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Biotechnology
DNA Cloning: An Overview
Bacterium
1 Gene inserted into
Cell containing gene
of interest
plasmid
Bacterial
chromosome
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
2
2 Plasmid put into
DNA of
chromosome
bacterial cell
Recombinant
bacterium
Gene cloning involves using bacteria to make multiple
copies of a gene
Fig. 20-2b
Recombinant
bacterium
3 Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Protein expressed
by gene of interest
Gene of
Interest
Copies of gene
Protein harvested
4 Basic research and
Basic
research
on gene
Gene for pest
resistance inserted
into plants
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
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. 20-3-1
Restriction site
DNA
1
5
3
3
5
Restriction enzyme
cuts sugar-phosphate
backbones.
Sticky end
Fig. 20-3-2
Restriction site
DNA
1
5
3
3
5
Restriction enzyme
cuts sugar-phosphate
backbones.
Sticky end
2
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
One possible combination
Fig. 20-3-3
Restriction site
DNA
1
5
3
3
5
Restriction enzyme
cuts sugar-phosphate
backbones.
Sticky end
2
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
One possible combination
3
DNA ligase
seals strands.
Recombinant DNA molecule
Fig. 20-UN3
Vector
DNA fragments from genomic DNA
or cDNA or copy of DNA obtained
by PCR
Cut by same restriction enzyme,
mixed, and ligated
Recombinant DNA plasmids
Amplifying DNA in Vitro:
The Polymerase Chain Reaction (PCR)
5
Target
sequence
3
Genomic DNA
1 Denaturation
PCR can make billions of a
specific DNA segment in
a few hours.
5
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
TECHNIQUE
3
PCR produces many copies of a
specific target segment of DNA
PCR Animation
PCR DNA Amplification
PCR Applications
30,000 y. o. woolly mammoth
In forensics, PCR requires only small
samples of DNA to analyze
DNA polymerase (Taq)
Fig. 20-9
TECHNIQUE
Gel Electrophoresis
One indirect method of rapidly
analyzing and comparing genomes
is gel electrophoresis
Mixture of
DNA molecules of
different
sizes
Power
source
– Cathode
Anode
+
Gel
1
Power
source
–
+
Longer
molecules
2
RESULTS
Shorter
molecules
TACGCACATTTACGTACGCGGATGCCGCGACTATGATC
ACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACT
human genome
CGACTAGCATGATCGATCAGCTACATGCTAGCACACYC
GTACATCGATCCTGACATCGACCTGCTCGTACATGCTA
CTAGCTACTGACTCATGATCCAGATCACTGAAACCCTA
GATCGGGTACCTATTACAGTACGATCATCCGATCAGAT
CATGCTAGTACATCGATCGATACTGCTACTGATCTAGC
TCAATCAAACTCTTTTTGCATCATGATACTAGACTAGC
TGACTGATCATGACTCTGATCCCGTAGATCGGGTACCT
ATTACAGTACGATCATCCGATCAGATCATGCTAGTACA
TCGATCGATACTGCTACTGATCTAGCTCAATCAAACTC
TTTTTGCATCATGATACTAGACTAGCTGACTGATCATG
ACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGA
TCATCCGATCAGATCATGCTAGTACATCGATCGATACT
Fig. 20-12
TECHNIQUE
Primer
DNA
(template strand)
DNA Sequencing
Deoxyribonucleotides
DNA
polymerase
DNA (template
strand)
Relatively short DNA fragments
can be sequenced using the dideoxy
chain termination method.
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
Labeled strands
Shortest
Direction
of movement
of strands
Longest
Longest labeled strand
Detector
Laser
RESULTS
Sequencing Video
Shortest labeled strand
Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand
Dideoxyribonucleotides
(fluorescently tagged)
DNA Sequencing
DNA Sequencer
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.
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
50 µm
Metagenomics
Genetic diversity is explored without isolating intact organisms.
From: National Academy of Science, 2009
Cloning organisms has the potential to generate
stem cells for research
• Organismal cloning produces one or more
organisms genetically identical to the “parent” that
donated the single cell
Fig. 20-16
Can a differentiated plant cell develop into a whole plant?
EXPERIMENT
RESULTS
Transverse
section of
carrot root
2-mg
fragments
Fragments were
cultured in nutrient medium;
stirring caused
single cells to
shear off into
the liquid.
Single
cells
began to
divide.
Embryonic
plant developed
from a cultured
single cell.
Plantlet was
cultured on
agar medium and
later, planted
in soil.
A single
somatic
carrot cell
developed
into a mature
carrot plant.
Can the nucleus from a differentiated animal cell direct development
of an organism?
Fig. 20-17
Frog egg cell Frog tadpole
EXPERIMENT Frog embryo
UV
Less differentiated cell
Fully differentiated
(intestinal) cell
Donor
nucleus
transplanted
Donor
nucleus
transplanted
Enucleated
egg cell
Egg with donor nucleus
activated to begin
development
RESULTS
Most develop
into tadpoles
Most stop developing
before tadpole stage
TECHNIQUE
Reproductive cloning of a mammal
Mammary
by nuclear transplantation
cell donor
Egg cell
donor
2
1
In 1997, Scottish researchers
announced the birth of Dolly
Egg cell
from ovary
3 Cells fused
Cultured
mammary cells 3
4 Grown in
Nucleus
removed
Nucleus from
mammary cell
culture
Early embryo
5 Implanted
in uterus
of a third
sheep
Surrogate
mother
6 Embryonic
development
RESULTS
Lamb (“Dolly”)
genetically identical to
mammary cell donor
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
A stem cell is a relatively unspecialized
cell that can reproduce itself indefinitely
and differentiate into specialized cells
of one or more types
Embryonic stem cells
Adult stem cells
Early human embryo
at blastocyst stage
(mammalian equivalent of blastula)
From bone marrow
in this example
Cells generating
all embryonic
cell types
Cells generating
some cell types
Cultured
stem cells
Different
culture
conditions
Stem cell animation
Nuclear implantation
Different
types of
differentiated
cells
Liver cells
Nerve cells
Blood cells
Fig. 20-22
Cloned
gene
Gene therapy
1
Insert RNA version of normal allele
into retrovirus.
Viral RNA
2
Retrovirus
capsid
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
3
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
4
Inject engineered
cells into patient.
Bone
marrow
Fig. 20-25
TECHNIQUE
Agrobacterium tumefaciens
Genetic engineering in plants
has been used to transfer many
useful genes
Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
RESULTS
Recombinant
Ti plasmid
Plant with new trait
Plant Breeding compared to Genetic Modification of Plants
GHOSTS
Fig. 20-11
TECHNIQUE
DNA + restriction enzyme
Restriction
fragments
I
II
III
Heavy
weight
Nitrocellulose
membrane (blot)
Gel
Sponge
I Normal
-globin
allele
II Sickle-cell
allele
III Heterozygote
1 Preparation of restriction fragments
Paper
towels
Alkaline
solution
2 Gel electrophoresis
3 DNA transfer (blotting)
Radioactively labeled
probe for -globin gene
I
II III
Probe base-pairs
with fragments
Fragment from
sickle-cell
-globin allele
Nitrocellulose blot
4 Hybridization with radioactive probe
Fragment from
normal -globin
allele
I
II III
Film
over
blot
5 Probe detection