Biotechnology Chapter 20

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BiotechnologyChapter 20
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Gene technology
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Biotechnology

• Manipulation of organisms to make useful products

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Genetic engineering

• Manipulation of genes
• Gene cloning
• Multiple copies of a single gene
• Produce a specific product

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Fig. 20-2
Cell containing geneof interest
Bacterium
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Gene inserted intoplasmid
Bacterialchromosome
Plasmid
Gene ofinterest
RecombinantDNA (plasmid)
DNA of chromosome
2
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in cultureto form a clone of
cellscontaining the clonedgene of interest
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Gene ofInterest
Protein expressedby gene of interest
Copies of gene
Protein harvested
Basic research andvarious applications
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Basicresearchon protein
Basicresearchon gene
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up
toxic waste
Protein dissolvesblood clots in heartattack
therapy
Human growth hor-mone treats stuntedgrowth
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Recombinant DNA

• 1970s
• Combining genes from different sources
• Even different species
• Combined into single DNA
• Example Bacteria and mammal

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Recombinant DNA

• Genetically modified bacteria
• Mass produce beneficial chemicals
• Insulin
• Growth hormone
• Cancer drugs
• Pesticides

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Plasmid
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Plasmid

• Small separate circular DNA
• Replicated same as main DNA
• Foreign DNA added to plasmid
• Replicated along with plasmid

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Recombinant DNA

• Nucleases
• Enzymes that degrade DNA
• Restriction endonulceases
• Restriction enzymes
• Cut DNA into fragments at specific points

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Recombinant DNA

• Restriction sites
• Places where DNA is cut
• Short DNA sequence

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Recombinant DNA

• Restriction enzyme recognizes short sequences in
  DNA
• Cuts at these sequences
• Staggered cut
• Leaves single-stranded ends
• Called sticky ends

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Recombinant DNA

• Sticky ends enables other DNA to join
• DNA fragments from other sources
• Match ends by base pairs (complementary
  sequences)
• DNA ligase
• Enzyme combines ends
• Forms a phosphodiester bond

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Recombinant DNA (Process)

• 1. Isolate gene of interest bacterial plasmid
• 2. Cut DNA plasmid into fragments
• 3. Mix DNA fragments with cut plasmid.
• Fragment with gene of interest is inserted into
  the plasmid
• 4. Recombinant plasmid is mixed with bacteria

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Recombinant DNA (Process)

• 5. Bacteria with recombinant DNA reproduce
• 6. Isolate bacterial clones that contain gene of
  interest
• Producing protein of interest
• 7. Grow large quantities of bacteria that produce
  the protein

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Fig. 20-4-4
Hummingbird cell
TECHNIQUE
Bacterial cell
lacZ gene
Restrictionsite
Gene of interest
Stickyends
Bacterial plasmid
ampR gene
Hummingbird DNA fragments
Nonrecombinant plasmid
Recombinant plasmids
Bacteria carryingplasmids
RESULTS
Colony carrying recombinant plasmid with
disrupted lacZ gene
Colony carrying non-recombinant plasmidwith
intact lacZ gene
One of manybacterial clones
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Recombinant DNA

• Vector
• DNA molecule that carry foreign DNA
• Enters replicates in the host
• Plasmids phages are common vectors
• Phages are larger than plasmid
• Can handle inserts up to 40 kilobases

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PCR

• Polymerase chain reaction
• Amplify DNA
• Makes large quantities of DNA
• 1985

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PCR

• Heated
• Denatured
• DNA primer
• Heat stable DNA polymerase
• Makes DNA

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Fig. 20-8
5?
3?
TECHNIQUE
Targetsequence
3?
5?
Genomic DNA
1
3?
5?
Denaturation
5?
3?
2
Annealing
Cycle 1yields 2 molecules
Primers
3
Extension
Newnucleo-tides
Cycle 2yields 4 molecules
Cycle 3yields 8 molecules2 molecules(in
whiteboxes)match targetsequence
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Gel electrophoresis

• Study DNA
• Polymer (gel)
• Restriction fragments
• Separates DNA based on charge size
• Nucleic acids negative charge (Phosphates)
• Migrate towards end (red)

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Fig. 20-9
TECHNIQUE
Powersource
Mixture ofDNA mol-ecules ofdifferentsizes
Anode
Cathode


Gel
1
Powersource


Longermolecules
2
Shortermolecules
RESULTS
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Fig. 20-10
Normal ?-globin allele
Normalallele
Sickle-cellallele
Large fragment
201 bp
175 bp
DdeI
DdeI
DdeI
Largefragment
DdeI
Sickle-cell mutant ?-globin allele
376 bp
201 bp175 bp
Large fragment
376 bp
DdeI
DdeI
DdeI
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
(a) DdeI restriction sites in normal and
sickle-cell alleles of ?-globin gene
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Cloning

• Multicellular organisms come from a single cell.
• Offspring are identical

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Cloning

• 1950
• Carrots
• Totipotent
• Mature cells that undifferentiated
• Give rise to any type of cells
• Common in plants

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Cloning

• Nuclear transplantation
• Nucleus of an unfertilized/fertilized egg is
  removed
• Replaced with nucleus of differentiated cell
• Direct development of cell into tissues etc.

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Cloning

• Removed nuclei from an egg
• Mammary cells
• Fused with egg cells
• Dolly, 1997, identical to mammary cell donor
• Died prematurely age 6
• Arthritis lung disease

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Fig. 20-18
TECHNIQUE
Mammarycell donor
Egg celldonor
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2
Egg cellfrom ovary
Nucleusremoved
Cells fused
Culturedmammary cells
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3
Nucleus frommammary cell
Grown inculture
4
Early embryo
Implantedin uterusof a thirdsheep
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Surrogatemother
Embryonicdevelopment
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Lamb (Dolly)genetically identical tomammary
cell donor
RESULTS
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Fig. 20-19
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Cloning

• Few develop normally
• Abnormalities
• Epigenetic changes to the chromatin
• More methylation of chromatin
• Reprogram chromatin of differentiated cell

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Stem cells

• Started 1998 at UW
• Early embryonic cells
• Potential to become any type of cell
• Master cell generates specialized cells
• Such as muscle cells, bone cells, or blood cells

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Stem cells

• Embryos
• Bone marrow
• Umbilical cord blood
• Blood stem cells
• ?? Turn skin cells into embryonic stem cells
• Therapeutic cloning

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Fig. 20-20
Adult stem cells
Embryonic stem cells
Early human embryoat blastocyst stage(mammalian
equiva-lent of blastula)
From bone marrowin this example
Cells generatingall embryoniccell types
Cells generatingsome cell types
Culturedstem cells
Differentcultureconditions
Differenttypes ofdifferentiatedcells
Blood cells
Nerve cells
Liver cells
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Medical applications

• Genetic markers
• Detect abnormal disease
• SNP
• Single nucleotide polymorphisms
• Single base pair site where variation is found
• RFLP
• Restriction fragment length polymorphisms

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Fig. 20-21
DNA
T
Normal allele
SNP
C
Disease-causingallele
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Medical applications

• Gene therapy
• Treat genetic defects
• Alters persons genes
• 2 girls with rare blood disease
• CF (vectors are viruses)
• SCID (immune disorder)
• Injected viral DNA with normal gene

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Fig. 20-22
Clonedgene
Insert RNA version of normal alleleinto
retrovirus.
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Viral RNA
Let retrovirus infect bone marrow cellsthat have
been removed from thepatient and cultured.
2
Retroviruscapsid
Viral DNA carrying the normalallele inserts into
chromosome.
3
Bonemarrowcell frompatient
Bonemarrow
Inject engineeredcells into patient.
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Medical applications

• Transgenic animal
• Gene from one animal is inserted into another
• Goat milk protein anti-thrombin
• Isolated from milk
• pharm animals

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Animals

• Transgenic animals engineered for specific traits
  
• Genetically create a racehorse
• Not have to breed
• Sheep with better wool??

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Agricultural applications

• Manipulate tomatoes
• Do not ripen as fast
• Flavr-Savr
• Slows down ethylene production

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Agricultural applications

• Introduce genes to plants
• Enable them to fix nitrogen
• Convert N2 to NH3
• Help eliminate use fertilizers
• Cut

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Agricultural applications

• Herbicide resistance
• Plant genetically resists the herbicide
• Insect resistance

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Agricultural applications

• Transgenic rice
• golden rice
• Rice with genes that code for better absorption
  of iron and beta carotene
• First of many genetically engineered foods
• Helps dietary deficiencies

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Forensics

• Genetic profile
• Individual genetic markers
• DNA fingerprint
• RFLP
• STR
• Short tandem repeats
• Occur in specific regions in genome
• Unique

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Fig. 20-24
(a)
This photo shows EarlWashington just before his
release in 2001,after 17 years in prison.
Source of sample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim
17, 19
13, 16
12, 12
Earl Washington
16, 18
14, 15
11, 12
17, 19
13, 16
12, 12
Kenneth Tinsley
(b)
These and other STR data exonerated Washington
andled Tinsley to plead guilty to the murder.
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Concerns over genetic engineering

• Genetically modified foods
• Harmful?
• Genetically engineered gametes
• Blonde and blue eyes??