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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
Overview: Understanding and Manipulating
Genomes
• Sequencing of the human genome was largely
completed by 2003
• 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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• 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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Concept 20.1: DNA cloning permits production of
multiple copies of a specific gene or other DNA segment
• To work directly with specific genes, scientists
prepare gene-sized pieces of DNA in identical
copies, a process called gene cloning
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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
• Cloned genes are useful for making copies of a
particular gene and producing a gene product
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-2
Bacterium
Gene inserted into
plasmid
Bacterial
chromosome
Cell containing gene
of interest
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
Plasmid put into
bacterial cell
DNA of
chromosome
Recombinant
bacterium
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
Copies of gene
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein harvested
Basic research and
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
Using Restriction Enzymes to Make Recombinant
DNA
• Bacterial restriction enzymes cut DNA molecules
at 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
• DNA ligase is an enzyme that seals the bonds
between restriction fragments
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-3
Restriction site
DNA 5
3
3
5
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
Sticky end
DNA fragment from another
source is added. Base pairing
of sticky ends produces
various combinations.
Fragment from different
DNA molecule cut by the
same restriction enzyme
One possible combination
DNA ligase
seals the strands.
Recombinant DNA molecule
Animation: Restriction Enzymes
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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 replicate there
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Producing Clones of Cells
• Cloning a human gene in a bacterial plasmid can
be divided into six steps:
1. Vector and gene-source DNA are isolated
2. DNA is inserted into the vector
3. Human DNA fragments are mixed with cut
plasmids, and base-pairing takes place
4. Recombinant plasmids are mixed with bacteria
5. The bacteria are plated and incubated
6. Cell clones with the right gene are identified
Animation: Cloning a Gene
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
Identifying 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
• An essential step in this process is denaturation of
the cells’ DNA, separation of its two strands
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-5
Master plate
Filter
Master plate
Probe
DNA
Radioactive
single-stranded
DNA
Solution
containing
probe
Colonies
containing
gene of
interest
Gene of
interest
Single-stranded
DNA from cell
Film
Filter lifted
and flipped over
Hybridization
on filter
A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.
The filter is treated to break
open the cells and denature
their DNA; the resulting
single-stranded DNA
molecules are treated so that
they stick to the filter.
The filter is laid
under photographic
film, allowing any
radioactive areas to
expose the film
(autoradiography).
After the
developed film is
flipped over, the
reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of
interest.
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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-6
or
Bacterial
clones
Foreign genome
cut up with
restriction
enzyme
Recombinant
plasmids
Recombinant
phage DNA
Plasmid library
Phage
clones
Phage library
• 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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Cloning and Expressing Eukaryotic Genes
• As an alternative to screening a DNA library,
clones can sometimes be screened for a desired
gene based on detection of its encoded protein
• After a gene has been cloned, its protein product
can be produced in larger amounts for research
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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 posttranslational
modifications that many proteins require
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• 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 microscopic needles
• Once inside the cell, the DNA is incorporated into
the cell’s DNA by natural genetic recombination
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-7
5
3
Target
sequence
Genomic DNA
Denaturation:
Heat briefly
to separate DNA
strands
Cycle 1
yields
2
molecules
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
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
3
5
5
3
3
5
Primers
New
nucleotides
Concept 20.2: Restriction fragment analysis
detects DNA differences that affect restriction sites
• Restriction fragment analysis detects differences
in the nucleotide sequences of DNA molecules
• Such analysis can rapidly provide comparative
information about DNA sequences
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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 nuclei acids or proteins by size
Video: Biotechnology Lab
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-8
Cathode
Power
source
Mixture
of DNA
molecules
of different sizes
Shorter
molecules
Gel
Glass
plates
Anode
Longer
molecules
• 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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-9
Normal b-globin allele
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
Electrophoresis of restriction fragments from normal
and sickle-cell alleles
• A technique called Southern blotting combines gel
electrophoresis 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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-10
Restriction
fragments
DNA + restriction enzyme
I
II
III
Heavy
weight
Nitrocellulose
paper (blot)
Gel
Sponge
I Normal
b-globin
allele
II Sickle-cell III Heterozygote
allele
Preparation of restriction fragments.
Radioactively
labeled probe
for b-globin
gene is added
to solution in
a plastic bag
I
II
III
Paper
towels
Alkaline
solution
Gel electrophoresis.
Probe hydrogenbonds to fragments
containing normal
or mutant b-globin
Blotting.
I
II
Fragment from
sickle-cell
b-globin allele
Paper blot
Hybridization with radioactive probe.
III
Film over
paper blot
Fragment from
normal b-globin
allele
Autoradiography.
Restriction Fragment Length Differences as
Genetic Markers
• Restriction fragment length polymorphisms
(RFLPs, or Rif-lips) are differences in DNA
sequences on homologous chromosomes that
result in restriction fragments of different lengths
• A RFLP can serve as a genetic marker for a
particular location (locus) in the genome
• RFLPs are detected by Southern blotting
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Concept 20.3: Entire genomes can be mapped at
the DNA level
• The most ambitious mapping project to date has
been the sequencing of the human genome
• Officially begun as the Human Genome Project in
1990, the sequencing was largely completed by
2003
• Scientists have also sequenced genomes of other
organisms, providing insights of general biological
significance
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Genetic (Linkage) Mapping: Relative Ordering of
Markers
• The first stage in mapping a large genome is
constructing a linkage map of several thousand
genetic markers throughout each chromosome
• The order of markers and relative distances
between them are based on recombination
frequencies
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-11
Chromosome
bands
Cytogenetic map
Genes located
by FISH
Genetic (linkage)
mapping
Genetic
markers
Physical mapping
Overlapping
fragments
DNA sequencing
Physical Mapping: Ordering DNA Fragments
• A physical map is constructed by cutting a DNA
molecule into many short fragments and arranging
them in order by identifying overlaps
• Physical mapping gives the actual distance in
base pairs between markers
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
DNA Sequencing
• Relatively short DNA fragments can be sequenced
by the dideoxy chain-termination method
• Inclusion of special dideoxyribonucleotides in the
reaction mix ensures that fragments of various
lengths will be synthesized
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-12
DNA
(template strand)
5
Primer
3
Deoxyribonucleotides
Dideoxyribonucleotides
(fluorescently tagged)
5
DNA
polymerase
3
5
DNA (template
strand)
Labeled strands
3
Direction
of movement
of strands
Laser
Detector
3
• Linkage mapping, physical mapping, and DNA
sequencing represent the overarching strategy of
the Human Genome Project
• An alternative approach to sequencing genomes
starts with sequencing random DNA fragments
• Computer programs then assemble overlapping
short sequences into one continuous sequence
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-13
Cut the DNA from
many copies of an
entire chromosome
into overlapping fragments short enough
for sequencing
Clone the fragments
in plasmid or phage
vectors
Sequence each fragment
Order the
sequences into one
overall sequence
with computer
software
Concept 20.4: Genome sequences provide clues to
important biological questions
• In genomics, scientists study whole sets of genes
and their interactions
• Genomics is yielding new insights into genome
organization, regulation of gene expression,
growth and development, and evolution
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Identifying Protein-Coding Genes in DNA
Sequences
• Computer analysis of genome sequences helps
identify sequences likely to encode proteins
• The human genome contains about 25,000 genes,
but the number of human proteins is much larger
• Comparison of sequences of “new” genes with
those of known genes in other species may help
identify new genes
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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
• In nonmammalian organisms, a simpler and faster
method, RNA interference (RNAi), has been used
to silence expression of selected genes
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Studying 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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-14
Tissue sample
Isolate mRNA.
Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
Apply the cDNA mixture to a
microarray, a microscope slide
on which copies of singlestranded DNA fragments from
the organism’s genes are fixed,
a different gene in each spot.
The cDNA hybridizes with any
complementary DNA on the
microarray.
Rinse off excess cDNA; scan
microarray for fluorescent.
Each fluorescent spot
(yellow) represents a gene
expressed in the tissue
sample.
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA
microarray
Size of an actual
DNA microarray
with all the genes
of yeast (6,400 spots)
Comparing Genomes of Different Species
• Comparative studies of genomes from related and
widely divergent species provide information in
many fields of biology
• The more similar the nucleotide sequences
between two species, the more closely related
these species are in their evolutionary history
• Comparative genome studies confirm the
relevance of research on simpler organisms to
understanding human biology
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Future Directions in Genomics
• Genomics is the study of entire genomes
• Proteomics is the systematic study of all proteins
encoded by a genome
• Single nucleotide polymorphisms (SNPs) provide
markers for studying human genetic variation
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
Medical Applications
• One benefit of DNA technology is identification of
human genes in which mutation plays a role in
genetic diseases
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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
• Even when a disease gene has not been cloned,
presence of an abnormal allele can be diagnosed
if a closely linked RFLP marker has been found
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-15
RFLP marker
DNA
Restriction
sites
Disease-causing
allele
Normal allele
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
• Gene therapy raises ethical questions, such as
whether human germ-line cells should be treated
to correct the defect in future generations
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
Forensic Evidence
• DNA “fingerprints” obtained by analysis of tissue or
body fluids can provide evidence in criminal and
paternity cases
• A DNA fingerprint is a specific pattern of bands of
RFLP markers on a gel
• The probability that two people who are not
identical twins have the same DNA fingerprint is
very small
• Exact probability depends on the number of
markers and their frequency in the population
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-17
Defendant’s
blood (D)
Blood from defendant’s
clothes
Victim’s
blood (V)
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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Agricultural Applications
• DNA technology is being used to improve
agricultural productivity and food quality
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Animal Husbandry and “Pharm” Animals
• Transgenic organisms are made by introducing
genes from one species into the genome of
another organism
• Transgenic animals may be created to exploit the
attributes of new genes (such as genes for faster
growth or larger muscles)
• Other transgenic organisms are pharmaceutical
“factories,” producers of large amounts of
otherwise rare substances for medical use
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-19
Agrobacterium tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
Recombinant
Ti plasmid
Plant with
new trait
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
• Most public concern about possible hazards
centers on genetically modified (GM) organisms
used as food
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings