biotechnology chapter 20. gene technology biotechnology manipulation of organisms to make useful...
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BiotechnologyBiotechnologyChapter 20Chapter 20
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Gene technologyGene technology
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BiotechnologyBiotechnology• Manipulation of organisms to make
useful products
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Genetic engineeringGenetic engineering• Manipulation of genes • Gene cloning: • Multiple copies of a single gene• Produce a specific product
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Fig. 20-2Fig. 20-2
DNA of chromosome
Cell containing geneof interest
Gene inserted intoplasmid
Plasmid put intobacterial cell
RecombinantDNA (plasmid)
Recombinantbacterium
Bacterialchromosome
Bacterium
Gene ofinterest
Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest
Plasmid
Gene ofInterest
Protein expressedby gene of interest
Basic research andvarious applications
Copies of gene Protein harvested
Basicresearchon gene
Basicresearchon protein
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 DNARecombinant DNA• 1970’s• Combining genes from different
sources• Even different species• Combined into single DNA• Example: Bacteria & mammal
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Recombinant DNARecombinant DNA• Genetically modified bacteria • Mass produce beneficial chemicals• Insulin• Growth hormone• Cancer drugs• Pesticides
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PlasmidPlasmid
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PlasmidPlasmid• Small separate circular DNA• Replicated same as main DNA• Foreign DNA added to plasmid • Replicated along with plasmid
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Recombinant DNARecombinant DNA• Nucleases:• Enzymes that degrade DNA• Restriction endonulceases: • Restriction enzymes• Cut DNA into fragments• Specific points
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Recombinant DNARecombinant DNA• Restriction sites: • Places where DNA is cut• Short DNA sequence
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Recombinant DNARecombinant 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 DNARecombinant DNA
D:\Chapter_20\A_PowerPoint_Lectures\20_Lecture_Presentation\2003RestrictionEnzymesA.html
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Recombinant DNARecombinant DNA• Sticky ends • Enables insertion of other DNA • 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)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)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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Recombinant DNA (Process)Recombinant DNA (Process)
D:\Chapter_20\A_PowerPoint_Lectures\20_Lecture_Presentation\2004CloningAGeneA.html
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Fig. 20-4-4Fig. 20-4-4
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Bacteria carryingplasmids
RESULTS
Colony carrying non-recombinant plasmidwith intact lacZ gene
One of manybacterialclones
Colony carrying recombinant plasmid with disrupted lacZ gene
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Recombinant DNARecombinant 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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PCRPCR• Polymerase chain reaction• Amplify DNA• Makes large quantities of DNA• 1985
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PCRPCR• Heated• Denatured • DNA primer• Heat stable DNA polymerase• Makes DNA
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Fig. 20-8Fig. 20-85
Genomic DNA
TECHNIQUE
Cycle 1yields
2molecules
Denaturation
Annealing
Extension
Cycle 2yields
4molecules
Cycle 3yields 8
molecules;2 molecules
(in whiteboxes)
match targetsequence
Targetsequence
Primers
Newnucleo-tides
3
3
3
3
5
5
51
2
3
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Gel electrophoresis 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. 20-9Fig. 20-9
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
TECHNIQUE
RESULTS
1
2
+
+
–
–
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Fig. 20-10Fig. 20-10
Normalallele
Sickle-cellallele
Largefragment
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
201 bp175 bp
376 bp
(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene
Normal -globin allele
Sickle-cell mutant -globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp175 bp
DdeIDdeI
DdeI DdeI DdeI DdeI
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Cloning Cloning • Multicellular organisms come from a
single cell.• Offspring are identical
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Cloning Cloning • 1950• Carrots• Totipotent: • Mature cells that undifferentiated• Give rise to any type of cells• Common in plants
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Cloning 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 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. 20-18Fig. 20-18TECHNIQUE
Mammarycell donor
RESULTS
Surrogatemother
Nucleus frommammary cell
Culturedmammary cells
Implantedin uterusof a thirdsheep
Early embryo
Nucleusremoved
Egg celldonor
Embryonicdevelopment Lamb (“Dolly”)
genetically identical tomammary cell donor
Egg cellfrom ovary
Cells fused
Grown inculture
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33
4
5
6
2
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Fig. 20-19Fig. 20-19
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Cloning Cloning • Few develop normally• Abnormalities• Epigenetic changes to the
chromatin• More methylation of chromatin• Reprogram chromatin of
differentiated cell
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Stem cellsStem 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 cellsStem cells• Embryos• Bone marrow• Umbilical cord blood• Blood stem cells• ?? Turn skin cells into embryonic
stem cells• Therapeutic cloning
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Fig. 20-20Fig. 20-20
Culturedstem cells
Early human embryoat blastocyst stage
(mammalian equiva-lent of blastula)
Differentcultureconditions
Differenttypes ofdifferentiatedcells
Blood cellsNerve cellsLiver cells
Cells generatingall embryoniccell types
Adult stem cells
Cells generatingsome cell types
Embryonic stem cells
From bone marrowin this example
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Medical applicationsMedical 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. 20-21Fig. 20-21
Disease-causingallele
DNA
SNP
Normal alleleT
C
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Medical applicationsMedical applications• Gene therapy• Treat genetic defects• Alters person’s 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. 20-22Fig. 20-22
Bonemarrow
Clonedgene
Bonemarrowcell frompatient
Insert RNA version of normal alleleinto retrovirus.
Retroviruscapsid
Viral RNA
Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.
Viral DNA carrying the normalallele inserts into chromosome.
Inject engineeredcells into patient.
1
2
3
4
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Medical applicationsMedical 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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AnimalsAnimals• Transgenic animals engineered for
specific traits • Genetically create a racehorse• Not have to breed• Sheep with better wool??
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Agricultural applicationsAgricultural applications• Manipulate tomatoes • Do not ripen as fast• “Flavr-Savr”• Slows down ethylene production
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Agricultural applicationsAgricultural applications• Introduce genes to plants• Enable them to “fix” nitrogen
• Convert N2 to NH3
• Help eliminate use fertilizers• Cut $$
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Agricultural applicationsAgricultural applications• Herbicide resistance• Plant genetically resists the herbicide• Insect resistance
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Agricultural applicationsAgricultural 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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ForensicsForensics• Genetic profile:• Individual genetic markers• “DNA fingerprint”• RFLP• STR• Short tandem repeats• Occur in specific regions in genome• Unique
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Fig. 20-24Fig. 20-24This photo shows EarlWashington just before his release in 2001,after 17 years in prison.
These and other STR data exonerated Washington andled Tinsley to plead guilty to the murder.
(a)
Semen on victim
Earl Washington
Source of sample
Kenneth Tinsley
STRmarker 1
STRmarker 2
STRmarker 3
(b)
17, 19
16, 18
17, 19
13, 16 12, 12
14, 15 11, 12
13, 16 12, 12
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Concerns over genetic Concerns over genetic engineeringengineering
• Genetically modified foods• Harmful?• Genetically engineered gametes • Blonde and blue eyes??