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Transcript of Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 20.1 Biotechnology...
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Figure 20.1
Biotechnology
Chapter 20
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
The DNA Toolbox
• Sequencing of the genomes of more than 7,000 species was under way in 2010
• Recombinant DNA nucleotide sequences from two different sources combined in vitro into the same DNA molecule
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Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Genetic engineering direct manipulation of genes for practical purposes
• Biotechnology manipulation of organisms or their genetic components to make useful products
© 2011 Pearson Education, Inc.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Foreign DNA inserted into plasmid* plasmid inserted into bacterial cell
• Reproduction in bacterial cell cloning of plasmid with foreign DNA
multiple copies of a single gene
*Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
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DNA Cloning
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Figure 20.2Bacterium
Bacterialchromosome
Plasmid
2
1
3
4
Gene inserted intoplasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene of interest
Plasmid put intobacterial cell
DNA ofchromosome(“foreign” DNA)
Recombinantbacterium
Host cell grown in culture toform a clone of cells containingthe “cloned” gene of interest
Gene of interest
Protein expressed fromgene of interest
Protein harvestedCopies of gene
Basic researchand variousapplications
Basicresearchon protein
Basic research on gene
Gene for pestresistance insertedinto plants
Gene used to alterbacteria for cleaningup toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growthhormone treatsstunted growth
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Using Restriction Enzymes to Make Recombinant DNA
• Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
• Yields restriction fragments
• Most useful restriction enzymes give staggered cut “sticky ends.”
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Animation: Restriction Enzymes
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• Sticky ends bond with complementary sticky ends of other fragments
• DNA ligase seals bonds between fragments
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Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Figure 20.3-1
Restriction enzymecuts sugar-phosphatebackbones.
Restriction site
DNA5
5
5
5
5
5
3
3
3
3
3
3
1
Sticky end
GAATTCCTTAAG
CTTAAG AATTC
G
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Figure 20.3-2
One possible combination
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
Restriction enzymecuts sugar-phosphatebackbones.
Restriction site
DNA5
5
5
5
5
5
5
5
55
5
5
55
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
1
Sticky end
GAATTCCTTAAG
CTTAAG AATTC
G
GGAATTC
CTTAA
GG
GG
AATT CAATT CC TTAA C TTAA
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Figure 20.3-3
Recombinant DNA molecule
One possible combinationDNA ligaseseals strands
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
Restriction enzymecuts sugar-phosphatebackbones.
Restriction site
DNA5
5
5
5
5
5
5
5
55
5
5
55
5
5
3
3
3
3
3
3
3
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3
3
3
3
3
3
3
3
2
3
1
Sticky end
GAATTCCTTAAG
CTTAAG AATTC
G
GGAATTC
CTTAA
GG
GG
AATT CAATT CC TTAA C TTAA
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Cloning a Eukaryotic Gene in a Bacterial Plasmid
• A cloning vector (original plasmid) DNA molecule that carries foreign DNA into a host cell
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Figure 20.4
Bacterial plasmidTECHNIQUE
RESULTS
ampR gene lacZ gene
Restrictionsite
Hummingbird cell
Sticky ends Gene of
interest
Humming-bird DNAfragments
Recombinant plasmids Nonrecombinant plasmid
Bacteria carryingplasmids
Colony carrying non-recombinant plasmidwith intact lacZ gene
Colony carrying recombinantplasmidwith disruptedlacZ gene
One of manybacterialclones
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Figure 20.4a-1
Bacterial plasmidTECHNIQUE
ampR gene lacZ gene
Restrictionsite
Hummingbird cell
Sticky ends Gene of
interest
Humming-bird DNAfragments
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Figure 20.4a-2
Bacterial plasmidTECHNIQUE
ampR gene lacZ gene
Restrictionsite
Hummingbird cell
Sticky ends Gene of
interest
Humming-bird DNAfragments
Recombinant plasmids Nonrecombinant plasmid
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Figure 20.4a-3
Bacterial plasmidTECHNIQUE
ampR gene lacZ gene
Restrictionsite
Hummingbird cell
Sticky ends Gene of
interest
Humming-bird DNAfragments
Recombinant plasmids Nonrecombinant plasmid
Bacteria carryingplasmids
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Figure 20.4b
RESULTS
Bacteria carryingplasmids
Colony carrying non-recombinant plasmidwith intact lacZ gene
Colony carrying recombinantplasmidwith disruptedlacZ gene
One of manybacterialclones
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Storing Cloned Genes in DNA Libraries
• A genomic library is made using plasmids or bacteriophages
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Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Figure 20.5
Foreign genome
Cut with restriction enzymes into eithersmallfragments
largefragments
or
Recombinantplasmids
Plasmidclone
(a) Plasmid library
(b) BAC clone
Bacterial artificialchromosome (BAC)
Largeinsertwithmanygenes
(c) Storing genome libraries
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• Made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell
• A cDNA library represents only the subset of genes transcribed into mRNA in the original cells
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Complementary DNA (cDNA) library
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Figure 20.6-1
DNA innucleus
mRNAs incytoplasm
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Figure 20.6-2
DNA innucleus
mRNAs incytoplasm
mRNA
Reversetranscriptase Poly-A tail
DNAstrand
Primer
55
33
A A A A A AT T T T T
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Figure 20.6-3
DNA innucleus
mRNAs incytoplasm
mRNA
Reversetranscriptase Poly-A tail
DNAstrand
Primer
55
55
33
33
A A A A A A
A A A A A A
T T T T T
T T T T T
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Figure 20.6-4
DNA innucleus
mRNAs incytoplasm
mRNA
Reversetranscriptase Poly-A tail
DNAstrand
Primer
DNA polymerase
55
55
55
33
33
33
A A A A A A
A A A A A A
T T T T T
T T T T T
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Figure 20.6-5
DNA innucleus
mRNAs incytoplasm
mRNA
Reversetranscriptase Poly-A tail
DNAstrand
Primer
DNA polymerase
cDNA
55
55
55
55
33
33
33
33
A A A A A A
A A A A A A
T T T T T
T T T T T
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Screening a Library for Clones Carrying a Gene of Interest
• Identified with a nucleic acid probe having a sequence complementary to the gene
• Process is called nucleic acid hybridization
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• A probe can be synthesized that is complementary to the gene of interest
• For example, if the desired gene is
– Then we would synthesize this probe
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5 3 CTCAT CACCGGC
53G A G T A G T G G C C G
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Figure 20.7
Radioactivelylabeled probemolecules Gene of
interestProbeDNA
Single-strandedDNA fromcell
Film
Location ofDNA with thecomplementarysequence
Nylonmembrane
Nylon membrane
Multiwell platesholding libraryclones
TECHNIQUE 5
53
3
GAGTAGTGGCCG CTCATCACCGGC
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Eukaryotic Cloning and Expression Systems
• Avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells, such as yeasts or cultured mammal cells, as hosts for cloning and expressing genes
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• Electroporation: applying a brief electrical pulse to create temporary holes in plasma membranes introduces recombinant DNA into eukaryotic cells
• Alternatively, scientists can inject DNA into cells using microscopically thin needles
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Cross-Species Gene Expression and Evolutionary Ancestry
• The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
• e.g., Pax-6 (gene that directs formation of vertebrate eye); also directs the formation of insect eye
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Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)
• Polymerase chain reaction, PCR produce many copies of a specific target segment of DNA
• Key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase.
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Figure 20.8
Genomic DNA
Targetsequence
Denaturation
Annealing
Extension
Primers
Newnucleotides
Cycle 1yields
2molecules
Cycle 2yields
4molecules
Cycle 3yields 8
molecules;2 molecules
(in white boxes)match target
sequence
5
5
5
5
3
3
3
3
2
3
1
TECHNIQUE
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Figure 20.8a
Genomic DNA
Targetsequence
5
5
3
3
TECHNIQUE
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Denaturation
Annealing
Extension
Primers
Newnucleo-tides
Cycle 1yields
2molecules
5
5
3
3
2
3
1
Figure 20.8b
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Figure 20.8c
Cycle 2yields
4molecules
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Figure 20.8d
Cycle 3yields 8
molecules;2 molecules
(in white boxes)match target
sequence
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Gel Electrophoresis and Southern Blotting
• Gel electrophoresis method of rapidly analyzing and comparing genomes
• Uses a gel as a molecular sieve to separate nucleic acids or proteins by size, electrical charge, and other properties
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Animation: Biotechnology Lab
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Figure 20.9
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Powersource
Longermolecules
Cathode Anode
Wells
Gel
Shortermolecules
TECHNIQUE
RESULTS
1
2
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Figure 20.9a
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Powersource
Longermolecules
Cathode Anode
Wells
Gel
Shortermolecules
TECHNIQUE
2
1
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Figure 20.9b
RESULTS
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• Restriction fragment analysis DNA fragments produced by restriction enzyme digestion sorted by gel electrophoresis
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• Variations in DNA sequence are called polymorphisms
• Sequence changes that alter restriction sites are called RFLPs (restriction fragment length polymorphisms)
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Figure 20.10
Normal -globin allele
Sickle-cell mutant -globin allele
Largefragment
Normalallele
Sickle-cellallele
201 bp175 bp
376 bp
(a) DdeI restriction sites in normal andsickle-cell alleles of the -globin gene
(b) Electrophoresis of restrictionfragments from normal andsickle-cell alleles
201 bp175 bp
376 bp
Large fragment
Large fragment
DdeI DdeI DdeI DdeI
DdeI DdeI DdeI
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Figure 20.10a
Normal -globin allele
Sickle-cell mutant -globin allele
(a) DdeI restriction sites in normal andsickle-cell alleles of the -globin gene
201 bp175 bp
376 bp
Large fragment
Large fragment
DdeI DdeI DdeI DdeI
DdeI DdeI DdeI
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Figure 20.10b
Largefragment
Normalallele
Sickle-cellallele
201 bp175 bp
376 bp
(b) Electrophoresis of restrictionfragments from normal andsickle-cell alleles
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• Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization
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Figure 20.11
DNA restriction enzyme
321
4
TECHNIQUE
I Normal-globinallele
II Sickle-cellallele
III Heterozygote
Restrictionfragments
Nitrocellulosemembrane (blot)
Heavyweight
Gel
Sponge
Alkalinesolution Paper
towels
III III
III III III III
Preparation ofrestriction fragments
Gel electrophoresis DNA transfer (blotting)
Radioactively labeledprobe for -globingene
Nitrocellulose blot
Probe base-pairswith fragments
Fragment from sickle-cell -globin allele
Fragment from normal - globin allele
Filmoverblot
Hybridization with labeled probe Probe detection5
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DNA Sequencing
• Relatively short DNA fragments can be sequenced by the dideoxy chain termination method, the first automated method to be employed
• Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths
• Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment
• The DNA sequence can be read from the resulting spectrogram
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Figure 20.12
DNA(template strand)
TECHNIQUE
5
3
C
C
C
C
T
TT
G
G
A
A
AA
GTT
T
DNApolymerase
Primer
5
3
P P P
OH
G
dATP
dCTP
dTTP
dGTP
Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)
P P P
H
G
ddATP
ddCTP
ddTTP
ddGTP
5
3
C
C
C
C
T
TT
G
G
A
A
AA
DNA (templatestrand)
Labeled strands
Shortest Longest5
3
ddCddG
ddAddA
ddA
ddG
ddG
ddTddC
GTT
TGTT
TC
GTT
TC
T T
G
GTT
TCT
GA
GTT
TCT
GAA
GTT
TCT
GAAG
GTT
TCT
GAAGT
GTT
TCT
GAAGTC
GTT
TCT
GAAGTCA
Directionof movementof strands
Longest labeled strand
Detector
LaserShortest labeled strand
RESULTS
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
G
G
G
A
AA
C
C
T
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Figure 20.12a
DNA(template strand)
TECHNIQUEPrimer Deoxyribonucleotides Dideoxyribonucleotides
(fluorescently tagged)
DNApolymerase
5
5
3
3
OH H
GG
dATP
dCTP
dTTP
dGTP
P P P P P P
ddATP
ddCTP
ddTTP
ddGTP
T
TT
G
G
G
C
C
C
CT
TT
A
A
AA
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Figure 20.12b
DNA (templatestrand)
Labeled strands
Shortest Longest
Directionof movementof strands
Longest labeled strand
Detector
LaserShortest labeled strand
TECHNIQUE (continued)
5
3
G
G
C
C
C
CT
TT
A
A
AA
T
TT
G
ddC
ddC
ddG
ddG
ddG
ddA ddA
ddA
ddT
3
5
T
TT
G
CT
TT
G
CG
T
TT
G
CGA
T
TT
G
CGAA
T
TT
G
CGAAG
T
TT
CGAAGT
T
TT
CGAAGTC
A
T
TT
CGAAGTC
G G G
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Figure 20.12c
RESULTS
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
G
G
G
A
AA
C
C
T
Directionof movementof strands
Longest labeled strand
Detector
LaserShortest labeled strand
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• Reverse transcriptase-polymerase chain reaction (RT-PCR)
• Reverse transcriptase + mRNA cDNA, which serves as a template for PCR amplification of the gene of interest
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Figure 20.13
cDNA synthesis
PCR amplification
Gel electrophoresis
mRNAs
cDNAs
Primers
-globingene
Embryonic stages1 2 3 4 5 6
2
3
1
RESULTS
TECHNIQUE
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Expression of Interacting Groups of Genes
• DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions
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Isolate mRNA.
2
1
3
4
TECHNIQUE
Make cDNA by reversetranscription, usingfluorescently labelednucleotides.
Apply the cDNA mixture to a microarray, a different genein each spot. The cDNA hybridizeswith any complementary DNA onthe microarray.
Rinse off excess cDNA; scan microarrayfor fluorescence. Each fluorescent spot(yellow) represents a gene expressedin the tissue sample.
Tissue sample
mRNA molecules
Labeled cDNA molecules(single strands)
DNA fragmentsrepresenting aspecific gene
DNA microarray
DNA microarraywith 2,400human genes
Figure 20.15
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Figure 20.15a
DNA microarraywith 2,400human genes
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Determining Gene Function
• in vitro mutagenesis mutations are introduced into a cloned gene, altering or destroying its function
• Mutated gene returned to the cell normal gene function determined by examining the mutant’s phenotype
• Gene expression can also be silenced using RNA interference (RNAi)
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• Genetic markers called SNPs (single nucleotide polymorphisms) occur on average every 100–300 base pairs
• SNPs can be detected by PCR, and any SNP shared by people affected with a disorder but not among unaffected people may pinpoint the location of the disease-causing gene
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Figure 20.16
DNA
SNPNormal allele
Disease-causingallele
T
C
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• Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell
Cloning
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Cloning Plants: Single-Cell Cultures
• Totipotent cell can generate a complete new organism
• Plant cloning is used extensively in agriculture
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Figure 20.17
Crosssection ofcarrot root
2-mgfragments
Fragments werecultured in nu-trient medium;stirring causedsingle cells toshear off intothe liquid.
Single cellsfree insuspensionbegan todivide.
Embryonicplant developedfrom a culturedsingle cell.
Plantlet wascultured onagar medium.Later it wasplanted in soil.
Adultplant
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Cloning Animals: Nuclear Transplantation
• Nucleus of an unfertilized egg cell is replaced with the nucleus of a differentiated cell
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Frog embryo Frog egg cell Frog tadpole
UV
Less differ-entiated cell
Donornucleustrans-planted
Enucleatedegg cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopment
Most developinto tadpoles.
Most stop developingbefore tadpole stage.
EXPERIMENT
RESULTS
Figure 20.18
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Reproductive Cloning of Mammals
• 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
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Figure 20.19
Mammarycell donor
21
3
4
5
6
TECHNIQUE
RESULTS
Culturedmammarycells
Eggcell fromovary
Egg cell donor
NucleusremovedCells fused
Grown in culture
Implanted in uterusof a third sheep
Embryonicdevelopment
Nucleus frommammary cell
Early embryo
Surrogatemother
Lamb (“Dolly”) geneticallyidentical to mammary cell donor
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Figure 20.19a
Mammarycell donor
21
3
TECHNIQUE
Culturedmammarycells
Eggcell fromovary
Egg cell donor
Nucleusremoved
Cells fused
Nucleus frommammary cell
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4
5
6
RESULTS
Grown in culture
Implanted in uterusof a third sheep
Embryonicdevelopment
Nucleus frommammary cell
Early embryo
Surrogatemother
Lamb (“Dolly”) geneticallyidentical to mammary cell donor
Figure 20.19b
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• Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs
• CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”
• Cloned animals do not always look or behave exactly the same
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Figure 20.20
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Stem Cells of Animals
• Relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types
• Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem (ES) cells; these are able to differentiate into all cell types
• The adult body also has stem cells, which replace nonreproducing specialized cells
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Figure 20.21
Culturedstem cells
Differentcultureconditions
Differenttypes ofdifferentiatedcells
Embryonicstem cells
Adultstem cells
Cells generatingall embryoniccell types
Cells generatingsome cell types
Livercells
Nervecells
Bloodcells
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Practical applications of DNA technology
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Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Medical Applications
• Identification of human genes in which mutation plays a role in genetic diseases
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Diagnosis and Treatment of Diseases
• Can diagnose PCR and sequence-specific primers, then sequencing the amplified product to look for the disease-causing mutation
• SNPs may be associated with a disease-causing mutation
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Human Gene Therapy
• The alteration of an afflicted individual’s genes
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Figure 20.23Cloned gene
2
1
3
4
Retroviruscapsid
Bonemarrowcell frompatient
Viral RNA
Bonemarrow
Insert RNA version of normal alleleinto retrovirus.
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.
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Pharmaceutical Products
• Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases
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Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Transgenic animals are made by introducing genes from one species into the genome of another animal
pharmaceutical “factories”
Protein Production by “Pharm” Animals
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Figure 20.24
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Forensic Evidence and Genetic Profiles
• An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids
• Can use PCR and/ or Southern Blotting/ RFLP
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Figure 20.25This photo showsWashington just beforehis release in 2001,after 17 years in prison.
(a)
(b)These and other STR data exonerated Washingtonand led Tinsley to plead guilty to the murder.
Semen on victim
Earl Washington
Kenneth Tinsley
17,19
16,18
17,19
13,16
14,15
13,16
12,12
11,12
12,12
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
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Environmental Cleanup
• Modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
• DNA technology is being used to improve agricultural productivity and food quality
• Genetic engineering of transgenic animals speeds up the selective breeding process
• Beneficial genes can be transferred between varieties of species
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• The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
• Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops
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Figure 20.26
Plant with new trait
RESULTS
TECHNIQUE
Tiplasmid
Site whererestrictionenzyme cuts
DNA withthe geneof interest
RecombinantTi plasmid
T DNA
Agrobacterium tumefaciens
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Safety and Ethical Questions Raised by DNA Technology
• Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
• Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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• Genetically modified (GM) organisms
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