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Biotechnology
DNA of chromosome
Cell containing geneof interest
Gene inserted intoplasmid
Plasmid put intobacterial cell
RecombinantDNA (plasmid)
Recombinantbacterium
Bacterialchromosome
Bacterium
Gene ofinterest
Plasmid
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2
Gene cloning involves using bacteria to make multiple copies of a gene
DNA Cloning: An Overview
Fig. 20-2b
Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest
Gene ofInterest
Protein expressedby gene of interest
Basic research andvarious applications
Copies of gene Protein harvested
Basicresearchon gene
Basicresearchon protein
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Recombinantbacterium
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
3
Restriction Enzymes
• Gene cloning was made possible by the discovery of restriction enzymes.
• Many different enzymes exist– named after the organism in which they are found
• EcoRI (E. coli), HindIII (Haemophilus influenza), PstI (Providencia stuartii)
Fig. 20-3-1Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
53
35
1
Fig. 20-3-2Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
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35
1
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
One possible combination
Fig. 20-3-3Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
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35
1
One possible combination
Recombinant DNA molecule
DNA ligaseseals strands.
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DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
Fig. 20-UN3
Cut by same restriction enzyme,mixed, and ligated
DNA fragments from genomic DNAor cDNA or copy of DNA obtainedby PCR
Vector
Recombinant DNA plasmids
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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
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3
3
3
5
5
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2
3
Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)
PCR can make billions of a specific DNA segment ina few hours.
PCR produces many copies of a specific target segment of DNA
PCR Animation
PC
R D
NA
Am
plifi
catio
n
PCR Applications
30,000 y. o. woolly mammoth
In forensics, PCR requires only smallsamples of DNA to analyze
DNA polymerase (Taq)
Fig. 20-9
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
TECHNIQUE
RESULTS
1
2
+
+
–
–
One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis
Gel Electrophoresis
TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACT
human genome
Fig. 20-12
DNA(template strand)
TECHNIQUE
RESULTS
DNA (template strand)
DNA polymerase
Primer Deoxyribonucleotides
Shortest
Dideoxyribonucleotides(fluorescently tagged)
Labeled strands
Longest
Shortest labeled strand
Longest labeled strand
Laser
Directionof movementof strands
Detector
Last baseof longest
labeledstrand
Last baseof shortest
labeledstrand
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP
DNA Sequencing
Relatively short DNA fragmentscan be sequenced using the dideoxychain termination method.
Sequencing Video
DN
A S
eque
ncin
g
DN
A S
eque
ncer
New approaches have accelerated the pace of genome sequencing
The Human Genome Project was proposed in 1986 to determine the normal sequence of all human DNA.
The history of sequencing• New Generation Sequencing
Millions of different fragments are sequenced at the same time. This is called massively parallel sequencing.
50 µm
Studying the Expression of Interacting Groups of Genes
DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions Microarray Video
From: National Academy of Science, 2009
Metagenomics
Genetic diversity is explored without isolating intact organisms.
• Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell
Cloning organisms has the potential to generate stem cells for research
Fig. 20-16
EXPERIMENT
Transversesection ofcarrot root
2-mgfragments
Fragments werecultured in nu-trient medium;stirring causedsingle cells toshear off intothe liquid.
Singlecellsbegan todivide.
Embryonicplant developedfrom a culturedsingle cell.
Plantlet wascultured onagar medium andlater, plantedin soil.
A singlesomaticcarrot celldevelopedinto a maturecarrot plant.
RESULTS
Can a differentiated plant cell develop into a whole plant?
Fig. 20-17
EXPERIMENT
Less differ-entiated cell
RESULTS
Frog embryo Frog egg cell
UV
Donornucleustrans-planted
Frog tadpole
Enucleated egg cell
Egg with donor nucleus activated to begin
development
Fully differ-entiated(intestinal) cell
Donor nucleus trans-planted
Most developinto tadpoles
Most stop developingbefore tadpole stage
Can the nucleus from a differentiated animal cell direct development of an organism?
TECHNIQUE
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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4
5
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2
Reproductive cloning of a mammal by nuclear transplantation
In 1997, Scottish researchers announced the birth of Dolly
Fig. 20-19
CC (for Carbon Copy) was the first cat cloned
The practical applications of DNA technology
• Many fields benefit from DNA technology and genetic engineering
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
A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types
Stem cell animation
Nuclear implantation
Fig. 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
Gene therapy
Fig. 20-25
Site whererestrictionenzyme cuts
T DNA
Plant with new trait
Tiplasmid
Agrobacterium tumefaciens
DNA withthe geneof interest
RecombinantTi plasmid
TECHNIQUE
RESULTS
Genetic engineering in plants has been used to transfer many useful genes
Plant Breeding compared to Genetic Modification of Plants
GHOSTS
Fig. 20-11TECHNIQUE
Nitrocellulosemembrane (blot)
Restrictionfragments
Alkalinesolution
DNA transfer (blotting)
Sponge
Gel
Heavyweight
Papertowels
Preparation of restriction fragments Gel electrophoresis
I II III
I II IIII II III
Radioactively labeledprobe for -globin gene
DNA + restriction enzyme
III HeterozygoteII Sickle-cellallele
I Normal-globinallele
Film overblot
Probe detectionHybridization with radioactive probe
Fragment fromsickle-cell-globin allele
Fragment fromnormal -globin allele
Probe base-pairswith fragments
Nitrocellulose blot
1
4 5
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