Biotechnology

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Biotechnology
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GENE TRANSFER

• Actually putting genes from one organism into
  another. This is the whole idea behind gene
  therapy to cure genetic disorders.

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• Alba was an albino rabbit engineered by splicing
  the green fluorescent protein (GFP) of a
  jellyfish into her genome.

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Lederburg and Tatum

• studied E. coli and discovered that they could
  combine genes from two different cells

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Bacteria Conjugation

• Hey, Ill give you some of my genes if you give
  me some of yours!

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Vector

• is a vehicle for delivering genetic material such
  as DNA to a cell.
• We will look at PLASMIDS as vectors.

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PLASMIDS circular DNA in bacteria
Regular DNA needed for mitosis
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Problem with R Plasmids

• Bacteria carry various kinds of Plasmids (F
  plasmid, R plasmid)
• R Plasmids are resistant to antibiotics such as
  penicillin and tetracycline

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Bacteria Lab

• We will put various antibiotic discs on a Petri
  dish inoculated with E. coli
• Will any be resistant?

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Petri Dishes

• Clear rings around antibiotic disk shows lack of
  bacterial growth

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Gene Transfer
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STEPS OF PLASMID CARRYING DNA TO ANOTHER CELL

• 1. Plasmid is
  isolated
• from a
  bacterial cell

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Isolate DNA from Another Cell

• 2. DNA is isolated from a gene of interest (ie.,
  insulin or enzyme to break down mucus in CF
  patient)

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Inserting DNA into a Plasmid

• 3. Gene is inserted into plasmid

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RECOMBINANT DNA

• 4. Plasmid is put into bacterial cell

Recombinant DNA A DNA molecule carrying genes
from another source
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• The bacterial cell takes up the DNA by a process
  called transformation.
• (remember Griffiths experiment with Pneumococcus
  R and S strains?)

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Cells Multiply

• 5. Cloned or produced genetically exact copies
  of itself

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If you put genes from one organism into a
different organism, then it is called a

• TRANSGENIC
• ORGANISM

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Transgenic Luciferase into tobacco

• Such as the glowing genes from a firefly into a
  tobacco plant

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Products Made with Transgenic Bacteria

• Human Insulin
• Human Growth Hormone
• Clotting factor VIII for hemophiliacs
• Human Lung Surfactant for premature babies
• Vaccines
• Inhibit ice crystals for crops

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• TOOLS FOR MAKING RECOMBINANT DNA

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HOW DOES DNA GET CUT OUT?

• Not with itty bitter scissors.
• But with RESTRICTION ENZYMES (they recognize
  specific 4 to 8 base regions of DNA and a cut
  will be made here)

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Restriction Enzymes

• Bacterial enzymes that cut DNA at specific
  regions (EXAMPLE GAATTC)

EcoR 1 was isolated from E. coli strain R and was
the 1st restriction enzyme to be isolated from
this bacterium.
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Various Restriction Enzymes
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Examples of Restriction Enzymes

• Enzyme Cutting Sites
• Bam HI GGATCC
• Hae III GGCC
• Pst ICT GCAGH
• inf I GANTC

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Restriction Enzymes
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Sticky Ends

• Single stranded section of complementary bases of
  two joining sections of DNA

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Recombinant DNA

• Ligase (enzyme) joins the DNA sticky ends

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Biotechnology Protest
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Do Chocolate Cherry Activity
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• Since 1987, forensic
• DNA analysis has made
  appearances in
  U.S. courtrooms. Originally known as "DNA
  fingerprinting," this type of analysis is now
  called "DNA profiling" or "DNA typing" to
  distinguish it from traditional skin
  fingerprinting.

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How is DNA Fingerprinting Done?

• First get your evidence sample
• Only need 1000 cells
• Blood, saliva, skin, semen, sweat

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How Reliable is DNA Fingerprinting?

• One chance in 100,00
• to
• one chance in 1,000,000,000
• Probability of two people having identical DNA

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Gel Electrophoresis

• Put DNA samples into wells

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• The gel is a matrix of cross-linked molecules
  that acts as a sieve through which nucleic acids
  can migrate.

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How does it work?

• Negatively-charged nucleic acids or proteins are
  repelled by a
  negative
  electric
  current.

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Gel Electrophoresis

• Sort by
• Electrical charge
• 2. Size

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RESULTS DNA Samples

• Lane       Tube
• 1 A Crime scene cut with Enzyme 12 B
  Crime scene cut with Enzyme 23 C
  Suspect 1 cut with Enzyme 14 D Suspect 1
  cut with Enzyme 25 E Suspect 2 cut with
  Enzyme 16 F Suspect 2 cut with Enzyme 2

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Electric Current Applied for Several Hours
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• The bigger molecules stay closer to the sample
  wells
• The smaller molecules travel farther down the gel

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Which lanes match?

• Larger
• Smaller

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Uses of Gel Electrophoresis

• Identify Criminals
• Identify Soldiers
• Identify Fathers of Babies
• (Hey, can you help me, Montel?)

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USES OF DNA

• For Forensic Identification
• Identify potential suspects whose DNA may match
  evidence left at crime scenes
• Exonerate persons wrongly accused of crimes
• Identify crime and catastrophe victims

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USES OF DNA

• Establish paternity and other family
  relationships
• Identify endangered and protected species as an
  aid to wildlife officials (could be used for
  prosecuting poachers)
• Authenticate consumables such as caviar and wine

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USES OF DNA

• Detect bacteria and other organisms that may
  pollute air, water, soil, and food
• Match organ donors with recipients in transplant
  programs
• Determine pedigree for seed or livestock breeds

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• CODIS enables federal, state, and local crime
  labs to exchange and compare DNA profiles
  electronically, thereby linking crimes to each
  other and to convicted offenders.

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• Just like no two people look exactly alike, no
  two people (except identical twins) have the
  exact same DNA.

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Genetic Marker

• Specific section of DNA
• Mark for detecting a genetic disease
• Specific restriction enzyme cutting site
• EX AFP marker (Alphafetoprotein)

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VNTR

• Variable Number Tandem Repeat.
• These genetic markers are commonly used for DNA
  fingerprinting.

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VNTR
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Whos Your Daddy?

• M mother
• C child
• F1 possible father
• F2 possible father
• (NOTE child has
• Markers from both parents)

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RFLP's--- Restriction Fragment Length Polymorphism

• -

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DO CRIME SCENE
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Cloning Dolly
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CLONINGHow is it done?
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How Dolly Was Cloned
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Cloning Humans
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Making Stem Cells

• Less than 8 days

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Flavr Savr
First GM food (Genetically Modified) 1994 Can
stay on vine
longer
with firmer skin
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What might be some problems with cloning humans?
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GEEP (sheep and goat)
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HGP

• Started 1990
• Took 13 years
• Goal to map the human genome
• Goal to correct genetic disorders

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• We share some genes with every creature on the
  planet. There is only 1 difference in 1000
  between you and the guy you wish your daughter
  wasn't going out with.

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• Who's genes did we usefor the Human Genome
  Project?

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• Grow your own spare parts! These cartilage ears
  and noses have been grown from a polymer
  scaffold.

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Is it ethical or legal? Gene therapy could be
used to create children who can think better and
run faster. It's controversial, but most
researchers believe super toddler's are on the
way.
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Human Genome Project

• 3 billion nucleotide pairs
• 20,000-25,000 genes (less than the predicted
  35,000)
• 5,000 Genetic Markers

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J. Craig Venter

• His company, Celera Genomics, helped to sequence
  90 of HGP

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• Can we clone dinosaurs?
• Probably notthe DNA is not a complete dinosaur
  and it is too degraded.

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MOVIE Jurassic Park
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Movie Duplicating Humans
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Movie Multiplicity
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X Files
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PCR

• Polymerase Chain Reaction making copies from a
  small amount of DNA

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Doubles with each cycle

• PCR Animation
• Another Good PCR
• Start with DNA molecule
• Some primers
• Can generate 100 billion molecules in a few hours

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Junk DNA

• Around 97 of the human genome
• BUT, we are finding uses for what we thought were
  non-coding DNA
• Promoters, enhancers, introns, suppressors,

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• JUNK DNA- May Not Be Junk After All

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Telomeres

• Complexes at chromosome ends
• Repetitive sequences
• Protects ends

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Indicates Age of Chromosome

• Each time a cell makes a copy of itself some
  repeats are lost. But it's no accident, this is
  your telomeres at work. Older cells have shorter
  telomeres. A baby has around 1700 repeats, but by
  70 years of age only about 300 repeats are left.

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• Also, maybe cancer cells are helped by abnormal
  lengthening of DNA

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Jumping Genes

• Also called transposons
• Barbara McClintock
• 1940s

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Jumping Genes
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Jumping Genes

• Note different color of kernels every now and
  then

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Good Thing?

• May Generate Genetic Diversity
• Could also be a bad thingdisrupt the normal
  protein synthesis

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Ti (tumor-inducing) plasmid

• Agrobacterium tumefaciens

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What is Ti used for?

• For gene transfers in plants.