Genetic Engineering and Recombinant DNA

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Genetic Engineering and Recombinant DNA
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Genetic Engineering and Recombinant DNA

• The Origin of Genetic Engineering
• Biotechnology - the use of living organisms for
  practical purposes.
• While many believe that biotechnology is a novel
  concept, it actually began about 10,000 years ago
  when human populations began selecting and
  breeding useful plants, animals, fungi, and
  microorganisms.

While the early biotechnology techniques were
relatively simple, modern genetic engineers move
genes among all kinds of organisms, including
humans, mice, tomatoes, yeasts, and bacteria.
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• Knowing Biochemical Pathways Helps Molecular
  Biologists Design Useful Organisms
• Today, the knowledge of biochemical pathways in
  some organisms allows biologists to predict what
  type of mutation will produce a desired trait.

With this in mind, molecular biologists have been
successful in designing useful organisms by
inserting or destroying genes that code for
proteins involved in specific biochemical
pathways.
What are some examples of this technology?
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• Knowing Biochemical Pathways Helps Molecular
  Biologists Design Useful Organisms
• For example, Calgene in central California
  deliberately damaged the gene that controls
  ethylene production in tomatoes.
• Eythlene is responsible for fruit ripening.
• Since these tomatoes do not produce ethylene,
  they will only ripen after the tomato distributor
  sprays them with ethylene.
• This prevents the tomatoes from being picked
  before they have developed their flavor
  components.

Such tomatoes are termed Flavor-Saver tomatoes.
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• How Do Restriction Enzymes Cut Up a Genome?
• DNA can be cut with special enzymes termed
  endonucleases.
• Endonucleases recognize specific sequences of
  nucleotides and sever the DNA at these sites.
• Endonucleases evolved in bacterial cells as a
  defense against bacteriophages (bacterial
  viruses).
• When phage DNA enters a bacteria, endonucleases
  break down the phage DNA (by cutting) in order to
  restrict viral replication.
• Since endonucleases restrict viral replication,
  they have become known as Restriction Enzymes.

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• How Do Restriction Enzymes Cut Up a DNA?
• Restriction enzymes recognize and cut DNA that
  is foreign to the bacterial cell.
• The DNA of the bacterial cell is chemically
  modified to prevent attack by restriction
  enzymes.
• Restriction Enzymes, therefore, chop-up
  foreign DNA, while leaving the DNA of the
  bacterial cell unaffected.

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• How Do Restriction Enzymes Recognize sites for
  severing?
• Since their discovery in 1962, hundreds of
  restriction enzymes have been identified and
  isolated from bacterial cells.
• These restriction enzymes are extremely specific
  and work by recognizing short nucleotide
  sequences in DNA molecules termed RECOGNITION
  SEQUENCES.
• Once these sequences are detected, the
  restriction enzyme severs the DNA at this point.

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• EXAMPLE
• Hae III cuts at the following recognition
  sequence
• GGCC
• CCGG
• Hae III will cut the DNA every time the above
  recognition sequence is detected.
• The result is a matching set of restriction
  fragments.
• Restriction fragments are pieces of DNA that
  begin and end with a restriction site.

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Hae What?
Hae III cuts the DNA each time the recognition
sequence repeats itself within a DNA sample.
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• Mapping
• A comparison of restriction fragment sizes
  allows biologists to construct a restriction map.
• Restriction maps demonstrate how the restriction
  sites are placed within a piece of DNA.
• More importantly, biologists can join these
  fragments into new combinations.
• For example, human and mouse fragments can be
  joined together.

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DNA Fingerprinting
Example Suppose Joes DNA has four restriction
sites for EcoR1
EcoR1 will, therefore, cut Joes DNA four times
_______________________________________________
5 fragments result from the action of EcoR1when
applied to Joes DNA What is the restriction site
for EcoR1?
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DNA Fingerprinting
Example Suppose Anisas DNA has 3 restriction
sites for EcoR1.
EcoR1 will, therefore, cut Anisas DNA three
times.
_______________________________________________
4 DNA segments result
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Restriction Enzyme DNA Fragments
EcoR1 cuts Joes DNA into 5 fragments and Anisas
into 4.
Joe
Anisa
Note In addition to differing in fragment
number, the size of the fragments differs as
well. Why is this significant?
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• These fragments can now be separated from one
  another using ELECTROPHORESIS
• DNA electrophoresis utilizes an agarose gel and
  a voltage current to separate the cut DNA
  fragments from one another.
• The DNA samples are placed into the agarose gel
  (a medium in which the DNA fragments will travel)
  and the voltage current separates the fragments.
• How?

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Gel Electrophoresis

-
Joes DNA
Anisas DNA
The current is applied and the fragments travel
to the end due to the negatively charged DNA
(phosphate).
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Analysis
Smaller DNA fragments will travel farther on the
gel than larger DNA fragments.
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Fingerprinting

• Since every individual has a unique sequence of
  bases in their DNA, a unique banding pattern will
  be generated by electrophoresis for each
  individual.
• This is known as a GENETIC FINGERPRINT.
• NOTE Even if two individuals have the same
  number of restriction sites in their DNA, the
  size of each fragment will differ and will,
  therefore, yield a unique banding pattern.
• The next slide presents an example

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Forensics
Who did it?
Where is the heaviest band?
Where is the lightest band?
All 4 samples are cut with the same restriction
enzyme.
How many restriction sites does the DNA
from suspect 1 have?
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• How Do Molecular Biologists Use Recombinant DNA?
• Recombinant DNA - a DNA molecule consisting of
  two or more DNA segments that are not found
  together in nature.
• For example, the next slide demonstrates how
  cells from a tobacco plant are infected with a
  plasmid carrying a gene for herbicide
  resistance. The herbicide resistant cells
  grow into mature plants which produce seeds
  containing the resistant gene.

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Genetic Engineering and Recombinant DNA
How Do Molecular Biologists Use Recombinant DNA?
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• How Do Molecular Biologists Use Recombinant DNA?
• Recombinant DNA has provided scientists with
• 1) a tool for studying structure, regulation and
  function of individual genes
• 2)a tool for unraveling the molecular bases of
  molecular diseases
• 3)the ability to turn organisms into factories
  that turn out vast quantities of product
  (protein or other substance) that these organisms
  would never make on their own.

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• How Do Molecular Biologists Join Restriction
  Fragments Together?
• Two pieces of DNA from different sources can be
  linked together by the enzyme DNA ligase.
• DNA ligase is normally used during DNA
  replication.
• DNA ligase is responsible for the linkage of
  separate pieces of DNA into one continuous strand.

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Ligase
This image demonstrates how ligase can be used to
link human and mouse DNA together as well
as the insertion of the human insulin gene into
a plasmid causing the bacteria to produce
insulin.
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• How Do Molecular Biologists Express Recombinant
  DNA in Bacteria and Other Hosts?
• Molecular biologists face two serious challenges
• 1) To produce large numbers of particular
  genes.
• 2) To induce host cells to express recombinant
  genes as usable proteins.

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• How Can Bacteria Be Induced To Make Great Numbers
  of Copies of a Gene?
• Biologists achieve this goal with the use of
  plasmids.
• Plasmids allow bacterial cells to produce large
  numbers of copies of a single gene.
• Using DNA ligase, researchers can link any gene
  to a plasmid which carries recombinant DNA into
  cells.
• Plasmids are an example of a vector.
• A vector is anything that spreads genes from one
  organism to another.

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• How Can Bacteria Be Induced To Make Eukaryotic
  Genes?
• Eukaryotic DNA contains introns which are base
  sequences in the pre-mRNA that are not expressed
  and normally removed by the eukaryotic cell
  before the mRNA is translated.
• Bacterial cells are prokaryotic and, therefore,
  do not have the required enzymes to recognize and
  remove the introns.
• If they cannot remove introns, they cannot make
  a mRNA molecule that is translateable and,
  therefore, cannot directly make eukaryotic genes.

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• How Can Bacteria Be Induced To Make Eukaryotic
  Genes?
• The solution of intron removal in bacterial
  cells comes from the action of retroviruses.
• Recall that retroviruses contain reverse
  transcriptase which allows the conversion of RNA
  to DNA.
• Researchers can take mature mRNA (introns have
  already been removed) and copy it back to DNA
  with the use of reverse transcriptase.
• The resulting DNA is termed complementary DNA
  (cDNA) and, unlike the genomic DNA, it has no
  introns.

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The image to the left demonstrates how reverse
transcriptase is used to copy mature insulin mRNA
into DNA.
This DNA can now be joined to a plasmid vector
and expressed by a bacterium.
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• Can Host Cells Be Induced To Express Polypeptides
  in a Usable Form?
• Unfortunately, not all eukaryotic genes can be
  expressed in bacteria.
• Such genes code for proteins that must be
  modified after translation.
• For example, most membrane proteins require
  modifications that can only be made in eukaryotic
  hosts.

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• How Do Researchers Make Multiple Copies of
  Recombinant DNA?
• Researchers need enormous quantities of a gene
  in order to sequence it, detect mutations or
  study how proteins interact with the gene to
  influence gene expression.
• Cloning and PCR (Polymerase Chain Reaction)
  allow researchers to make millions of copies of a
  particular gene.

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How Do Researchers Make Multiple Copies of
Recombinant DNA?
Cloning simply involves the introduction of a
single recombinantDNA (gene and plasmid) molecule
into a bacterial host cell.
The plasmid can induce the host cell to make many
copies of the gene it carries and, in addition,
researchers can induce the bacterial cell to
divide rapidly.
As the bacteria divide, the recombinant DNA
multiplies.
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• PCR allows researchers to produce multiple
  numbers of individual DNA sequences in a very
  short period of time.
• In PCR
• The selected DNA segment is heated causing the
  two strands to separate.
• The DNA is cooled and two short nucleotide
  sequences termed primers bind to the
  complementary DNA strands.
• DNA polymerase then copies each strand until the
  researcher stops the reaction by again raising
  the temperature.
• Increasing the temperature repeats the process.

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• How Do Biologists Find the Right DNA Sequence in
  a Recombinant DNA Library?
• A gene library is a collection of restriction
  fragments from a single genome.
• Such a library is only useful to researchers if
  they can find the gene they are interested in.

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• Two tools are used to find specific genes
• 1) hybridization probes - short segments of
  single stranded DNA that binds to and detects the
  gene in question.
• 2) antibodies - detect and bind with specific
  proteins in colonies of bacteria containing
  recombinant DNA.

The following slides demonstrates the use of
each technique.
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Genetic Engineering and Recombinant DNA
Note that the hybridization probe locates
specific DNA sequences while antibodies locate
the protein product of the same sequence.
Figure 13-4
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• Genetically Engineered Bacteria and Eukaryotic
  Cells Can Make Useful Proteins
• Genetic reprogramming using recombinant DNA
  technology allows the production of an
  extraordinary number of products.
• For example
• insulin
• growth hormone
• ingredients for processed foods
• enzymes used to produce valuable molecules or
  destroy pollutants
• enzymes in laundry soap
• Vaccines
• New proteins researchers are currently
  developing new antibodies that can interfere
  with disease processes

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• Gene Therapy
• Products of Recombinant DNA Can Be Released
  Directly into the Body from Engineered Somatic
  Cells
• Gene Therapy - The insertion of therapeutic
  genes into an individual so that their products
  act to modulate a particular phenotype.
• One strategy associated with gene therapy
  involves the removal of cells from the body,
  engineering them to produce the desired effect,
  and then implanting them back into the body of
  the individual.
• For example, researchers are now experimenting
  with the insertion of genes for clotting factor
  into cells that are then implanted into
  individuals suffering from hemophilia.
• This allows the body to produce clotting factor
  and alleviate symptoms associated with
  hemophilia.

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• RECOMBINANT DNA CAN GENETICALLY ALTER ANIMALS AND
  PLANTS
• Organisms that carry recombinant DNA are termed
  transgenic organisms and the added DNA is termed
  a transgene.

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• How Do Researchers Produce a Transgenic Mammal?
• For a gene to be expressed, researchers must put
  the transgene into the zygote before the
  beginning of embryonic development.
• If this is performed successfully, all of the
  cells of the organism will contain the desired
  DNA.
• To date, researchers have been successful in
  producing transgenic mice, pigs, goats, and sheep.

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• How Do Researchers Produce a Transgenic Mammal?
• The engineering of transgenic animals faces
  serious obstacles
• 1) they must be made one at a time
• 2) in knockouts (animals in which a particular
  gene has been inactivated), recombinant genes are
  inserted at random and may not function as
  researchers hope.
• In spite of these obstacles, such animals can
  provide clues about how previously mysterious
  proteins function in the body.

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• The Genetic Engineering of Plants Is Easier Than
  That of Animals
• Plant advantages
• 1) they are easier to clone than animal cells
• 2) they can be grown in vast fields which allows
  massive production of desired products
• 3) they have the potential to be extremely
  lucrative.
• ex If the Flavor-Saver tomato becomes popular,
  the inventors will gain a virtual monopoly in the
  tomato market.
• Molecular biologists can genetically engineer
  plants that can
• synthesize animal or plant proteins
• resist herbicides
• resist infection by plant viruses.

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• What Are the Environmental Risks of Recombinant
  DNA?
• The long-term consequences are unknown.
• Some argue that severe ecological effects will
  result.
• For example, genetically engineered plants may
  eventually transfer their engineered genes into
  other plants.
• Will pesticide resistant genes inserted into a
  crop plant be transferred to unrelated pest
  plants creating herbicide resistant weeds?

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• The Application of Recombinant DNA Technology
  Poses Moral Questions for Society
• Diagnosis of genetic disease is far in advance
  of treatment.
• Under such a situation, people may know that they
  have a genetic disease, but will not be able to
  do anything about it.
• Will biologists try to modify genes that affect
  characteristics other than those responsible for
  disease?
• Will future societies try to produce more
  intelligent citizens?
• Will future societies try to produce fewer
  aggressive people?