20_Lecture_Presentation_0.ppt

Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings
PowerPoint® Lecture Presentations for
Biology
Eighth Edition
Neil Campbell and Jane Reece
Lectures by Chris Romero, updated by Erin Barley with contributions from Joan Sharp
Chapter 20
Biotechnology

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Overview: The DNA Toolbox
• Sequencing of the human genome was
completed by 2007
• DNA sequencing has depended on advances
in technology, starting with making recombinant
DNA
• In recombinant DNA, nucleotide sequences
from two different sources, often two species,
are combined in vitro into the same DNA
molecule

• Methods for making recombinant DNA are
central to genetic engineering, the direct
manipulation of genes for practical purposes
• DNA technology has revolutionized
biotechnology, the manipulation of organisms
or their genetic components to make useful
products
• An example of DNA technology is the
microarray, a measurement of gene expression
of thousands of different genes

Concept 20.1: DNA cloning yields multiple copies
of a gene or other DNA segment
• To work directly with specific genes, scientists
prepare gene-sized pieces of DNA in identical
copies, a process called DNA cloning

DNA Cloning and Its Applications: A Preview
• Most methods for cloning pieces of DNA in the
laboratory share general features, such as the
use of bacteria and their plasmids
• Plasmids are small circular DNA molecules
that replicate separately from the bacterial
chromosome
• Cloned genes are useful for making copies of a
particular gene and producing a protein product

• Gene cloning involves using bacteria to make
multiple copies of a gene
• Foreign DNA is inserted into a plasmid, and the
recombinant plasmid is inserted into a bacterial
cell
• Reproduction in the bacterial cell results in
cloning of the plasmid including the foreign
DNA
• This results in the production of multiple copies
of a single gene

Fig. 20-2
DNA of
chromosome
Cell containing gene
of interest
Gene inserted into
plasmid
Plasmid put into
bacterial cell
Recombinant
DNA (plasmid)
Recombinant
bacterium
Bacterial
chromosome
Bacterium
Gene of
interest
Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Plasmid
Gene of
Interest
Protein expressed
by gene of interest
Basic research and
various applications
Copies of gene Protein harvested
Basic
research
on gene
Basic
research
on protein
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Human growth hor-
mone treats stunted
growth
2
4
1
3

Fig. 20-2a
DNA of
chromosome
Cell containing gene
of interest
Gene inserted into
plasmid
Plasmid put into
bacterial cell
Recombinant
DNA (plasmid)
Recombinant
bacterium
Bacterial
chromosome
Bacterium
Gene of
interest
Plasmid
2
1
2

Fig. 20-2b
Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
Interest
Protein expressed
by gene of interest
Basic research and
various applications
Copies of gene Protein harvested
Basic
research
on gene
Basic
research
on protein
4
Recombinant
bacterium
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Human growth hor-
mone treats stunted
growth
3

Using Restriction Enzymes to Make Recombinant
DNA
• 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” that bond with complementary
sticky ends of other fragments
Animation: Restriction Enzymes

• DNA ligase is an enzyme that seals the bonds
between restriction fragments

Fig. 20-3-1
Restriction site
DNA
Sticky end
Restriction enzyme
cuts sugar-phosphate
backbones.
5
3
3
5
1

Fig. 20-3-2
Restriction site
DNA
Sticky end
Restriction enzyme
backbones.
5
3
3
5
1
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
2
One possible combination

Fig. 20-3-3
Restriction site
DNA
Sticky end
Restriction enzyme
backbones.
5
3
3
5
1
One possible combination
Recombinant DNA molecule
DNA ligase
seals strands.
3
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
2

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 host cell and replicate
there

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
Animation: Cloning a Gene

– 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
Bacterial cell
Bacterial
plasmid
lacZ gene
Hummingbird
cell
Gene of interest
Hummingbird
DNA fragments
Restriction
site
Sticky
ends
ampR gene
TECHNIQUE

Fig. 20-4-2
Bacterial cell
Bacterial
plasmid
lacZ gene
Hummingbird
cell
Gene of interest
Hummingbird
DNA fragments
Restriction
site
Sticky
ends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant
plasmid

Fig. 20-4-3
Bacterial cell
Bacterial
plasmid
lacZ gene
Hummingbird
cell
Gene of interest
Hummingbird
DNA fragments
Restriction
site
Sticky
ends
ampR gene
TECHNIQUE
Nonrecombinant
plasmid
Bacteria carrying
plasmids

Fig. 20-4-4
Bacterial cell
Bacterial
plasmid
lacZ gene
Hummingbird
cell
Gene of interest
Hummingbird
DNA fragments
Restriction
site
Sticky
ends
ampR gene
TECHNIQUE
Nonrecombinant
plasmid
Bacteria carrying
plasmids
RESULTS
Colony carrying non-
recombinant plasmid
with intact lacZ gene
One of many
bacterial
clones
Colony carrying recombinant
plasmid with disrupted lacZ gene

Storing Cloned Genes in DNA Libraries
• A genomic library that is made using bacteria
is the collection of recombinant vector clones
produced by cloning DNA fragments from an
entire genome
• A genomic library that is made using
bacteriophages is stored as a collection of
phage clones

Fig. 20-5
Bacterial
clones
Recombinant
plasmids
Recombinant
phage DNA
or
Foreign genome
cut up with
restriction
enzyme
(a) Plasmid library (b) Phage library (c) A library of bacterial artificial
chromosome (BAC) clones
Phage
clones
Large plasmid
Large insert
with many genes
BAC
clone

Fig. 20-5a
Bacterial
clones
Recombinant
plasmids
Recombinant
phage DNA
or
Foreign genome
cut up with
restriction
enzyme
(a) Plasmid library (b) Phage library
Phage
clones

• A bacterial artificial chromosome (BAC) is a
large plasmid that has been trimmed down and
can carry a large DNA insert
• BACs are another type of vector used in DNA
library construction

Fig. 20-5b
(c) A library of bacterial artificial
chromosome (BAC) clones
Large plasmid
Large insert
with many genes
BAC
clone

• A complementary DNA (cDNA) library is
made by cloning DNA made in vitro by reverse
transcription of all the mRNA produced by a
particular cell
• A cDNA library represents only part of the
genome—only the subset of genes transcribed
into mRNA in the original cells

Fig. 20-6-1
DNA in
nucleus
mRNAs in
cytoplasm

Fig. 20-6-2
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
transcriptase Poly-A tail
DNA
strand
Primer
mRNA

Fig. 20-6-3
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
DNA
strand
Primer
mRNA
Degraded
mRNA

Fig. 20-6-4
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
DNA
strand
Primer
mRNA
Degraded
mRNA
DNA
polymerase

Fig. 20-6-5
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
DNA
strand
Primer
mRNA
Degraded
mRNA
DNA
polymerase
cDNA

Screening a Library for Clones Carrying a Gene of
Interest
• A clone carrying the gene of interest can be
identified with a nucleic acid probe having a
sequence complementary to the gene
• This process is called nucleic acid
hybridization

• 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
G
5 3
… …
G G
C C C
T TT
AA A
C
3 5
C C
G G G
A AA
TT T

• The DNA probe can be used to screen a large
number of clones simultaneously for the gene
of interest
• Once identified, the clone carrying the gene of
interest can be cultured

Fig. 20-7
Probe
DNA
Radioactively
labeled probe
molecules
Film
Nylon membrane
Multiwell plates
holding library
clones
Location of
DNA with the
complementary
sequence
Gene of
interest
Single-stranded
DNA from cell
Nylon
membrane
TECHNIQUE
•

Expressing Cloned Eukaryotic Genes
• After a gene has been cloned, its protein
product can be produced in larger amounts for
research
• Cloned genes can be expressed as protein in
either bacterial or eukaryotic cells

Bacterial Expression Systems
• Several technical difficulties hinder expression
of cloned eukaryotic genes in bacterial host
cells
• To overcome differences in promoters and
other DNA control sequences, scientists
usually employ an expression vector, a
cloning vector that contains a highly active
prokaryotic promoter

Eukaryotic Cloning and Expression Systems
• The use of cultured eukaryotic cells as host
cells and yeast artificial chromosomes
(YACs) as vectors helps avoid gene
expression problems
• YACs behave normally in mitosis and can carry
more DNA than a plasmid
• Eukaryotic hosts can provide the post-
translational modifications that many proteins
require

• One method of introducing recombinant DNA
into eukaryotic cells is electroporation,
applying a brief electrical pulse to create
temporary holes in plasma membranes
• Alternatively, scientists can inject DNA into
cells using microscopically thin needles
• Once inside the cell, the DNA is incorporated
into the cell’s DNA by natural genetic
recombination

Amplifying DNA in Vitro: The Polymerase Chain
Reaction (PCR)
• The polymerase chain reaction, PCR, can
produce many copies of a specific target
segment of DNA
• A three-step cycle—heating, cooling, and
replication—brings about a chain reaction that
produces an exponentially growing population
of identical DNA molecules

Fig. 20-8
5
Genomic DNA
TECHNIQUE
Cycle 1
yields
2
molecules
Denaturation
Annealing
Extension
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Target
sequence
Primers
New
nucleo-
tides
3
3
3
3
5
5
5
1
2
3

Fig. 20-8a
5
Genomic DNA
TECHNIQUE
Target
sequence
3
3 5

Fig. 20-8b
Cycle 1
yields
2
molecules
Denaturation
Annealing
Extension
Primers
New
nucleo-
tides
3 5
3
2
5 3
1

Fig. 20-8c
Cycle 2
yields
4
molecules

Fig. 20-8d
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence

Concept 20.2: DNA technology allows us to study
the sequence, expression, and function of a gene
• DNA cloning allows researchers to
– Compare genes and alleles between
individuals
– Locate gene expression in a body
– Determine the role of a gene in an organism
• Several techniques are used to analyze the
DNA of genes

Gel Electrophoresis and Southern Blotting
• One indirect method of rapidly analyzing and
comparing genomes is gel electrophoresis
• This technique uses a gel as a molecular sieve
to separate nucleic acids or proteins by size
• A current is applied that causes charged
molecules to move through the gel
• Molecules are sorted into “bands” by their size
Video: Biotechnology Lab

Fig. 20-9
Mixture of
DNA mol-
ecules of
different
sizes
Power
source
Power
source
Longer
molecules
Shorter
molecules
Gel
Anode
Cathode
TECHNIQUE
RESULTS
1
2
+
+
–
–

Fig. 20-9a
Mixture of
DNA mol-
ecules of
different
sizes
Power
source
Longer
molecules
Shorter
molecules
Gel
Anode
Cathode
TECHNIQUE
1
2
Power
source
– +
+
–

• In restriction fragment analysis, DNA fragments
produced by restriction enzyme digestion of a
DNA molecule are sorted by gel
electrophoresis
• Restriction fragment analysis is useful for
comparing two different DNA molecules, such
as two alleles for a gene
• The procedure is also used to prepare pure
samples of individual fragments

Fig. 20-10
Normal
allele
Sickle-cell
allele
Large
fragment
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
201 bp
175 bp
376 bp
(a) DdeI restriction sites in normal and
sickle-cell alleles of -globin gene
Normal -globin allele
Sickle-cell mutant -globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp
175 bp
DdeI
DdeI
DdeI DdeI DdeI DdeI

• A technique called Southern blotting
combines gel electrophoresis of DNA
fragments with nucleic acid hybridization
• Specific DNA fragments can be identified by
Southern blotting, using labeled probes that
hybridize to the DNA immobilized on a “blot” of
gel

Fig. 20-11
TECHNIQUE
Nitrocellulose
membrane (blot)
Restriction
fragments
Alkaline
solution
DNA transfer (blotting)
Sponge
Gel
Heavy
weight
Paper
towels
Preparation of restriction fragments Gel electrophoresis
I II III
I II III
I II III
Radioactively labeled
probe for -globin gene
DNA + restriction enzyme
III Heterozygote
II Sickle-cell
allele
I Normal
-globin
allele
Film
over
blot
Probe detection
Hybridization with radioactive probe
Fragment from
sickle-cell
-globin allele
Fragment from
normal -globin
allele
Probe base-pairs
with fragments
Nitrocellulose blot
1
4 5
3
2

Fig. 20-11a
TECHNIQUE
Nitrocellulose
membrane (blot)
Restriction
fragments
Alkaline
solution
DNA transfer (blotting)
Sponge
Gel
Heavy
weight
Paper
towels
Preparation of restriction fragments Gel electrophoresis
I II III
DNA + restriction enzyme
III Heterozygote
II Sickle-cell
allele
I Normal
-globin
allele
1 3
2

Fig. 20-11b
I II III
I II III
Film
over
blot
Probe detection
Hybridization with radioactive probe
Fragment from
sickle-cell
-globin allele
Fragment from
normal -globin
allele
Probe base-pairs
with fragments
Nitrocellulose blot
4 5
Radioactively labeled
probe for -globin gene

DNA Sequencing
• Relatively short DNA fragments can be
sequenced by the dideoxy chain termination
method
• 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

Fig. 20-12
DNA
(template strand)
TECHNIQUE
RESULTS
DNA (template
strand)
DNA
polymerase
Primer Deoxyribonucleotides
Shortest
Dideoxyribonucleotides
(fluorescently tagged)
Labeled strands
Longest
Shortest labeled strand
Longest labeled strand
Laser
Direction
of movement
of strands
Detector
Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP

Fig. 20-12a
DNA
(template strand)
TECHNIQUE
DNA
polymerase
Primer Deoxyribonucleotides Dideoxyribonucleotides
(fluorescently tagged)
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP

Fig. 20-12b
TECHNIQUE
RESULTS
DNA (template
strand)
Shortest
Labeled strands
Longest
Shortest labeled strand
Longest labeled strand
Laser
Direction
of movement
of strands
Detector
Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand

Analyzing Gene Expression
• Nucleic acid probes can hybridize with mRNAs
transcribed from a gene
• Probes can be used to identify where or when
a gene is transcribed in an organism

Studying the Expression of Single Genes
• Changes in the expression of a gene during
embryonic development can be tested using
– Northern blotting
– Reverse transcriptase-polymerase chain
reaction
• Both methods are used to compare mRNA
from different developmental stages

• Northern blotting combines gel
electrophoresis of mRNA followed by
hybridization with a probe on a membrane
• Identification of mRNA at a particular
developmental stage suggests protein function
at that stage

• Reverse transcriptase-polymerase chain
reaction (RT-PCR) is quicker and more
sensitive
• Reverse transcriptase is added to mRNA to
make cDNA, which serves as a template for
PCR amplification of the gene of interest
• The products are run on a gel and the mRNA of
interest identified

Fig. 20-13
TECHNIQUE
RESULTS
Gel electrophoresis
cDNAs
-globin
gene
PCR amplification
Embryonic stages
Primers
1 2 3 4 5 6
mRNAs
cDNA synthesis
1
2
3

• In situ hybridization uses fluorescent dyes
attached to probes to identify the location of
specific mRNAs in place in the intact organism

Studying the Expression of Interacting Groups of
Genes
• Automation has allowed scientists to measure
expression of thousands of genes at one time
using DNA microarray assays
• DNA microarray assays compare patterns of
gene expression in different tissues, at different
times, or under different conditions

Fig. 20-15
TECHNIQUE
Isolate mRNA.
Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
Apply the cDNA mixture to a
microarray, a different gene in
each spot. The cDNA hybridizes
with any complementary DNA on
the microarray.
Rinse off excess cDNA; scan
microarray for fluorescence.
Each fluorescent spot represents a
gene expressed in the tissue sample.
Tissue sample
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA fragments
representing
specific genes
DNA microarray
with 2,400
human genes
DNA microarray
1
2
3
4

Determining Gene Function
• One way to determine function is to disable the
gene and observe the consequences
• Using in vitro mutagenesis, mutations are
introduced into a cloned gene, altering or
destroying its function
• When the mutated gene is returned to the cell,
the normal gene’s function might be
determined by examining the mutant’s
phenotype

• Gene expression can also be silenced using
RNA interference (RNAi)
• Synthetic double-stranded RNA molecules
matching the sequence of a particular gene are
used to break down or block the gene’s mRNA

• Organismal cloning produces one or more
organisms genetically identical to the “parent”
that donated the single cell
Concept 20.3: Cloning organisms may lead to
production of stem cells for research and other
applications

Cloning Plants: Single-Cell Cultures
• One experimental approach for testing
genomic equivalence is to see whether a
differentiated cell can generate a whole
organism
• A totipotent cell is one that can generate a
complete new organism

Fig. 20-16
EXPERIMENT
Transverse
section of
carrot root
2-mg
fragments
Fragments were
cultured in nu-
trient medium;
stirring caused
single cells to
shear off into
the liquid.
Single
cells
free in
suspension
began to
divide.
Embryonic
plant developed
from a cultured
single cell.
Plantlet was
cultured on
agar medium.
Later it was
planted
in soil.
A single
somatic
carrot cell
developed
into a mature
carrot plant.
RESULTS

Cloning Animals: Nuclear Transplantation
• In nuclear transplantation, the nucleus of an
unfertilized egg cell or zygote is replaced with
the nucleus of a differentiated cell
• Experiments with frog embryos have shown
that a transplanted nucleus can often support
normal development of the egg
• However, the older the donor nucleus, the
lower the percentage of normally developing
tadpoles

Fig. 20-17
EXPERIMENT
Less differ-
entiated cell
RESULTS
Frog embryo Frog egg cell
UV
Donor
nucleus
trans-
planted
Frog tadpole
Enucleated
egg cell
Egg with donor nucleus
activated to begin
development
Fully differ-
entiated
(intestinal) cell
Donor
nucleus
trans-
planted
Most develop
into tadpoles
Most stop developing
before tadpole stage

Reproductive Cloning of Mammals
• In 1997, Scottish researchers announced the
birth of Dolly, a lamb cloned from an adult
sheep by nuclear transplantation from a
differentiated mammary cell
• 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

Fig. 20-18
TECHNIQUE
Mammary
cell donor
RESULTS
Surrogate
mother
Nucleus from
mammary cell
Cultured
mammary cells
Implanted
in uterus
of a third
sheep
Early embryo
Nucleus
removed
Egg cell
donor
Embryonic
development Lamb (“Dolly”)
genetically identical to
mammary cell donor
Egg cell
from ovary
Cells fused
Grown in
culture
1
3
3
4
5
6
2

• 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”

Problems Associated with Animal Cloning
• In most nuclear transplantation studies, only a
small percentage of cloned embryos have
developed normally to birth
• Many epigenetic changes, such as acetylation
of histones or methylation of DNA, must be
reversed in the nucleus from a donor animal in
order for genes to be expressed or repressed
appropriately for early stages of development

Stem Cells of Animals
• A stem cell is a 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
cells; these are able to differentiate into all cell
types
• The adult body also has stem cells, which
replace nonreproducing specialized cells

Fig. 20-20
Cultured
stem cells
Early human embryo
at blastocyst stage
(mammalian equiva-
lent of blastula)
Different
culture
conditions
Different
types of
differentiated
cells
Blood cells
Nerve cells
Liver cells
Cells generating
all embryonic
cell types
Adult stem cells
Cells generating
some cell types
Embryonic stem cells
From bone marrow
in this example

• The aim of stem cell research is to supply cells
for the repair of damaged or diseased organs

Concept 20.4: The practical applications of DNA
technology affect our lives in many ways
• Many fields benefit from DNA technology and
genetic engineering

Medical Applications
• One benefit of DNA technology is identification
of human genes in which mutation plays a role
in genetic diseases

Diagnosis of Diseases
• Scientists can diagnose many human genetic
disorders by using PCR and primers
corresponding to cloned disease genes, then
sequencing the amplified product to look for the
disease-causing mutation
• Genetic disorders can also be tested for using
genetic markers that are linked to the disease-
causing allele

• Single nucleotide polymorphisms (SNPs)
are useful genetic markers
• These are single base-pair sites that vary in a
population
• When a restriction enzyme is added, SNPs
result in DNA fragments with different lengths,
or restriction fragment length
polymorphism (RFLP)

Fig. 20-21
Disease-causing
allele
DNA
SNP
Normal allele
T
C

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
specific types of cells, for example bone marrow
• Gene therapy raises ethical questions, such as
whether human germ-line cells should be
treated to correct the defect in future
generations

Fig. 20-22
Bone
marrow
Cloned
gene
Bone
marrow
cell from
patient
Insert RNA version of normal allele
into retrovirus.
Retrovirus
capsid
Viral RNA
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Viral DNA carrying the normal
allele inserts into chromosome.
Inject engineered
cells into patient.
1
2
3
4

Pharmaceutical Products
• Advances in DNA technology and genetic
research are important to the development of
new drugs to treat diseases

• The drug imatinib is a small molecule that
inhibits overexpression of a specific leukemia-
causing receptor
• Pharmaceutical products that are proteins can
be synthesized on a large scale
Synthesis of Small Molecules for Use as Drugs

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

• Transgenic animals are made by introducing
genes from one species into the genome of
another animal
• Transgenic animals are pharmaceutical
“factories,” producers of large amounts of
otherwise rare substances for medical use
• “Pharm” plants are also being developed to
make human proteins for medical use
Protein Production by “Pharm” Animals and
Plants

Forensic Evidence and Genetic Profiles
• An individual’s unique DNA sequence, or
genetic profile, can be obtained by analysis of
tissue or body fluids
• Genetic profiles can be used to provide
evidence in criminal and paternity cases and to
identify human remains
• Genetic profiles can be analyzed using RFLP
analysis by Southern blotting

• Even more sensitive is the use of genetic
markers called short tandem repeats (STRs),
which are variations in the number of repeats
of specific DNA sequences
• PCR and gel electrophoresis are used to
amplify and then identify STRs of different
lengths
• The probability that two people who are not
identical twins have the same STR markers is
exceptionally small

Fig. 20-24
This photo shows Earl
Washington just before
his release in 2001,
after 17 years in prison.
These and other STR data exonerated Washington and
led Tinsley to plead guilty to the murder.
(a)
Semen on victim
Earl Washington
Source of
sample
Kenneth Tinsley
STR
marker 1
STR
marker 2
STR
marker 3
(b)
17, 19
16, 18
17, 19
13, 16 12, 12
14, 15 11, 12
13, 16 12, 12

Environmental Cleanup
• Genetic engineering can be used to modify the
metabolism of microorganisms
• Some modified microorganisms can be used to
extract minerals from the environment or
degrade potentially toxic waste materials
• Biofuels make use of crops such as corn,
soybeans, and cassava to replace fossil fuels

Agricultural Applications
• DNA technology is being used to improve
agricultural productivity and food quality

Animal Husbandry
• Genetic engineering of transgenic animals
speeds up the selective breeding process
• Beneficial genes can be transferred between
varieties or species

Genetic Engineering in Plants
• Agricultural scientists have endowed a number
of crop plants with genes for desirable traits
• 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

Fig. 20-25
Site where
restriction
enzyme cuts
T DNA
Plant with new trait
Ti
plasmid
Agrobacterium tumefaciens
DNA with
the gene
of interest
Recombinant
Ti plasmid
TECHNIQUE
RESULTS

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

• Most public concern about possible hazards
centers on genetically modified (GM)
organisms used as food
• Some are concerned about the creation of
“super weeds” from the transfer of genes from
GM crops to their wild relatives

• As biotechnology continues to change, so does
its use in agriculture, industry, and medicine
• National agencies and international
organizations strive to set guidelines for safe
and ethical practices in the use of
biotechnology

Fig. 20-UN3
Cut by same restriction enzyme,
mixed, and ligated
DNA fragments from genomic DNA
or cDNA or copy of DNA obtained
by PCR
Vector
Recombinant DNA plasmids

Fig. 20-UN4
Aardvark DNA
Plasmid
5
3
3
TCCATGAATTCTAAAGCGCTTATGAATTCACGGC
5
AGGTACTTAAGATTTCGCGAATACTTAAGTGCCG
A

You should now be able to:
1. Describe the natural function of restriction
enzymes and explain how they are used in
recombinant DNA technology
2. Outline the procedures for cloning a
eukaryotic gene in a bacterial plasmid
3. Define and distinguish between genomic
libraries using plasmids, phages, and cDNA
4. Describe the polymerase chain reaction
(PCR) and explain the advantages and
limitations of this procedure

5. Explain how gel electrophoresis is used to
analyze nucleic acids and to distinguish
between two alleles of a gene
6. Describe and distinguish between the
Southern blotting procedure, Northern blotting
procedure, and RT-PCR
7. Distinguish between gene cloning, cell
cloning, and organismal cloning
8. Describe how nuclear transplantation was
used to produce Dolly, the first cloned sheep

9. Describe the application of DNA technology to
the diagnosis of genetic disease, the
development of gene therapy, vaccine
production, and the development of
pharmaceutical products
10.Define a SNP and explain how it may produce
a RFLP
11.Explain how DNA technology is used in the
forensic sciences

12.Discuss the safety and ethical questions
related to recombinant DNA studies and the
biotechnology industry

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