the current uses of
genetically modified crops or animals
oModified DNA can be introduced into dairy cows and
sheep so that they
 Examples of medically important proteins that have
been produced in transgenic mammals include:
• Blood clotting Factor VIII to
• Alpha-1 antitrypsin (AAT protein) protects lungs,
without it the alveoli are susceptible to damage and
can lead to lung disease
• http://learn.genetics.utah.edu/content/disorders/what
aregd/a1ad/
• Rainbow trout and salmon that are given
can
4.4.9 – State two examples of the current uses
of genetically modified crops or animals
•Transgenic plants
oPlants have been genetically altered to
receive
(several strains of cotton)
 Makes them
oSome crop plants are being engineered to
resist infectious pathogens and pest insects –
4.4.9 – State two examples of the current uses of
genetically modified crops or animals
oFirst genetically engineered fruits approved by the
FDA for human consumption were
 Researchers isolated gene responsible for ripening
 They prepared a gene who's template strand had a base
sequence complementary to the normal gene – an
antisense version of the gene
 When spliced into the DNA of a tomato plant, the
antisense gene is transcribed into RNA that is
complementary to the ripening gene’s mRNA
 the antisense RNA binds to the normal mRNA, blocking
the synthesis of the enzyme causing ripening and
spoilage
Videos
Genetically modifying humans
 http://www.pbs.org/wgbh/nova/genome/media/2809_q056_15.html
Spider goats (2:39):
 http://science.discovery.com/videos/kapow-superhero-science-spider-silkgene-goats.html
Jelly pig:
 http://news.bbc.co.uk/player/nol/newsid_4600000/newsid_4609200/4609202.
stm?bw=nb&mp=wm&news=1&ms3=6&ms_javascript=true&bbcws=2
Transgenic rabbits (4:04) – doesn’t work at school
 http://video.google.com/videoplay?docid=6349294947234590009&ei=mVp1
Sd3-DJy4qAPB15CpBA&q=transgenic+&hl=en
Classical vs. transgenic breeding
 http://www.teachersdomain.org/resource/tdc02.sci.life.gen.breeding/
4.4.10 – Discuss the potential benefits and possible
harmful effects of one example of genetic modification
See handouts and
Clegg pg. 127-128
4.4.11 – Define clone.
Cloning
• Clone –
• Gene cloning –
oDonor gene inserted into a bacterium is copied
every time the plasmid containing it replicates –
genes can be cloned by growing genetically
engineered bacteria
4.4.1 – Outline the use of polymerase chain reaction
(PCR) to copy and amplify minute quantities of DNA.
Polymerase Chain Reaction (PCR)
• Cloning a gene through genetic engineering can
be time-consuming and requires an adequate
DNA sample as starting material
• PCR technique
• PCR is useful in
4.4.1 – Outline the use of polymerase chain reaction
(PCR) to copy and amplify minute quantities of DNA.
•How does it work?
• DNA polymerase uses nucleotides and primers
to replicate a DNA sequence in vitro, thereby
producing two molecules
• Two strands of each molecule are then
separated by heating and replicated again, so
then there are four, double-stranded molecules
• After the next cycle of heating and replication
there are eight molecules, and so on
• Number of molecules doubles with each cycle
• http://www.dnalc.org/resources/animations/pcr.ht
ml
Cloning organisms
• Cloning sometimes occurs naturally (
)
• Organisms can be cloned artificially
o Sheep, rabbits, toads and other sexually
reproducing animals have been cloned by
dividing up an embryo and transplanting them
into surrogate mothers
4.4.12 – Outline a technique for cloning using
differentiated animal cells.
• Cloning of Dolly
1) Sheep cloned from a
2) Cell taken from
and cultured in lab for 6 days
3) Unfertilized egg taken
–
4) Egg without nucleus is fused with donor cell using a
5) Embryo resulting from fusion of udder cell and egg is
transferred into the uterus of a third sheep who acts as
the
6) Surrogate mother gives birth to lamb – lamb is
genetically identical to
The Process of Nuclear Transplantation (1:22)
http://streaming.discoveryeducation.com/searc
h/assetDetail.cfm?guidAssetId=30B3BAF149D1-48FC-B959-47148A6FD7CE
Cloning Adult Animals (3:36)
Stem Cells
• Stem cells are cells that
• Plants contain stem cells in their
(reason why a cutting can grow into a new plant)
• Embryonic stem cells are pluripotent –
• Adult stem cells can divide to form
(i.e. blood stem cells)
•Therapeutic Uses of Stem Cells
oEmbryonic stem cells are the most flexible
and can grow into any type of mature cell
Parkinson’s and Alzheimer’s Disease can be
potentially treated by implanting stem cells that
could replace the damaged cells
4.4.13 – Discuss the ethical issues of therapeutic
cloning in humans.
o Ethical issues surrounding
therapeutic cloning:
 Therapeutic Cloning is the creation of an
embryo to supply embryonic stem cells
for medical use
• Raises issue of whether it is right or wrong to
generate a new human embryo for medical
research
4.4.13 – Discuss the ethical issues of therapeutic
cloning in humans.
 Two distinct forms of cloning:
1. Reproductive cloning –
2. Therapeutic cloning –
 Opinions vary about whether both
forms are right/wrong or if one or the
other is acceptable
4.4.6 – Outline three outcomes of the sequencing of
the complete human genome.
Human Genome Project
• A project that involved mapping the entire
human genome –
• Completed in June of 2000.
4.4.6 – Outline three outcomes of the sequencing of
the complete human genome.
• Outcomes of the HGP:
1. Determined how many individual genes we have and
how they work (30,000 to 45,000 genes)
2. Locating and determining the cause of genetic
disorders.
3. Development of gene therapies to treat genetic
disorders.
•
•
Supplying body with missing gene’s product
Supply body cells with missing gene to permanently fix it
4. Comparing genetic makeup of human populations to
determine ancestries and how humans have migrated
and mixed their genes with other populations over
time.