Lecture outline

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Lecture outline

• Gene cloning
• DNA analysis
• Microarrays
• Techniques for amplifying and studying DNA
• DNA fingerprinting
• Gene therapy
• Genomics
• Human Genome Project
• The study of whole genomes (Genomics)
• GMOs genetically modified organisms

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Recombinant DNA technology

• Recombinant DNA DNA in which genes from two
  different sources are combined in vitro into the
  same DNA molecule.
• Gene cloning the production of multiple
  identical copies of a piece of DNA that is
  carrying a gene.
• Bacteria (often E. coli--not the infectious
  strain) are often used for manipulating genes in
  the lab.
• Because they easily take up plasmid DNA that can
  carry almost any gene and be replicated in the
  bacterial cell.
• Gene cloning in bacteria can be used to
  mass-produce many useful substances, from cancer
  drugs to insulin to treat diabetes to pest
  resistance proteins for agriculture.

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Overview of gene cloning in bacteria

• 1. Plasmid is isolated
• from bacteria.
• 2. Gene of interest is
• isolated from a cell.
• 3) Gene is inserted
• into plasmid to create
• recombinant DNA
• plasmid.
• 4) Bacterial cell takes
• up recombinant plasmid
• by transformation.
• 5) This bacterial cell
• reproduces to form a
• clone of identical cells
• that all carry the plasmid

Figure 12.1
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Gene cloning in bacteria, cont.

• As the recombinant bacteria multiply into a clone
  of cells, the gene of interest is also
  multiplied.
• Cloned gene can be used to manufacture proteins.
• Or, biologist can learn more about the normal
  function of a protein by generating different
  mutations in the gene and examining whether the
  resulting proteins function normally.

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Enzymes are used to cut and paste DNA

• How did the gene of interest get out of the
  original cell it came from and into the bacterial
  plasmid?
• It was cut out of the original cells chromosome
  and pasted into the bacterial plasmid.
• The DNA cutting tools are restriction enzymes
  enzymes that originated in bacteria to cut up
  foreign DNA as a defense mechanism.
• There are hundreds of different restriction
  enzymes, and each one is very specific for
  recognizing and cutting within a certain DNA
  sequence.
• DNA ligase an enzyme that functions as the DNA
  pasting tool.

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Enzymes are used to cut and paste DNA

• Restriction enzymes cut DNA containing gene of
  interest as well as DNA that it will be pasted
  into (i.e. bacterial plasmid).
• These pieces of DNA cut by restriction enzymes
  are called restriction fragments.
• Restriction enzymes usually make staggered cuts
  that leave single-stranded DNA overhangs called
  sticky ends
• Called sticky b/c these overhanging ends can
  stick together by hydrogen bonding across the
  base pairs.
• DNA ligase seals the pieces of DNA together into
  one recombinant DNA molecule.

Figure 12.2
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Cloning a gene into a recombinant plasmid a
closer look

• Biologist at a biotech company discovers a human
  gene that codes for protein V that kills
  certain viruses.
• Biologist isolates gene V from human cells and
  clones gene V into a bacterial plasmid (the
  plasmid is called a vector, or gene carrier).

Figure 12.3
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Genomic libraries store cloned genes

• Genomic library the entire collection of all the
  cloned DNA fragments from a genome.
• Whole genome is cut with restriction enzymes and
  the fragments are pasted into a vector.
• Bacterial plasmids are one kind of vector for a
  genomic library.
• Phages (viruses that infect bacteria) are another
  type of vector for a genomic library.

Figure 12.4
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Reverse transcriptase allows the cloning of a
gene from mRNA

• Allows a researcher to focus on cloning a genes
  that are transcribed in a given cell type.
• Total mRNA in the cell is isolated.
• Reverse transcriptase
• an enzyme (originally from retroviruses) that
  makes complementary single-stranded DNA (cDNA)
  from mRNA.
• Used here to make cDNAs of all the mRNAs that
  were transcribed by the cell.
• cDNA pieces are now cloned into a vector such as
  a plasmid just like the whole genes were in the
  previous example.

Figure 12.5
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Recombinant cells and organisms can mass-produce
gene products
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DNA technology in the pharmaceutical industry
medicine

• Therapeutic hormones
• Humulin (human insulin produced in E. coli) was
  the first recombinant DNA drug approved
  by the FDA in 1982--people w/diabetes
  depend on it.
• before this, insulin came from pig and cattle
  tissues from slaughterhouses,
  and b/c it wasnt human insulin, had
  some harmful side effects
• Human growth hormone (HGH) before recombinant
  HGH was available, it was obtained from cadavers
  and was in very scarce supply.
• Diagnosis and treatment of disease
• Can idenify alleles associated w/genetic disease.
• Can identify infection by viruses (HIV, HPV,
  etc.) by detecting viral DNA.

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DNA technology in the pharmaceutical industry
medicine

• Vaccine a harmless variant or protein from a
  pathogen (i.e. a bacteria or virus) that prevents
  infection by the pathogen by stimulating the
  immune system to develop long-lasting defenses.
• Many viral diseases have no drug treatment
  (antibiotics wont work against viruses). For
  these diseases, prevention by vaccines is the
  only medical approach.
• Different types of vaccines
• 1) genetically engineered cells/organisms are
  used to produce large amounts of a protein that
  is found on the pathogens surface (used for
  Hepatitis B vaccine).
• 2) A harmless mutant of the pathogen is made by
  altering one or more of its genes so that it is
  not infectious but still triggers the immune
  response.

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Some of the vaccine strategies currently being
tested against HIV

• Peptide virus made of tiny pieces of proteins
  from the HIV virus
• Recombinant protein vaccines made of bigger
  pieces of proteins that are on the surface of the
  HIV virus
• DNA vaccines a few HIV genes are inserted into
  plasmids that will produce some of the HIV
  proteins.
• Live vector virus (or psuedovirion) artificial
  vector that resembles the HIV virus but is not
  harmful vector carries either HIV proteins or
  genes that will produce proteins found on the
  surface of the virus.

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Techniques for molecular biology and genetic
engineering

• Nucleic acid probes
• DNA microarrays
• Gel electrophoresis
• Restriction fragment length polymorphisms (RFLPs)
• Polymerase chain reaction (PCR)

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Nucleic acid probes detect the presence of a
specific DNA sequence

• Nucleic acid probe
• a synthetic piece of single-stranded DNA or RNA
• is labeled with radioactivity or fluorescent dye
• Sequence of probe is complementary to the
  sequence of the DNA being tested for (could also
  be used to test for RNA).
• How it works
• 1) DNA sample to be tested
  is treated w/heat or
  strong
  base to separate the 2
  strands (to allow probe
  
  to bind).
• 2) Labeled probe is added
  and tags the DNA of
  
  interest by base-pairing.

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An example of how nucleic acid probes could be
used

• Imagine you have a genomic library with genes
  stored on plasmids in bacteria.
• You need to find out which bacterial clone
  contains the gene youre interested in.
• Plate the bacterial colonies in rows on an agar
  plate.
• Press a piece of filter paper against the
  colonies to pick up some cells from each
  bacterial colony.
• Soak the filter paper in solution containing
  labeled probe that is complementary to your gene
  of interest.
• Probe will bind to gene of interest.
• Detect radioactivity on film to identify which
  colony has the gene of interest.

Filter paper blotted against bacterial colonies
Filter paper soaking in solution w/labeled probe
Probe detects one bacterial colony containing gene
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DNA microarrays

• DNA microarray glass slide carrying thousands of
  different genes arranged in an array (a grid).
• Uses nucleic acid probes to perform large-scale
  analyses of all the genes that are expressed in a
  particular cell type or condition.

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DNA microarrays

• Example of an application of this technology
• Perform microarray analysis on breast cancer
  tumor cells and on normal breast tissue cells to
  look at differences in gene expression--results
  in better understanding of breast cancer and
  better treatments.

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Gel electrophoresis sorts DNA molecules by size

• Gel electrophoresis technique that uses a thin
  gel as a molecular sieve to separate nucleic
  acids or proteins according to size or electrical
  charge.
• DNA is negatively charged b/c of phosphate
  groups, so DNA molecules migrate through the gel
  to the positive electrode.
• The shorter the molecule, the faster it can move
  through the polymer of the gel. Result DNA
  molecules are separated into bands each band
  is group of DNA molecules of the same size,
  w/ shorter molecules at the bottom.

Figure 12.10
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Restriction fragment length polymorphisms (RFLPs)

• restriction fragment the DNA fragments
  produced by cutting DNA with restriction enzymes.
• length polymorphism polymorphism several
  forms, so this means that there can be DNA
  fragments of different lengths in the population.
• To perform RFLP analysis
• Specific DNA segments are cut with different
  restriction enzymes to produce DNA fragments.
• The DNA fragments are sorted by gel
  electrophoresis, and the patterns of fragments
  (show up as bands on the gel) are compared.
• Usually, the DNA segments analyzed are from
  noncoding areas of the genome--this is where
  there are the most differences (polymorphisms)
  among people.

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Restriction fragment length polymorphisms (RFLPs)

• Here, the suspects DNA does not match DNA found
  at the crime scene!
• DNA evidence has been used to free innocent
  people years after conviction.

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Heres an example where the defendant will need
to explain why the victims blood is all over the
defendants clothes
Figure 12.12
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2 more examples of forensic DNA evidence from
crime scenes
0

• Q Which suspects DNA were a match with the DNA
  found at the crime scene?

Crime 1
Crime 2
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Using RFLPs and DNA probes to detect harmful
alleles

• DNA of an individual who is symptom-free is
  tested to see if they carry
• A harmful recessive allele
• Ex cystic fibrosis, tay-sachs
• A harmful dominant allele that isnt expressed
  until later in life
• Ex huntingtons disease
• Disease allele usually has 1 or more restriction
  sites that differs from the normal allele.
• Need to use a nucleic acid probe to focus on the
  bands coming solely from the disease allele
  marker DNA.
• (Same for crime scene DNA)

Figure 12.11C
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DNA testing can be used to verify family
relationships

• Paternity testing
• Historical analysis
• DNA fingerprinting provided strong evidence that
  Thomas Jefferson or one of his close male
  relatives fathered at least one child with his
  slave, Sally Hemmings.
• Reuniting families
• Baby 81, found after the tsunami in Sri Lanka
  was claimed by nine different families--DNA
  testing ensured he went to the proper home.

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Gene therapy to treat disease

• Alteration of an individuals genes to treat a
  disease.
• For disorders that are due to a single defective
  gene.
• Replace or supplement the defective gene w/the
  normal allele
• Picture shows one example for a disease where
  bone marrow cells dont produce a vital protein
  due to a defective gene.
• Normal gene put into a retrovirus that has been
  made harmless
• Bone marrow cells are taken from the patient,
  infected with virus, then put back into patients
  bone marrow
• If successful, infected bone marrow cells will
  multiply and produce enough protein to cure the
  disease.

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Gene therapy slow progress, ethical questions

• Procedure in previous picture was attempted in a
  human trial to treat SCID, an immune disorder.
• 10 children w/SCID treated by this procedure, and
  9/10 showed improvement in SCID symptoms
• But 2 patients developed leukemia b/c insertion
  of DNA had affected another gene involved in cell
  proliferation.
• Technical issues
• how to make sure that the inserted gene makes the
  right amount of protein, at the right time, and
  in the right part of the body?
• How to ensure that inserted gene doesnt harm
  other cell functions?
• Ethical questions
• Should gene therapy only be used for
  life-threatening diseases, or might it be used to
  make designer people?
• Gene therapy is technically most promising in
  germ cells or zygotes--Should we try to eliminate
  genetic defects in our children before theyre
  born?

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PCR is used to amplify DNA sequences

• PCR polymerase chain reaction
• Can amplify (make large quantities of) a specific
  segment of DNA even if sample is impure
• This amplification step is usually necessary for
  applications of genetic testing we discussed
  before (crime scene analysis, detection of
  harmful alleles, paternity testing, etc.)
• Procedure
• Mix DNA polymerase, nucleotides, and a DNA
  sample, plus some DNA fragments that tell DNA
  polymerase which segment of DNA to amplify.
• Expose this mixture to multiple cycles of heat
  (to separate the 2 strands of DNA) and cooling.
• PCR uses a special DNA polymerase enzyme that can
  withstand the heat at the start of each cycle.

Figure 12.14
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The Human Genome Project

• HGP determining the entire nucleotide sequence
  of human DNA--was mostly complete after 13 yrs
  (1990 - 2003)
• Initial publicly funded approach involved 3
  stages
• 1) linkage mapping to map 5000 genetic markers
  to get a low-resolution big picture map.
• 2) physical mapping cutting each chromosome into
  overlapping fragments and ordering the fragments
• 3) DNA sequencing of each fragment mapped in
  stage 2.
• Privately funded approach by Celera Genomics used
  whole genome shotgun approach
• Went directly to sequencing of small fragments
  and relied on computer software to determine the
  order of the fragments.
• Competition between the public and private groups
  hastened the project.

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The Human Genome Project

• Whose DNA was sequenced?
• Public group much of the DNA sequenced
  supposedly comes from one anonymous male donor
  from New York.
• Private group DNA from 5 different individuals
  was used (one was Craig Venter, president
  founder of Celera).
• Venter wanted to privatize the genome and sell
  information for a fee.
• Clinton declared genome patenting illegal in
  2000.
• f.y.i. Venter started his career at community
  college in San Mateo.
• Potential benefits
• Insight into development, evolution
• Diagnosis, treatment, and prevention of disease
• Still to do
• Figuring out what all these genes (as well as the
  noncoding DNA) do.

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the Human genome

• Biggest surprise the small of human
  genes--only about 20,000-25,000 genes in the
  whole genome!
• Basically the same as the roundworm C. elegans, a
  much less complex organism!
• Scientists speculate that a lot of the increased
  complexity in humans comes from alternative RNA
  splicing.
• Most of the genome doesnt consist of genes.
• Noncoding DNA is about 97 of the DNA in the
  genome.
• Noncoding DNA includes
• Gene control sequences such as promoters,
  enhancers, etc.
• Introns
• Repetitive DNA--nucleotide sequences present in
  many copies in the genome (may play a role in
  chromosome structure)
• Repetitive DNA at the ends of chromosomes (called
  telomeres) is essential for cell survival and
  proliferation--also plays a major role in cancer.
• Transposons (jumping genes) DNA segments that
  can move or be copied from one location in the
  genome to another.

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The science of genomics compares whole genomes

• As of 2005, 150 species genomes have been
  sequenced.
• Comparative genomics provides clues about what
  genes in humans do.
• If a gene in another organism is similar in
  sequence to a human gene, its likely also
  similar in function.
• Comparative genomics also allows the evaluation
  of evolutionary relationships among species.
• The more similar two organisms are in the
  sequence of their genomes, the more closely
  related they are in their evolutionary history.
• Genomics also looks at the overall organization
  of the genome to look at patterns of gene
  expression, growth, development.

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• Others mosquito, dog, rat, chicken, frog

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Next proteomics

• Proteomics similar systematic study of the full
  sets of proteins (proteomes) encoded by genomes
• The of proteins in humans far exceeds the
  number of genes.
• It is proteins, not genes, that carry out the
  activities of the cell.
• Therefore, scientists want to understand when and
  where proteins are produced, and which proteins
  interact with one another.

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Genetically modified organisms (GMOs) (also
called transgenic organisms--contain genes from a
different species)
Tobacco plant expressing a gene from fireflies
Transgenic chickens produce therapeutic human
proteins in their eggs
Obese mouse Animal model to study
obesity.Mice are missing genes Involved in
sensing fullness
Enviropigs Produce less Phosphorus waste in
their manure b/c they express an enzyme to
break down phosphate better
Alba, glowing bunny commissioned by French artist
Eduardo Kac as transgenic art (jellyfish green
fluorescent protein)
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Genetically modified organisms (GMOs) most are
plant crops

• GMO an organism that has acquired one or more
  genes by aritifical means (not traditional
  breeding methods)
• New gene may or may not be from a different
  species
• Most common vector is a plasmid from soil
  bacterium Agrobacterium tumefaciens called the Ti
  plasmid.
• Gene is inserted into Ti plasmid plasmid is then
  inserted into plant cell in culture, and the
  plant is grown.

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GMOs potential advantages

• 1) Pest resistance
• GM crops resistant to attack by pests could
  eliminate application of harmful pesticides.
• 2) Herbicide tolerance
• Farmers can control weeds by using herbicide
  and not worry about
  the herbicide killing the
  crop in theory, allows reduced use
  of the herbicide.
• 3) Disease resistance
• Would prevent crops from being wiped out by
  specific bacterial, fungal, and viral plant
  diseases.
• 4) Tolerance for extreme environments (cold, dry,
  high-salt, etc.)
• Allows land previously unsuited for agriculture
  to be used to grow crops.
• 5) Nutrition, vaccines
• Could treat malnutrition in developing countries
  by adding vitamins and nutrients to crops that
  are already staples in those countries (e.g.
  rice).
• Bananas engineered to carry vaccines against Hep
  B other diseases.

golden rice 50 RDA Vit.A
kid eats banana gets Hep B immunity
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Restrictions on GMOs

• Production of GMOs is banned in Mendocino, Marin,
  and Trinity counties in CA.
• European Union (EU), Japan, Korea, Sri Lanka, and
  other countries have imposed restrictions against
  genetically modified food imports from the U.S.
• China recently restricted import of products
  containing GM ingredients
• Japan, Australia, and New Zealand have mandatory
  labeling restrictions

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GMOs Health concerns

• 1) Allergy issues adding a new gene into a crop
  may transfer allergens, create a new allergen or
  cause allergic reactions in susceptible
  individuals.
• 2) Antibiotic resistance genes used in the
  creation of GMOs may be released into the
  environment and cause those antibiotics to become
  ineffective.
• may become resistant to crops genetically
  engineered to produce their own pesticides.
• 3) Unforseen health effects the Showa Denko
  story
• S.D. produced tryptophan (an amino acid) as a
  dietary supplement by genetically engineering
  bacteria to produce high concentrations of
  it--when concentrated this high in the bacteria,
  the tryptophan reacted with itself, forming new
  compounds that proved toxic, killing 37 people
  and permanently disabling 1500 more (late
  1980s).
• Flavr Savr tomato caused stomach ulcers and
  lesions in rats in 2 out of 3 studies
  performed--not clear why.

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GMOs Environmental concerns

• 1) Unintended harm to other organisms
• Bt corn, a GM corn modified to kill insect larvae
  pests that kill the corn, also caused high
  mortality in monarch butterfly caterpillars.
• 2) Reduced effectiveness of pesticides
• may become resistant to crops genetically
  engineered to produce their own pesticides.
• 3) Increased use of herbicides
• Knowing that their GM crops are resistant to an
  herbicide, farmers might use more herbicide to
  kill weeds.
• 4) Gene transfer to non-target species
• If herbicide-resistance gene crosses with weeds,
  super-weeds would be created that are also
  resistant to herbicides
• 5) Widespread crop failure
• GM crops are patented, therefore all the seeds
  are genetically identical. If there is a pest
  that can attack these seeds all of these crops
  would be vulnerable to crop failure.
• 6) Unforseen effects
• Can we possibly understand enough about natural
  ecosystems to experiment with them?

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GMOs Economic concerns

• Increasing dependence on industralized nations by
  developing countries.
• Domination of world food production by a few
  companies.
• Reduced export of food products to countries with
  increasing restrictions on GMO food product
  imports.
• Starlink crisis GM corn called StarLink that
  wasnt yet approved for human food use leaked
  into corn shipments destined for food consumption
  in US and also Japan (which has banned GM
  foods)--this caused export of all U.S. produced
  corn products to Japan and other countries to
  plummet.
• Farmers whose crops are contaminated with GM
  seeds have been sued by agribusiness companies
  for patent infringement.
• In Feb 2006, a coalition of farmers, consumers,
  and environmental activists sued the USDA for
  approving Monsantos GM herbicide-resistant
  alfalfa seed, claiming that the USDA failed to
  analyze the health, environmental, and economic
  risks.