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Chapter 20
DNA Technology
and Genomics
PowerPoint Lectures for
Biology, Seventh Edition
Neil Campbell and Jane Reece
Lectures by Chris Romero
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Using Restriction Enzymes to Make Recombinant
DNA
Restriction site
• Bacterial restriction enzymes DNA 53
cut DNA molecules at DNA Restriction enzyme cuts
sugar-phosphate
sequences called restriction the
backbones at each arrow.
sites
– fragments with “sticky
ends”
3
5
Sticky end
DNA fragment from another
source is added. Base pairing Fragment from different
of sticky ends produces
DNA molecule cut by the
various combinations.
same restriction enzyme
One possible combination
DNA ligase
seals the strands.
Animation: Restriction Enzymes
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Recombinant DNA molecule
Cloning a Eukaryotic Gene in a Bacterial Plasmid
• In gene cloning, the original plasmid is called a
cloning vector
• A cloning vector is a DNA molecule that can carry
foreign DNA into a cell and be replicated.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-4_1
Bacterial cell
Isolate plasmid DNA
and human DNA.
lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Cut both DNA samples with
the same restriction enzyme.
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids
LE 20-4_2
Bacterial cell
Isolate plasmid DNA
and human DNA.
lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Cut both DNA samples with
the same restriction enzyme.
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria
LE 20-4_3
Bacterial cell
Isolate plasmid DNA
and human DNA.
lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Cut both DNA samples with
the same restriction enzyme.
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria
Plate the bacteria on agar
containing ampicillin and X-gal.
Incubate until colonies grow.
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid with
disrupted lacZ gene
Bacterial
clone
Amplifying DNA in Vitro: The Polymerase Chain
Reaction (PCR)
5
3
Target
sequence
• The polymerase
chain reaction, PCR,
can produce many
copies of a specific
target segment of
DNA
Genomic DNA
Denaturation:
Heat briefly
to separate DNA
strands
Cycle 1
yields
2
molecules
Extension:
DNA polymerase
adds nucleotides to
the 3 end of each
primer
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
3
5
5
3
3
5
Primers
New
nucleotides
Gel Electrophoresis and Southern Blotting
• Gel electrophoresis - technique uses a gel as a
molecular sieve to separate nucleic acids or
proteins by size
Cathode
Power
source
Mixture
of DNA
molecules
of different sizes
Shorter
molecules
Gel
Glass
plates
Anode
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Video:
Biotechnology Lab
Longer
molecules
Normal b-globin allele
• Restriction fragment
analysis is useful for
comparing two
different DNA
molecules, such as
two alleles for a
gene
175 bp
Ddel
201 bp
Ddel
Large fragment
Ddel
Ddel
Sickle-cell mutant b-globin allele
376 bp
Ddel
Large fragment
Ddel
Ddel
Ddel restriction sites in normal and sickle-cell alleles of
b-globin gene
Normal
allele
Sickle-cell
allele
Large
fragment
376 bp
201 bp
175 bp
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Electrophoresis of restriction fragments from normal
and sickle-cell alleles
Forensic Evidence
• DNA “fingerprints”
obtained by analysis of
tissue or body fluids can
provide evidence in criminal
and paternity cases
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Defendant’s
blood (D)
Blood from defendant’s
clothes
Victim’s
blood (V)
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Concept 20.5: The practical applications of DNA
technology affect our lives in many ways
• Many fields benefit from DNA technology and
genetic engineering
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Human Gene Therapy
• Gene therapy is the alteration of an afflicted
individual’s genes
• Gene therapy holds great potential for treating
disorders traceable to a single defective gene
• Vectors are used for delivery of genes into cells
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-16
Cloned gene
Insert RNA version of normal allele
into retrovirus.
Viral RNA
Retrovirus
capsid
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
Inject engineered
cells into patient.
Bone
marrow
Pharmaceutical Products
• Some pharmaceutical applications of DNA
technology:
– Large-scale production of human hormones
and other proteins with therapeutic uses
– Production of safer vaccines
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Environmental Cleanup
• Some modified
microorganisms can be
used to extract
minerals from the
environment or
degrade potentially
toxic waste materials
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Genetic Engineering in Plants
Agrobacterium tumefaciens
• Agricultural scientists
have endowed a
number of crop plants
with genes for
desirable traits
Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
Recombinant
Ti plasmid
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Plant with
new trait