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9.1 Manipulating DNA 9.1 Manipulating DNA

KEY CONCEPT What steps does DNA Technology Involve?

Biotechnology relies on cutting DNA at specific
places.

9.1 Manipulating DNA 9.1 Manipulating DNA

Scientists use several techniques to manipulate DNA.
• Chemicals, computers, and bacteria are used to work
with DNA.
• Scientists use these tools in genetics research and
biotechnology.
Restriction enzymes cut DNA.
• Restriction enzymes act as “molecular scissors.”
– come from various types of bacteria
– allow scientists to more easily study and manipulate
genes
– cut DNA at a specific nucleotide sequence called a
restriction site

9.1 Manipulating DNA 9.1 Manipulating DNA

• Different restriction enzymes cut DNA in different – some cut straight across and leave “blunt ends”
ways.
– some make staggered cuts and leave “sticky ends”
– each enzyme has a different restriction site

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Restriction maps show the lengths of DNA fragments.

• Gel electrophoresis is used to separate DNA fragments
by size.
– A DNA sample is cut with restriction enzymes.
– Electrical current pulls DNA fragments through a gel.

9.1 Manipulating DNA 9.1 Manipulating DNA

– Smaller fragments move faster and travel farther • A restriction map shows the lengths of DNA fragments
than larger fragments. between restriction sites.
– Fragments of different – only indicate size, not
sizes appear as bands DNA sequence
on the gel. – useful in genetic
engineering
– used to study
mutations

DNA
Electrophoresis
Animation

9.1 Manipulating DNA 9.1 Manipulating DNA

Examples KEY CONCEPT

The polymerase chain reaction rapidly copies
segments of DNA.
• When is the technique used to separate DNA,
– To get a DNA fingerprint for forensic purposes
– To get a DNA fingerprint for paternity testing
– To get a DNA fingerprint so that you can look for
evolutionary relationships among organisms
• When is the technique used to separate proteins
– Many know you can separate DNA fragments – thank
you TV and movies – but you can also separate
proteins if the correct gel is used
– Think about it – do all proteins contain the same
number of amino acids?

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PCR uses polymerases to copy DNA segments. PCR is a three-step process.

• PCR makes many copies of a specific DNA sequence in a • PCR uses four materials.
few hours. – DNA to be copied
– DNA polymerase
– A, T, C, and G nucleotides
– two primers

DNA strands

target sequence of DNA primer 1

polymerase

• PCR amplifies DNA samples.

• PCR is similar to DNA replication. nucleotides
primer 2

9.1 Manipulating DNA 9.1 Manipulating DNA

• The three steps of PCR occur in a cycle. • Each PCR cycle doubles the number of DNA molecules.
– heat is used to separate double-stranded DNA
molecules
– primers bind to each DNA strand on opposite ends of
the segment to be copied
– DNA polymerase binds nucleotides together to form
new strands of DNA

DNA strands

primer 1

polymerase

primer 2
nucleotides

9.1 Manipulating DNA 9.1 Manipulating DNA

Uses of PCR

• PCR can isolate a single gene our of hundreds of thousands. (just to

give you an idea- a gene of 3000 bp (or less) can readily be fished
out of a human genome of 3 billion bp)
• PCR can also be used to introduce mutations into genes, which can
then be studied to understand the effects of these mutations on the
function of the encoded protein.
• Since it is possible to amplify large amounts of DNA from tiny traces,
it is used by forensic labs to get sufficient DNA from evidence at
crime scenes to help identify "whodunnit".
• For the same reason as above, PCR can be used to detect the
presence of infectious agents,for example, the AIDS virus (HIV) in
blood, long before it can be detected by other means
available today.

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Examples KEY CONCEPT
• Disease DNA fingerprints identify people at the molecular
– Tissue typing for organ transplants level.
– Prenatal testing – are you are carrier for a genetic disease
– Epidemics - ie strain in wildlife/human
• Forensics
– Fingerprint testing – samples to crime scene or close
relative identification
• Research
– Can get known segments of genetic disease mutation
– Phylogenic analysis to link to ancient sources –
mammoths, and neanderthals OH MY!
– Patterns of gene expression – ie through different
developmental stages

9.1 Manipulating DNA 9.1 Manipulating DNA

A DNA fingerprint is a type of restriction map.

• DNA fingerprints are based on parts of an individual’s
DNA that can by used for identification.
– based on noncoding regions of DNA
– noncoding regions have repeating DNA sequences
– number of repeats differs between people
– banding pattern on a gel is a DNA fingerprint

9.1 Manipulating DNA 9.1 Manipulating DNA

DNA fingerprinting is used for identification. • DNA fingerprinting is used in several ways.
• DNA fingerprinting depends on the probability of a – evidence in criminal
match. (mother) (child 1) (child 2) (father)
cases
– Many people have the – paternity tests
same number of
– immigration requests
repeats in a certain
region of DNA. – studying biodiversity
– The probability that two – tracking genetically
people share identical modified crops
numbers of repeats in
several locations is
Cleared by
very small.
DNA list
Innocence
project

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KEY CONCEPT Entire organisms can be cloned.

DNA sequences of organisms can be changed.
• A clone is a genetically identical copy of a gene or of an
organism.

9.1 Manipulating DNA 9.1 Manipulating DNA

• Cloning occurs in nature.
– bacteria (binary fission)
– some plants (from roots)
– some simple animals (budding, regeneration)
• Mammals can be cloned through a process called nuclear
transfer.
– nucleus is removed from an egg cell
– nucleus of a cell from the animal to be cloned is
implanted in the egg

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• Cloning has potential benefits.

– organs for transplant into humans
– save endangered species
• Cloning raises concerns.
– low success rate ( Dolly was the only one out of 277
embyros)
– clones “imperfect” and less healthy than original animal
– decreased biodiversity

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• Carp
9.1 Manipulating DNA 9.1 Manipulating DNA
• Sheep
Animals Clones • Mouse
• Cows
New genes can be added to an organism’s DNA.
• Goats
• Genetic engineering involves changing an organism’s
• Pigs
DNA to give it new traits.
• Mouflon
• Rhesus Monkey • Genetic engineering is based on the use of
• Guar recombinant DNA.
• Rabbit • Recombinant DNA contains genes from more than one
• Cat
organism.
• Rat
• Mule
• Horse
• African Wildcat
• Deer
• Ferret
• Buffalo
• Dog
• Wolves (bacterial DNA)

9.1 Manipulating DNA 9.1 Manipulating DNA

Genetic engineering produces organisms with new

traits.
• A transgenic organism has one or more genes from
another organism inserted into its genome.

9.1 Manipulating DNA 9.1 Manipulating DNA

• Transgenic bacteria can be used to produce human Transgenic Animals????

proteins.
• In cases like insulin production, the gene can be inserted
– gene inserted into plasmid
into a plasmid and replicated in bacteria and grown in
– plasmid inserted into bacteria vats
– bacteria express the gene • Sometimes, mammalian cells are needed so they use
– Can also create a transgenic animal in cases where diary animals like cows and goats – why MILK!!!!
bacteria aren’t possible – Can just milk the animal and harness large amounts of
• Transgenic plants are common in agriculture. the protein produced.
– transgenic bacteria – Gene is given an on/off switch that only turns on in
infect a plant mammary cells => only in milk
– plant expresses – Treatments for hemophilia, blood clotting disorders,
foreign gene – Cystic fibrosis, and anemia are some being studied
– many crops are now
Video on how
genetically modified
it is done
(GMO)

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GMOs Oh My

Pros Cons
• WHO, American • Rise of superweeds
Academy of Sciences, • Health Problems
and AMA say they are • Frankenfoods
safer than traditional
– Inadvertent creation of
methods other products due to
• More food introduction of genes and
bacteria
• Less stress on
environment • Other organisms could be
harmed in an ecosystem
• Better products

9.1 Manipulating DNA 9.1 Manipulating DNA

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• Transgenic animals are used to study diseases and

gene functions.
– transgenic mice used to study development and
disease
– gene knockout mice used to study gene function

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Genetically modified cattle treating Ebola??? • Scientists have concerns about some uses of genetic
• Using cloned, genetically engineered cattle – identical, part engineering.
human – possible long-term health effects of eating GM foods
• Incubating antibodies against ebola – will be in blood plasma
– possible effects of GM plants on ecosystems and
• Genetically engineered to produce human antibodies biodiversity
INSTEAD of cattle antibodies
• Vaccinated against deadly disease to make them produce
antibodies which can then be removed and used to treat
humans
• Similar to transfusing patients with survivor serum
• 30-60L of plasma enough = 500-1,000 doses/month/animal
• Currently being used on mice – can protect if only exposed
one day with one dose, beyond that working on everyday
doses
• Next monkeys and then onto humans
• Hantavirus, flu, MERS as well – 50 cattle currently

9.1 Manipulating DNA 9.1 Manipulating DNA

KEY CONCEPT Genomics involves the study of genes, gene functions,

Entire genomes are sequenced, studied, and and entire genomes.
compared. • Genomics is the study of genomes.
– can include the sequencing of the genome
– comparisons of genomes within and across species

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• Gene sequencing is determining the order of DNA • The Human Genome Project has sequenced all of the
nucleotides in genes or in genomes. DNA base pairs of human chromosomes.
• The genomes of several – analyzed DNA from a
different organisms have few people
been sequenced. – still working to identify
and map human genes

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Technology allows the study and comparison of both
genes and proteins.
• Bioinformatics is the use of computer databases to
organize and analyze biological data.
• DNA microarrays are used to study the expression of
many genes at once.

• Proteomics is the study and comparison of proteins.

9.1 Manipulating DNA 9.1 Manipulating DNA

DNA Microarray DNA Microarray

• A DNA micorarray allows scientists to perform an
experiment on thousands of genes at the same time.
• Each spot on a microarray contains multiple identical
strands of DNA.
• The DNA sequence on each spot is unique.
• Each spot represents one gene.
• Thousands of spots are arrayed in orderly rows and
columns on a solid surface (usually glass).
• The precise location and sequence of each spot is
recorded in a computer database.
• Microarrays can be the size of a microscope slide, or
even smaller.
http://learn.genetics.utah.edu/content/labs/microarray/ http://learn.genetics.utah.edu/content/labs/microarray/

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DNA Microarray – how it works DNA Microarray - Applications

• Gene Discovery: DNA Microarray technology helps in the
• Blood sample is taken from the patient and a normal identification of new genes, know about their functioning and
control is uses expression levels under different conditions.
• Disease Diagnosis:
• DNA is denatured so the two strands come apart
– DNA Microarray technology helps researchers learn more about
• DNA is cut into fragments and each fragment is labeled different diseases such as heart diseases, mental illness,
with a fluorescent dye – patient green, normal red infectious disease and especially the study of cancer.
• Both sets of DNA are placed on a chip and allowed to – Until recently, different types of cancer have been classified on
the basis of the organs in which the tumors develop. Now, with
bind to the synthesized DNA on the chip
the evolution of microarray technology, it will be possible for the
• IF NO MUTATION – patient and control DNA will bind to researchers to further classify the types of cancer on the basis of
the same sequences on the chip the patterns of gene activity in the tumor cells.
• IF PATIENT HAS A MUTATION – those sequences – This will tremendously help the pharmaceutical community to
develop more effective drugs as the treatment strategies will be
containing the mutation will bind to the mutated targeted directly to the specific type of cancer.
sequences on the chip

9.1 Manipulating DNA 9.1 Manipulating DNA

DNA Microarray - Applications EXAMPLE

• Drug Discovery:
– Microarray technology has extensive application
in Pharmacogenomics. Pharmacogenomics is the study of
correlations between therapeutic responses to drugs and
the genetic profiles of the patients.
– Comparative analysis of the genes from a diseased and a
normal cell will help the identification of the biochemical
constitution of the proteins synthesized by the diseased
genes. The researchers can use this information to
synthesize drugs which combat with these proteins and
reduce their effect.
• Toxicological Research: Microarray technology provides a
robust platform for the research of the impact of toxins on the
cells and their passing on to the progeny.

9.1 Manipulating DNA 9.1 Manipulating DNA

KEY CONCEPT Genetic screening can detect genetic disorders.

Genetics provides a basis for new medical
• Genetic screening involves the testing of DNA.
treatments.
– determines risk of having DMD N
or passing on a genetic
disorder
– used to detect specific
genes or proteins
– can detect some genes
related to an increased
risk of cancer
– can detect some genes
known to cause genetic
disorders

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Gene therapy is the replacement of faulty genes. • Several experimental techniques are used for gene
• Gene therapy replaces defective or missing genes, or therapy.
adds new genes, to treat a disease. – genetically engineered viruses used to “infect” a
patient’s cells
– insert gene to stimulate immune system to attack
cancer cells
– insert “suicide” genes into cancer cells that activate
a drug

9.1 Manipulating DNA 9.1 Manipulating DNA

Examples Parkinsons
• Gene therapy is being used in many ways. For example,
to: • Patients with Parkinson's disease gradually lose cells in
– Replace missing or defective genes; the brain that produce the signaling molecule dopamine.
As the disease advances, patients lose the ability to
– Deliver genes that speed the destruction control their movements.
of cancer cells;
• A small group of patients with advanced Parkinson's
– Supply genes that cause cancer cells to revert back to disease were treated with a retroviral vector to introduce
normal cells; three genes into cells in a small area of the brain. These
– Deliver bacterial or viral genes as a form genes gave cells that don't normally make dopamine the
of vaccination; ability to do so. After treatment, all of the patients in the
– Provide genes that promote or impede the growth of trial had improved muscle control.
new tissue; and;
– Deliver genes that stimulate the healing of damaged
tissue.

9.1 Manipulating DNA 9.1 Manipulating DNA

Hemophilia

• People with hemophilia are missing proteins that help

their blood form clots. Those with the most-severe forms
of the disease can lose large amounts of blood through
internal bleeding or even a minor cut.
• In a small trial, researchers successfully used an adeno-
associated viral vector to deliver a gene for Factor IX, the
missing clotting protein, to liver cells. After treatment,
most of the patients made at least some Factor IX, and
they had fewer bleeding incidents.

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• Gene therapy has many technical challenges.

– inserting gene into correct cells
– controlling gene expression
– determining effect on other genes

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