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19
DNA Technology
Lecture Presentation by
Nicole Tunbridge and
Kathleen Fitzpatrick
Detector
Laser
Shortest labeled strand
Results
Last nucleotide G
of longest A
labeled strand C
T
G
Last nucleotide A
A
of shortest G
labeled strand C
Technique
DNA Primer Deoxyribo- Dideoxyribonucleotides
(template strand) T
3′ nucleotides (fluorescently tagged)
5′ C G
dATP ddATP
T T
G T dCTP ddCTP
A 5′
C DNA dTTP ddTTP
T
T polymerase dGTP ddGTP
C
G
A P P P P P P
C G G
A
3′ A
Technique
Technique
Direction
of movement Longest labeled strand
of strands
Detector
Laser
Shortest labeled strand
Results
Last nucleotide G
of longest A
C
labeled strand T
G
A
Last nucleotide A
of shortest G
labeled strand C
4-mer A
T
2 Each fragment is isolated with G
3-mer
a bead. C
2-mer
3 Using PCR, 106 copies of each
1-mer
fragment are made, each attached
to the bead by 5′ end.
Template
C C C C
strand
C C C C
A of DNA A dTTP A dCTP
A dGTP
A dATP A A A
T T T T
G G G GC PPi
TA PPi TA TA TA
DNA GC GC GC GC
polymerase GC GC GC GC
AG Primer AG AG AG
TA TA TA TA
Technique
A T GC A T GC
Template
C strand C
C C
of DNA
A A dTTP
A dATP A
T T
G G
TA PPi TA
DNA GC GC
polymerase GC GC
AG Primer AG
TA TA
Technique
A T GC A T GC
C C
C C
A dGTP A dCTP
A A
T T
G GC PPi
TA TA
GC GC
GC GC
AG AG
TA TA
Results
4-mer A
T
3-mer G
C
2-mer
1-mer
Recombinant
bacterium
3 Host cell grown in culture to form a clone of
cells containing the “cloned” gene of interest
Gene of
interest Protein expressed
from gene of interest
4 Basic research
Gene for pest resistance Human growth hormone
and various
inserted into plants treats stunted growth
applications
1 Gene inserted
into plasmid
Bacterial Plasmid
chromosome DNA of
Gene of chromosome
Recombinant
interest (“foreign” DNA)
DNA (plasmid)
2 Plasmid put into
bacterial cell
Recombinant
bacterium
3 Host cell grown in culture to form a
clone of cells containing the “cloned”
gene of interest
Gene of
interest Protein expressed
from gene of interest
© 2015 Pearson Education Ltd.
Figure 19.5b
Gene of
interest Protein expressed
from gene of interest
4 Basic research
and various
applications
Restriction site
5′ 3′
G AAT T C
DNA
C T T AAG
3′ 5′
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
3′ 5′
5′ 3′
5′ 3′
3′ 5′
Sticky end
5′
3′
2 Base pairing of sticky
ends produces various 3′
combinations. 5′
Fragment from different DNA molecule
cut by the same restriction enzyme
5′ 3′ 5′ 3′ 5′ 3′
G AAT T C G AAT T C
C T TA A G C TTAA G
3′ 5′ 3′ 5′ 3′ 5′
One possible combination
3 DNA ligase
seals the strands.
5′ 3′
Recombinant
plasmid
Bacterial
plasmid
Restriction site
5′ 3′
G A AT T C
DNA C T TA A G
3′ 5′
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
3′ 5′
5′ 3′
5′ 3′
3′ 5′
Sticky end
© 2015 Pearson Education Ltd.
Figure 19.6b
3′ 5′
5′ 3′
5′ 3′
3′ 5′
Sticky end
5′
3′
2 Base pairing of sticky
ends produces various 3′
5′
combinations.
Fragment from different
DNA molecule cut by the
same restriction enzyme
5′ 3′ 5′ 3′ 5′ 3′
G AATT C G AATT C
C TTAA G C TTAA G
3′ 5′ 3′ 5′ 3′ 5′
One possible combination
© 2015 Pearson Education Ltd.
Figure 19.6c
5′ 3′ 5′ 3′ 5′ 3′
G AATT C G AATT C
C TTAA G C TTAA G
3′ 5′ 3′ 5′ 3′ 5′
One possible combination
3 DNA ligase
seals the strands
5′ 3′
Recombinant
plasmid
Restriction fragments
(size standards)
(b) Shorter molecules are slowed down less than
longer ones, so they move faster through the gel.
© 2015 Pearson Education Ltd.
Figure 19.7a
Mixture of Power
DNA mol- source
ecules of Cathode Anode
different
sizes
Wells
Gel
Restriction fragments
(size standards)
(b) Shorter molecules are slowed down less
than longer ones, so they move faster
through the gel.
1 Denaturation 5′ 3′
3′ 5′
2 Annealing
Cycle 1
yields
2 Primers
molecules
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3 yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Technique
5′ 3′
Target
sequence
Genomic DNA 3′ 5′
Technique
1 Denaturation 5′ 3′
3′ 5′
Cycle 1
yields
2
molecules
Technique
1 Denaturation 5′ 3′
3′ 5′
2 Annealing
Cycle 1
yields
2 Primers
molecules
Technique
1 Denaturation 5′ 3′
3′ 5′
2 Annealing
Cycle 1
yields
2 Primers
molecules
3 Extension
New
nucleotides
Technique
Cycle 2
yields
4
molecules
Cycle 3 yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Cloning vector
(bacterial plasmid)
Mix and ligate
Recombinant
DNA plasmid
50 µm T1 T2 T3 A1 A2 A3 A4 A5
Segment
boundary
50 µm T1 T2 T3 A1 A2 A3 A4 A5
© 2015 Pearson Education Ltd.
Figure 19.10b
Head Thorax Abdomen
50 µm T1 T2 T3 A1 A2 A3 A4 A5
Segment
boundary
50 µm T1 T2 T3 A1 A2 A3 A4 A5
Reverse transcriptase
mRNA Poly-A tail
5′ A A A A A A 3′
3′ T T T T T 5′
DNA Primer
strand (poly-dT)
Reverse transcriptase
mRNA Poly-A tail
5′ A A A A A A 3′
3′ T T T T T 5′
DNA Primer
strand (poly-dT)
5′ A A A A A A 3′
3′ T T T T T 5′
Reverse transcriptase
mRNA Poly-A tail
5′ A A A A A A 3′
3′ T T T T T 5′
DNA Primer
strand (poly-dT)
5′ A A A A A A 3′
3′ T T T T T 5′
5′ 3′
3′ 5′
DNA
polymerase
Reverse transcriptase
mRNA Poly-A tail
5′ A A A A A A 3′
3′ T T T T T 5′
DNA Primer
strand (poly-dT)
5′ A A A A A A 3′
3′ T T T T T 5′
5′ 3′
3′ 5′
DNA
polymerase
5′ 3′
3′ 5′
cDNA
© 2015 Pearson Education Ltd.
Figure 19.12
Technique
1 cDNA synthesis mRNAs
cDNAs
Primers
2 PCR amplification
Specific
gene
3 Gel electrophoresis
Genes expressed
in second tissue.
Genes expressed
in both tissues.
Genes expressed
► in neither tissue.
DNA microarray
(actual size)
A
DNA
T
Normal allele
SNP
C
G
Disease-causing
allele
Cross section
of carrot root
Small
fragments
Fully differ-
Less differ- entiated
entiated cell (intestinal) cell
Donor Donor
nucleus Enucleated nucleus
trans- egg cell trans-
planted Egg with donor nucleus planted
activated to begin
Results development
1 2
Egg cell
from ovary Nucleus
removed
Cultured 3 Cells fused
mammary
cells
Nucleus from
mammary cell
4 Grown in culture
Early embryo
5 Implanted in uterus
of a third sheep
Surrogate
mother
6 Embryonic
development
Lamb (“Dolly”)
Results genetically identical to
mammary cell donor
© 2015 Pearson Education Ltd.
Figure 19.17a
Technique
1 2
Egg cell
from ovary Nucleus
removed
Cultured 3 Cells fused
mammary
cells
Nucleus from
mammary cell
© 2015 Pearson Education Ltd.
Figure 19.17b
Technique
Nucleus from
mammary cell
4 Grown in culture
Early embryo
5 Implanted in uterus
of a third sheep
Surrogate
mother
6 Embryonic
development
Lamb (“Dolly”)
Results genetically identical to
mammary cell donor
© 2015 Pearson Education Ltd.
a) Since 1997, cloning has been demonstrated in many
mammals, including mice, cats, cows, horses, mules,
pigs, and dogs
b) CC (for Carbon Copy) was the first cat cloned;
however, CC differed somewhat from her
female “parent”
c) Cloned animals do not always look or behave exactly
the same
Stem cell
Cell division
Different culture
conditions
Different types of
differentiated cells
Skin
Oct3/4
Sox2 fibroblast
cell
Four “stem cell” master regulator
genes were introduced, using
the retroviral cloning vector.
c-Myc
Klf4
Induced pluripotent
stem (iPS) cell
© 2015 Pearson Education Ltd.
Concept 19.4: The practical applications of DNA-based
biotechnology affect our lives in many ways
Viral RNA
Bone
marrow
cell from
patient
5′ CUCAUCACCGGC 3′
3′ G A G TA G T G G C C G 5′
Treatments
A B C
A B C
A B C
A B C
5′ 3′ 5′ 3′
G A AT T C
C T TA A G
3′ 5′ 3′ 5′
Sticky end
5′ G A AT T C TA A A G C G C T TAT G A AT T C 3′
3′ C T TA A G AT T T C G C G A ATA C T TA A G 5′
Aardvark DNA
Plasmid
© 2015 Pearson Education Ltd.
Figure 19.UN07