Topic 2 DNA Tools - Lecturer - 201801

BIOLOGY TENTH EDITION
Global Edition
Topic 2 Biotechnology Campbell • Reece • Urry • Cain • Wasserman • Minorsky • Jackson
19
DNA
Technology
Lecture Presentation by
Nicole Tunbridge and
Kathleen Fitzpatrick
© 2015 Pearson Education Ltd

Learning outcomes:
1. Relate the natural function of restriction

enzymes to how they are used in recombinant
DNA technology
2. Outline the procedures for cloning a eukaryotic
gene in a bacterial plasmid
3. Describe the polymerase chain reaction (PCR)
and explain the advantages and limitations of
this procedure

4. Explain how gel electrophoresis is used to
analyze nucleic acids and to distinguish between
two alleles of a gene
5. Describe various DNA screening methods eg.
6. Describe the application of DNA technology in
medical, forensic and agriculture science.

Cloning Applications
Purposes of DNA Cloning Overall Idea of DNA Cloning
Cloning Organism
Cloning vector Gene of Interest
e.g. Plasmid amplification
PCR
RE Digestion & Ligation

(RE: restriction endonucleases)
Transformation
Host cells: bacteria, animal cells OR plant cells
Selection of transformed bacteria X-gal digestion
Antibiotic resistant gene
Screening of positive results (clone/ recombinant DNA)

Other Usage Gel Electrophoresis of
Restriction i.Digested clone
fragment analysis ii.PCR products Example
Steps in Gene cloning (host cells carrying foreign cells) :
1) Isolation of target DNA from chromosome & vector DNA

2) PCR / Amplification - gene is replicated using PCR so that
more copies are available to produce a protein for the
cell's use.
3) Restriction enzyme digestion
4) Recombination & Ligation – insertion of target DNA into
vector DNA
5) Transformation of host cells - uptake of recombinant
vector DNA by the host cells
6) Screening – of cell clones carrying the gene of interest
(PCR, gel electrophoresis, southern blotting, DNA
sequencing, X-gal screening)
• Biotechnology is the manipulation of organisms or
their components to make useful products
• The applications of DNA technology affect
everything from agriculture, to criminal law,
to medical research

Concept 19.1: DNA sequencing and DNA cloning are
valuable tools for genetic engineering and biological
inquiry
• Genetic engineering is the direct manipulation of
genes for practical purposes
• To work directly with specific genes, scientists prepare
well-defined DNA segments in multiple identical copies
by a process called DNA cloning
o One common approach is use bacteria e.g. E.coli.
o E.coli chromosome is a large circular molecule of DNA
o E.coli and many other bacteria also have plasmids

Cloning a Eukaryotic Gene in a Bacterial Plasmid
Plasmids are small circular DNA molecules that

replicate separately from the bacterial
chromosome
o Has only a small number of genes that may
be useful when the bacterium is in a
particular environment but may not be
required for survival or reproduction under
most conditions

Cloning a Eukaryotic Gene in a Bacterial Plasmid
o A plasmid used to clone a foreign gene is called a

cloning vector
o A cloning vector is a DNA molecule that can carry
foreign DNA into a host cell and replicate there
o Bacterial plasmids are widely used as cloning
vectors because they are readily obtained, easily
manipulated, easily introduced into bacterial cells,
and once in the bacteria they multiply rapidly
(high reproduction rate)

Bacterium Cell containing gene
of interest
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
Copies of gene Protein harvested
4 Basic research
Gene for pest resistance Human growth hormone
and various
inserted into plants treats stunted growth
applications
Gene used to alter bacteria Protein dissolves blood clots

for cleaning up toxic waste in heart attack therapy

Making Multiple Copies of a Gene or Other DNA
Segment
• Researchers can insert DNA into plasmids to produce

recombinant DNA, a molecule with DNA from two
different sources
• Reproduction of a recombinant plasmid in a bacterial
cell results in cloning of the plasmid including the
foreign DNA
• This results in the production of multiple copies of a
single gene
• The production of multiple copies of a single gene is a
type of DNA cloning called gene cloning
Gene Cloning Procedures
© 2015 Pearson Education Ltd.

of interest
1 Gene inserted
into plasmid
Bacterial Plasmid
chromosome DNA of
Gene of chromosome
Recombinant
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
Copies of gene Protein harvested
4 Basic research
Gene for pest resistance Human growth hormone
and various
inserted into plants treats stunted growth
applications
Gene used to alter bacteria Protein dissolves blood clots

for cleaning up toxic waste in heart attack therapy

of interest
1 Gene inserted
into plasmid
Bacterial Plasmid
chromosome DNA of
Gene of chromosome
Insert DNA Recombinant
from another DNA (plasmid)
source (foreign 2 Plasmid put into
DNA) into bacterial cell
plasmid. The
resulting
plasmid is Recombinant
called bacterium
Recombinant
3 Host cell grown in culture to form a
DNA
clone of cells containing the “cloned”
(containing
gene of interest
DNA from 2
different
sources) Gene of
Using Restriction Enzymes to Make a Recombinant
DNA Plasmid
Restriction enzymes
• Bacterial restriction enzymes cut DNA molecules at
specific DNA sequences called restriction sites
• A restriction enzyme usually makes many cuts,
yielding restriction fragments
• The most useful restriction enzymes cut DNA
in a staggered way, producing fragments with
“sticky ends”
• Sticky ends can hydrogen bond with complementary
sticky ends of other fragments cuts with the same
enzyme
Results
Results of Type
from II Digestion
Restriction Enzymes Digestion
1) Sticky ends
•Enzymes with staggered cuts  complementary ends
a) Enzymes that cut at same position on both strands
leave “blunt” ends
•HindIII - leaves 5´ overhangs (“sticky”)
5’ --AAGCTT-- 3’
b) SmaI 5’ --A AGCTT--3’
3’ --TTCGAA-- 5’ 3’ --TTCGA A--5’
5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’

•KpnI leaves 3´ overhangs (“sticky”)
3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’
5’--GGTACC-- 3’ 5’ --GGTAC C-- 3’

3’--CCATGG-- 5’ 3’ --C CATGG-- 5’

2) Blunt ends
• Enzymes that cut at same position on both
strands leave “blunt” ends
• SmaI
5’ --CCCGGG-- 3 5’ --CCC GGG-- 3’

3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’

Figure 19.6
Bacterial
plasmid
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 T TAA G
3′ 5′ 3′ 5′ 3′ 5′
One possible combination
3 DNA ligase
seals the strands.
5′ 3′
3′ Recombinant DNA molecule 5′
Recombinant
plasmid

Figure 19.6a
Restriction
enzyme (EcoR1) Bacterial
recognizes a plasmid
specific 6-base
sequence, the
restriction site,
present at one Restriction site
place in this 5′ 3′
plasmid 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
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′
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′
3 DNA ligase
seals the strands
5′ 3′
3′ Recombinant DNA molecule 5′
Recombinant
plasmid

Ligation
• DNA ligase is an enzyme that seals the bonds
between restriction fragments
• DNA ligase catalyzes the formation of covalent
bonds that close up the sugar-phosphate backbones
of DNA strands

of interest
1 Gene inserted
into plasmid
Bacterial Plasmid
chromosome DNA of
Gene of chromosome
Plasmid is Recombinant
then returned DNA (plasmid)
to a bacterial 2 Plasmid put into
cell (host cell), bacterial cell
producing a
recombinant
bacterium. Recombinant
bacterium
Transformation 3 Host cell grown in culture to form a
- uptake of clone of cells containing the “cloned”
recombinant gene of interest
DNA by the
host cells Gene of
Transformation : insertion of
recombinant DNA into host cell
• Recombinant DNA are likely to be inserted into
bacterial cell (eg. E.coli) if the cells walls are
altered
• These cells are called competent cells
• Cells are made competent by a process that uses
calcium chloride and heat shock
• Competent cells can be transformed using
electroporation or heat-shock method.

Transformation
1. Electroporation
• Brief electrical pulse applied to a solution containing cells
creates temporary holes in their plasma membrane, through
which DNA can enter

Transformation
2. Heat – shock
• By exposing cells to a sudden increase in temperature, or
heat shock, a pressure difference between outside and
inside of the cell is created, that induces formation of pores,
through which supercoiled plasmid DNA can enter
• After returning the cells to a more normal temperature, the
cell wall will self-heal.
• Video: http://www.jove.com/science-
education/5059/bacterial-transformation-the-heat-shock-
method

How to select clones that
carry the plasmid?

Figure 19.9
DNA fragments obtained
by PCR with restriction
sites matching those in
the cloning vector
Cut with same restriction

enzyme used on cloning
A gene that makes bacterial
vector
cells resistant to an antibiotic
is present on the plasmid.
Cloning vector
(bacterial plasmid)
Mix and ligate
Recombinant
DNA plasmid
Only cells that take up

a plasmid will survive

Figure 19.5a
of interest
1 Gene inserted
into plasmid
Bacterial Plasmid
chromosome DNA of
This cell Gene of chromosome
Recombinant
reproduce interest (“foreign” DNA)
DNA (plasmid)
through
repeated cell 2 Plasmid put into
divisions to bacterial cell
form a clone
of cells, a Recombinant
population of bacterium
genetically
3 Host cell grown in culture to form a
identical clone of cells containing the “cloned”
cells gene of interest
Gene of
Figure 19.5b
Purpose of gene Gene of

cloning interest Protein expressed
• To make from gene of interest
many copies Copies of gene Protein harvested
of gene
• amplify a
particular 4 Basic research
genes to and various
• produce a applications
protein
product for Gene for pest resistance Human growth hormone
research, inserted into plants treats stunted growth
medical, or
other
purposes
Gene used to alter Protein dissolves

bacteria for cleaning blood clots in heart
up toxic waste attack therapy
Producing Clones of Cells Carrying Recombinant
Plasmids
Several steps are required to clone the

hummingbird β-globin gene in a bacterial
plasmid:
• The hummingbird genomic DNA and a bacterial plasmid
are isolated
• Both are digested with the same restriction enzyme
• The fragments are mixed, and DNA ligase is added to
bond the fragment sticky ends

• Some recombinant plasmids now contain
hummingbird DNA
• The DNA mixture is added to bacteria that have been
genetically engineered to accept it
• The bacteria are plated on a type of agar that selects for
the bacteria with recombinant plasmids
• This results in the cloning of many hummingbird DNA
fragments, including the β-globin gene

Fig. 20-4-1
TECHNIQUE Hummingbird
cell
Bacterial cell
lacZ gene
Restriction Sticky Gene of interest

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments
1) Obtained engineered plasmid DNA & DNA from bird cells. DNA
Hummingbirds contains gene of interest.
• Isolate hummingbirds genomic DNA from hummingbird cells
• Vector = bacterial plasmid from E.coli cells
• Plasmid has been engineered to carry 2 genes :
o ampR ampicillin resistance – makes E.coli cells resistant to
antibiotic ampicillin
o lacZ lactose breakdown – encodes the enzyme b-galactosidase
(hydrolyze X-gal molecule form blue product)
• Plasmid contains only one copy of the restriction site recognized by
restriction enzyme
2) Cut both DNA samples with same restriction enzyme, one that makes
a single cut with the lacZ gene & many cuts within the hummingbird DNA
Fig. 20-4-2
cell
Bacterial cell
lacZ gene

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
3) Mix cut plasmids with DNA fragments

•Add DNA ligase - covalent bond the sugar-P backbones of
fragments whose sticky ends have base-paired them
together.
•Products obtained:
o Recombinant plasmids = The gene of interest was
inserted into a plasmid. This disrupts the lacZ gene
and becomes nonfunctional.
o Nonrecombinant plasmids - plasmid may recircularize
without any insert
Fig. 20-4-3
cell
Bacterial cell
lacZ gene

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments
Nonrecombinant
plasmid
Bacteria carrying
plasmids
4) DNA mixture is added to bacteria that have been genetically

engineered to accept it. Some cells take up a recombinant
plasmid or other DNA molecule by transformation

Fig. 20-4-4
cell
Bacterial cell
lacZ gene

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments
5). The bacteria are Nonrecombinant

plasmid
plated on a type of
agar that selects for
the bacteria with
recombinant plasmids Bacteria carrying
•Agar medium + plasmids
ampicillin + X-gal
•Incubate until colonies
RESULTS
grow
•This results in the Colony carrying non- Colony carrying recombinant
cloning of many recombinant plasmid
with intact lacZ gene
plasmid with disrupted lacZ gene
hummingbird DNA
One of many
fragments, including bacterial
the β-globin gene clones

a) Cell + non recombinant plasmid
o Intact LacZ gene encodes b-galactosidase
o b-galactosidase cleave lactose into glucose and galactose
o X-gal is a colourless analog of lactose that may be cleaved by
β-galactosidase to form blue colonies
b) Cell + recombinant plasmid (plasmid + ampR gene)
o LacZ is disrupted, no b-galactosidase is produced
o Form white colonies because enzyme cannot hydrolyze X-gal
Polymerase chain reaction,
PCR

Amplifying DNA: The Polymerase Chain Reaction
(PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction, PCR, can produce

many copies of a specific target segment of DNA
b) A three-step cycle—heating, cooling, and
replication—brings about a chain reaction that
produces an exponentially growing population of
identical DNA molecules

Figure 19.8a
Technique
5′ 3′
Target
sequence
Genomic DNA 3′ 5′
PCR requires:
1)double-stranded DNA containing the target sequence
2)Two 15-20 nucleotide DNA strands that serve as
primers (specific sequence to be amplified).
o One primer is complementary to one end of the target
sequence on one strand, the second primer is
complementary to the other end of the sequence on
the target strand

3) All four nucleotides
4) A heat-resistant DNA polymerase called Taq
polymerase
o Named after bacterial species, Thermus aquaticus
from which it was first isolated. This bacteria lives in
hot springs

Figure 19.8
Technique 5′ 3′
Target
sequence
Genomic DNA 3′ 5′
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

Figure 19.8b-1
Technique
3′ 5′
Cycle 1 1) Heat briefly to separate

yields
2 double-stranded DNA
molecules

Figure 19.8b-2
Technique
3′ 5′
2 Annealing
Cycle 1
yields
2 Primers
molecules
2) Cool to allow primers (short single-stranded

DNA) to form hydrogen bonds with ends of target
sequence
Figure 19.8b-3
Technique
3) DNA polymerase (heat stable)
1 Denaturation 5′ adds 3′
nucleotides/extend primers in 5’ to 3’ direction
3′ 5′
2 Annealing
Cycle 1
yields
2 Primers
molecules
3 Extension
New
nucleotides

Figure 19.8c
Technique
Cycle 2
yields
4
molecules
Cycle 3 yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Number of DNA = 2n where n = the number of cycles

Results After 30 more cycles, over 1 billion (109) molecules
match the target sequence.

• PCR amplification occasionally incorporates errors
into the amplified strands. So cannot substitute for
gene cloning in cells when large amounts of genes
are required and limit the number of good copies and
the length of DNA fragments that can be copied.
• But PCR is used to provide a supply of the specific
DNA fragment for cloning.
o PCR primers are synthesized/designed to include a
restriction site at each end of the DNA fragment that
matches the site in the cloning vector.
o The resulting clones are sequenced and error-free
inserts selected

Gel Electrophoresis &
Southern Blotting

Gel Electrophoresis and Southern Blotting
a)One indirect method of rapidly analyzing and

comparing genomes is gel electrophoresis
b)This technique uses a gel as a molecular sieve to
separate nucleic acids or proteins by size,
electrical charge, and other properties
c) A current is applied that causes charged
molecules to move through the gel
d)Molecules are sorted into “bands” by their size

Each sample, a mixture of DNA molecules, is placed in a
separate well near one end of the gel.
The gel is set into a plastic support, immersed in a solution in

a tray, with electrode at each end.

When the current is turned on, the negatively charged DNA
molecules moves toward the +ve electrode (anode).
Shorter molecules move faster than longer molecules.

(bands are not visible at this time)

Fig. 20-9b
RESULTS
If all the samples were
initially cut with the
same restriction
enzyme, then the
different band patterns
indicate that they came
from different sources.
After the current is turned off, a DNA-binding dye is added.

This dye flouresces in UV light, revealing the separated
bands.
In the gel above, the pink bands correspond to DNA

fragments of different lengths separated by electrophoresis.
a)In restriction fragment analysis, DNA fragments
produced by restriction enzyme digestion of a DNA
molecule are sorted by gel electrophoresis
b)Restriction fragment analysis can be used to
compare two different DNA molecules, such as
two alleles for a gene, if the nucleotide difference
alters a restriction site
©©
2015 Pearson
2011 Education
Pearson Ltd
Education, Inc.
Normal b-globin allele
175 bp 201 bp Large fragment
DdeI DdeI DdeI DdeI
Sickle-cell mutant b-globin allele
376 bp Large fragment
DdeI DdeI DdeI

(a) DdeI restriction sites in normal and
sickle-cell alleles of the b-globin gene
Restriction fragment analysis is used to distinguish the

normal and sickle-cell alleles of human b-globin gene.
Samples of each alleles were cut using the same restriction

enzyme (DdeI), and then subjected to gel electrophoresis.

Normal Sickle-cell
allele allele
Large
fragment
376 bp
201 bp
175 bp
(b) Electrophoresis of restriction

fragments from normal and
sickle-cell alleles
The sickle cell mutation destroys one of the DdeI restriction

site within the gene, resulting in 2 bands for the sickle cell
allele and 3 bands for normal allele.

a)A technique called Southern blotting combines
gel electrophoresis of DNA fragments with nucleic
acid hybridization
b)Specific DNA fragments can be identified by
Southern blotting, using labeled probes that
hybridize to the DNA immobilized on a “blot” of gel
©©
2015 Pearson
2011 Education
Pearson Ltd
Education, Inc.
TECHNIQUE Heavy
DNA  restriction enzyme Restriction I II III weight
Nitrocellulose
fragments
membrane (blot)
Gel
Sponge
I Normal II Sickle-cell III Heterozygote Alkaline

b-globin allele solution Paper
allele towels
1 Preparation of 2 Gel electrophoresis 3 DNA transfer (blotting)
restriction fragments
Probe base-pairs
I II III with fragments I II III
Radioactively labeled Fragment from

probe for b-globin sickle-cell
gene b-globin allele Film
over
Fragment from
blot
normal b- globin
Nitrocellulose blot allele
4 Hybridization with labeled probe 5 Probe detection

DNA Sequencing

DNA Sequencing
a) Researchers can exploit
the principle of
complementary base
pairing to determine a
gene’s complete
nucleotide sequence,
called DNA (a) Standard sequencing machine
sequencing
b) The first automated
procedure was based
on a technique called
dideoxy or chain
termination sequencing,
developed by Sanger
© 2015 Pearson Education Ltd (b) Next-generation sequencing machines
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
DNA (template Labeled strands

5′ C
strand) dd G 3′
T dd A A
G dd C C C
A dd T T T T
C dd G G G G G
T dd A A A A A A
T dd A A A A A A A
C dd G G G G G G G G
G dd C 3′ C C C C C C C C
A T T T T T T T T T
C G G G G G G G G G
A T T T T T T T T T
3′ A T 5′ T T T T T T T T 5′
Shortest Longest

Technique
Direction
of movement Longest labeled strand
of strands
through gel
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

a) “Next-generation sequencing” techniques use a
single template strand that is immobilized and
amplified to produce an enormous number of identical
fragments
b) Thousands or hundreds of thousands of fragments
(400–1,000 nucleotides long) are sequenced in
parallel
c) This is a type of “high-throughput” technology

Technique
1 Genomic DNA is fragmented.
2 Each fragment is isolated with

a bead.
3 Using PCR, 106 copies of each

fragment are made, each attached
to the bead by 5′ end.
4 The bead is placed into a well with

DNA polymerases and primers.
Template strand
of DNA
3′
5′ 3′ 5′
Primer A T GC
5 A solution of each of the four nucleotides

is added to all wells and then washed off.
The entire process is then repeated.
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
6 If a nucleotide is joined to 7 If a nucleotide is not

a growing strand, PPi is complementary to the
released, causing a flash next template base,
of light that is recorded. no PPi is released, and
no flash of light is recorded.

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
8 The process is repeated until every

fragment has a complete complementary
strand. The pattern of flashes reveals the
sequence.

Results
4-mer A
T
3-mer G
C
2-mer
1-mer

a) In “third-generation sequencing,” the techniques used
are even faster and less expensive than
the previous

Concept 19.3: Cloned organisms and stem cells are
useful for basic research and other applications
a) Organismal cloning produces one or more organisms

genetically identical to the “parent” that donated the
single cell
b) A stem cell is a relatively unspecialized cell that can
reproduce itself indefinitely, or under certain conditions
can differentiate into one or more types of specialized
cells

Cloning Plants: Single-Cell Cultures
a) In plants, cells can dedifferentiate and then

give rise to all the specialized cell types of
the organism
b) A totipotent cell, such as this, is one that can
generate a complete new organism
c) Plant cloning is used extensively in agriculture

Cross section
of carrot root
Small
fragments
Fragments Single Embryonic Plantlet was Adult plant

were cultured cells free in plant cultured on
in nutrient suspension developed agar medium.
medium; began to from a Later it was
stirring divide. cultured planted in soil.
caused single single cell.
cells to shear
off into the
liquid.
Cloning Animals: Nuclear Transplantation
a) In nuclear transplantation, the nucleus of an

unfertilized egg cell or zygote is replaced with the
nucleus of a differentiated cell
b) Experiments with frog embryos have shown that
a transplanted nucleus can often support normal
development of the egg
c) However, the older the donor nucleus, the lower the
percentage of normally developing tadpoles

Experiment Frog embryo Frog egg cell Frog tadpole
UV
Fully differ-
Less differentiated
entiated cell (intestinal) cell
Donor Donor
nucleus Enucleated nucleus
trans- egg cell trans-
planted Egg with donor nucleus planted
activated to begin
Results development
Most develop Most stop developing

into tadpoles. before tadpole stage.
Reproductive Cloning of Mammals
a) In 1997, Scottish researchers announced the birth of

Dolly, a lamb cloned from an adult sheep by nuclear
transplantation from a differentiated mammary cell
b) Dolly’s premature death in 2003, as well as her
arthritis, led to speculation that her cells were not as
healthy as those of a normal sheep, possibly
reflecting incomplete reprogramming of the original
transplanted nucleus

Technique
Mammary Egg cell

cell donor donor
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
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 Cells of Animals
a) Stem cells are relatively unspecialized cells that can

both reproduce indefinitely and, under certain
conditions, differentiate into one or more specialized
cell types

Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bone or White

cells blood cells

Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capable

of giving rise to differentiated embryonic cells of
any type
b) In culture, these embryonic stem cells reproduce
indefinitely
c) Depending on culture conditions, they can be made to
differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell
types and are used in the body to replace
nonreproducing cells as needed

Embryonic Adult stem
stem cells cells
Cells that can generate Cells that generate a limited

all embryonic cell types number of cell types
Cultured stem cells
Different culture
conditions
Liver cells Nerve cells Blood cells
Different types of
differentiated cells

a) Embryonic stem (ES) cells are pluripotent, capable
of differentiating into many different
cell types
b) The ultimate aim of research with stem cells
is to supply cells for the repair of damaged or
diseased organs
c) ES cells present ethical and political issues

Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells, and

reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies
of four stem cell master regulatory genes to produce
induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions of
ES cells
d) iPS cells can be used as models for study of certain
diseases and potentially as replacement cells for
patients
Experiment Stem cell Precursor cell
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
Concept 19.4: The practical applications of DNA-based
biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic

engineering

Medical Applications
a) One benefit of DNA technology is identification of

human genes in which mutation plays a role in
genetic diseases
b) Researchers use microarray assays or other tools to
identify genes turned on or off in particular diseases
c) The genes and their products are then potential
targets for prevention or therapy

Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic

disorders using PCR and sequence-specific primers,
then sequencing the amplified product to look for the
disease-causing mutation
b) SNPs may be associated with a disease-causing
mutation
c) SNPs may also be correlated with increased risks for
conditions such as heart disease or certain types of
cancer

Human Gene Therapy
a) Gene therapy is the alteration of an afflicted

individual’s genes
b) Gene therapy holds great potential for treating
disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific
types of cells, for example bone marrow
d) Gene therapy provokes both technical and ethical
questions

Cloned
gene 1 Insert RNA version of normal allele
into retrovirus or other viral vector.
Viral RNA
2 Let virus infect bone marrow cells

Viral that have been removed from the
capsid patient and cultured.
3 Viral DNA carrying the normal

allele inserts into chromosome.
Bone
marrow
cell from
patient
4 Inject engineered Bone

cells into patient. marrow
Pharmaceutical Products
a) Advances in DNA technology and genetic research

are important to the development
of new drugs to treat diseases

Synthesis of Small Molecules for Use as Drugs
•The drug imatinib is a small molecule that inhibits
overexpression of a specific leukemia-causing receptor
•This approach is feasible for treatment of cancers in
which the molecular basis is well-understood

Protein Production in Cell Cultures
a)Host cells in culture can be engineered to secrete a
protein as it is made, simplifying the task of purifying it
b)This is useful for the production of insulin, human
growth hormones, and vaccines

Protein Production by “Pharm” Animals
a)Transgenic animals are made by introducing genes
from one species into the genome of another animal
b)Transgenic animals are pharmaceutical “factories,”
producers of large amounts of otherwise rare
substances for medical use

Forensic Evidence and Genetic Profiles
a) An individual’s unique DNA sequence, or genetic

profile, can be obtained by analysis of tissue or body
fluids
b) DNA testing can identify individuals with a high
degree of certainty
c) Genetic profiles are currently analyzed using genetic
markers called short tandem repeats (STRs)

a) STRs are variations in the number of repeats of
specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and
then identify STRs of different lengths
c) The probability that two people who are not identical
twins have the same STR markers is exceptionally
small
d) As of 2013 more than 300 innocent people have been
released from prison as a result of STR analysis of
old DNA evidence

(a) Earl Washington just
before his release in 2001,
after 17 years in prison.
Source of STR STR STR

sample marker 1 marker 2 marker 3
Semen on victim 17,19 13,16 12,12
Earl Washington 16,18 14,15 11,12
Kenneth Tinsley 17,19 13,16 12,12
(b) These and other STR data (not shown) exonerated

Washington and led Tinsley to plead guilty to the murder.
Environmental Cleanup
a) Genetic engineering can be used to modify the
metabolism of microorganisms
b) Some modified microorganisms can be used to
extract minerals from the environment or degrade
potentially toxic waste materials

Agricultural Applications
a) DNA technology is being used to improve agricultural
productivity and food quality
b) Genetic engineering of transgenic animals speeds up the
selective breeding process
c) Beneficial genes can be transferred between varieties or
species

a) Agricultural scientists have endowed a number of
crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for
introducing new genes into plant cells
c) 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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BIOLOGY TENTH EDITION

Topic 2 Biotechnology Campbell • Reece • Urry • Cain • Wasserman • Minorsky • Jackson

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1. Relate the natural function of restriction

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RE Digestion & Ligation

Antibiotic resistant gene

Screening of positive results (clone/ recombinant DNA)

1) Isolation of target DNA from chromosome & vector DNA

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Plasmids are small circular DNA molecules that

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o A plasmid used to clone a foreign gene is called a

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Copies of gene Protein harvested

Gene used to alter bacteria Protein dissolves blood clots

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• Researchers can insert DNA into plasmids to produce

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Copies of gene Protein harvested

Gene used to alter bacteria Protein dissolves blood clots

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5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’

5’--GGTACC-- 3’ 5’ --GGTAC C-- 3’

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5’ --CCCGGG-- 3 5’ --CCC GGG-- 3’

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3′ Recombinant DNA molecule 5′

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3′ Recombinant DNA molecule 5′

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Cut with same restriction

Only cells that take up

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Purpose of gene Gene of

Gene used to alter Protein dissolves

Several steps are required to clone the

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Restriction Sticky Gene of interest

Restriction Sticky Gene of interest

3) Mix cut plasmids with DNA fragments

Restriction Sticky Gene of interest

4) DNA mixture is added to bacteria that have been genetically

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Restriction Sticky Gene of interest

5). The bacteria are Nonrecombinant

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a) The polymerase chain reaction, PCR, can produce

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Cycle 1 1) Heat briefly to separate

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