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Chapter 12
DNA Technology and Genomics
PowerPoint Lectures for
Biology: Concepts & Connections, Sixth Edition
Campbell, Reece, Taylor, Simon, and Dickey
Lecture by Mary C. Colavito
Copyright © 2009 Pearson Education, Inc.
GENE CLONING
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12.1 Genes can be cloned in recombinant plasmids
 Genetic engineering involves manipulating
genes for practical purposes
– Gene cloning leads to the production of multiple
identical copies of a gene-carrying piece of DNA
– Recombinant DNA is formed by joining DNA
sequences from two different sources
– One source contains the gene that will be cloned
– Another source is a gene carrier, called a vector
– Plasmids (small, circular DNA molecules
independent of the bacterial chromosome) are often
used as vectors
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12.1 Genes can be cloned in recombinant plasmids
 Steps in cloning a gene
1. Plasmid DNA is isolated
2. DNA containing the gene of interest is isolated
3. Plasmid DNA is treated with restriction enzyme that
cuts in one place, opening the circle
4. DNA with the target gene is treated with the same
enzyme and many fragments are produced
5. Plasmid and target DNA are mixed and associate with
each other
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12.1 Genes can be cloned in recombinant plasmids
6. Recombinant DNA molecules are produced when
DNA ligase joins plasmid and target segments
together
7. The recombinant DNA is taken up by a bacterial cell
8. The bacterial cell reproduces to form a clone of
cells
Animation: Cloning a Gene
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E. coli bacterium
Plasmid
Bacterial
chromosome
1
Cell with DNA
containing gene
of interest
Isolate
plasmid
Isolate
DNA
2
3
Cut plasmid
with enzyme
DNA
Gene of interest
4
Cut cell’s DNA
with same enzyme
Gene
of interest
5
6
Recombinant
DNA
plasmid
Combine targeted fragment
and plasmid DNA
Examples of
gene use
Add DNA ligase,
which closes
the circle with
covalent bonds
Genes may be inserted
into other organisms
Gene
of interest
9
7
Put plasmid
into bacterium
by transformation
Recombinant
bacterium
8
Clone
of cells
Allow bacterium
to reproduce
Genes or proteins
are isolated from the
cloned bacterium
Harvested
Examples of
proteins
protein use
may be
used directly
12.2 Enzymes are used to “cut and paste” DNA
 Restriction enzymes cut DNA at specific
sequences
– Each enzyme binds to DNA at a different restriction
site
– Many restriction enzymes make staggered cuts that
produce restriction fragments with single-stranded
ends called “sticky ends”
– Fragments with complementary sticky ends can
associate with each other, forming recombinant DNA
 DNA ligase joins DNA fragments together
Animation: Restriction Enzymes
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Restriction enzyme
recognition sequence
1
DNA
Restriction enzyme
cuts the DNA into
fragments
2
Sticky end
Addition of a DNA
fragment from
another source
3
Two (or more)
fragments stick
together by
base-pairing
4
DNA ligase
pastes the strands
5
Recombinant
DNA molecule
12.3 Cloned genes can be stored in genomic
libraries
 A genomic library is a collection of all of the
cloned DNA fragments from a target genome
 Genomic libraries can be constructed with
different types of vectors
– Plasmid library: genomic DNA is carried by plasmids
– Phage library: genomic DNA is incorporated into
bacteriophage DNA
– Bacterial artificial chromosome (BAC) library:
specialized plasmids can carry large DNA sequences
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Genome cut up with
restriction enzyme
Recombinant
plasmid
Recombinant
phage DNA
or
Bacterial
clone
Plasmid library
Phage
clone
Phage library
12.4 Reverse transcriptase can help make genes
for cloning
 Complementary DNA (cDNA) is used to clone
eukaryotic genes
– mRNA from a specific cell type is the template
– Reverse transcriptase produces a DNA strand
from mRNA
– DNA polymerase produces the second DNA strand
 Advantages of cloning with cDNA
– Study genes responsible for specialized
characteristics of a particular cell type
– Obtain gene sequences without introns
– Smaller size is easier to handle
– Allows expression in bacterial hosts
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Cell nucleus
DNA of
eukaryotic
gene
Exon Intron
Exon
Intron Exon
1 Transcription
RNA
transcript
2 RNA splicing
mRNA
3 Isolation of mRNA
Test tube
Reverse transcriptase
cDNA strand
being synthesized
and addition of reverse
transcriptase; synthesis
of DNA strand
4 Breakdown of RNA
5 Synthesis of second
DNA strand
cDNA of gene
(no introns)
12.5 Nucleic acid probes identify clones carrying
specific genes
 Nucleic acid probes bind to cloned DNA
– Probes can be DNA or RNA sequences
complementary to a portion of the gene of interest
– A probe binds to a gene of interest by base pairing
– Probes are labeled with a radioactive isotope or
fluorescent tag for detection
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12.5 Nucleic acid probes identify clones carrying
specific genes
 Screening a gene library
– Bacterial clones are transferred to filter paper
– Cells are lysed and DNA is separated into single
strands
– A solution containing the probe is added, and binding
to the DNA of interest is detected
– The clone carrying the gene of interest is grown for
further study
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Radioactive
DNA probe
Mix with singlestranded DNA from
genomic library
Single-stranded
DNA
Base pairing
indicates the
gene of interest
GENETICALLY MODIFIED
ORGANISMS
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12.6 Recombinant cells and organisms can
mass-produce gene products
 Cells and organisms containing cloned genes are
used to manufacture large quantities of gene
products
 Capabilities of the host cell are matched to the
characteristics of the desired product
– Prokaryotic host: E. coli
– Can produce eukaryotic proteins that do not require posttranslational modification
– Has many advantages in gene transfer, cell growth, and
quantity of protein production
– Can be engineered to secrete proteins
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12.6 Recombinant cells and organisms can
mass-produce gene products
 Capabilities of the host cell are matched to the
characteristics of the desired product
– Eukaryotic hosts
– Yeast: S. cerevisiae
– Can produce and secrete complex eukaryotic
proteins
– Mammalian cells in culture
– Can attach sugars to form glycoproteins
– “Pharm” animals
– Will secrete gene product in milk
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12.7 CONNECTION: DNA technology has
changed the pharmaceutical industry and
medicine
 Products of DNA technology
– Therapeutic hormones
– Insulin to treat diabetes
– Human growth hormone to treat dwarfism
– Diagnosis and treatment of disease
– Testing for inherited diseases
– Detecting infectious agents such as HIV
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12.7 CONNECTION: DNA technology has
changed the pharmaceutical industry and
medicine
 Products of DNA technology
– Vaccines
– Stimulate an immune response by injecting
– Protein from the surface of an infectious agent
– A harmless version of the infectious agent
– A harmless version of the smallpox virus containing
genes from other infectious agents
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12.7 CONNECTION: DNA technology has
changed the pharmaceutical industry and
medicine
 Advantages of recombinant DNA products
– Identity to human protein
– Purity
– Quantity
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12.8 CONNECTION: Genetically modified
organisms are transforming agriculture
 Genetically modified (GM) organisms contain
one or more genes introduced by artificial means
 Transgenic organisms contain at least one
gene from another species
 GM plants
– Resistance to herbicides
– Resistance to pests
– Improved nutritional profile
 GM animals
– Improved qualities
– Production of proteins or therapeutics
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Agrobacterium tumefaciens
Plant cell
DNA containing
gene for desired trait
1
Ti
plasmid
Insertion of gene
into plasmid
3
2
Recombinant
Ti plasmid
Introduction
into plant
cells
Regeneration
of plant
DNA carrying new gene
Restriction site
Plant with new trait
12.9 Genetically modified organisms raise concerns
about human and environmental health
 Scientists use safety measures to guard against
production and release of new pathogens
 Concerns related to GM organisms
– Can introduce allergens into the food supply
– FDA requires evidence of safety before approval
– Exporters must identify GM organisms in food shipments
– May spread genes to closely related organisms
– Hybrids with native plants may be prevented by modifying
GM plants
 Regulatory agencies address the safe use of
biotechnology
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12.10 CONNECTION: Gene therapy may
someday help treat a variety of diseases
 Gene therapy aims to treat a disease by
supplying a functional allele
 One possible procedure
– Clone the functional allele and insert it in a retroviral
vector
– Use the virus to deliver the gene to an affected cell
type from the patient, such as a bone marrow cell
– Viral DNA and the functional allele will insert into the
patient’s chromosome
– Return the cells to the patient for growth and division
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12.10 CONNECTION: Gene therapy may
someday help treat a variety of diseases
 SCID (severe combined immune deficiency) was
the first disease treated by gene therapy
– First trial in 1990 was inconclusive
– Second trial in 2000 led to the development of
leukemia in some patients due to the site of gene
insertion
 Challenges
– Safe delivery to the area of the body affected by the
disease
– Achieving a long-lasting therapeutic effect
– Addressing ethical questions
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Cloned gene
(normal allele)
1
Insert normal gene
into virus
Viral nucleic acid
Retrovirus
2
Infect bone marrow
cell with virus
3
Viral DNA inserts
into chromosome
Bone marrow
cell from patient
Bone
marrow
4
Inject cells
into patient
DNA PROFILING
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12.11 The analysis of genetic markers can
produce a DNA profile
 DNA profiling is the analysis of DNA fragments
to determine whether they come from a particular
individual
– Compares genetic markers from noncoding regions
that show variation between individuals
– Involves amplification (copying) of markers for
analysis
– Sizes of amplified fragments are compared
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Crime scene
1 DNA isolated
2 DNA of selected
markers amplified
3 Amplified DNA
compared
Suspect 1
Suspect 2
12.12 The PCR method is used to amplify DNA
sequences
 Polymerase chain reaction (PCR) is a method
of amplifying a specific segment of a DNA
molecule
 Relies upon a pair of primers
– Short DNA molecules that bind to sequences at each
end of the sequence to be copied
– Used as a starting point for DNA replication
 Repeated cycle of steps for PCR
– Sample is heated to separate DNA strands
– Sample is cooled and primer binds to specific target
sequence
– Target sequence is copied with heat-stable DNA
polymerase
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12.12 The PCR method is used to amplify DNA
sequences
 Advantages of PCR
– Can amplify DNA from a small sample
– Results are obtained rapidly
– Reaction is highly sensitive, copying only the target
sequence
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Cycle 1
yields 2 molecules
Genomic
DNA
3
1
3
5
3
Target
sequence
5
5
5
3
Cycle 2
yields 4 molecules
5
5
2 Cool to allow
3
Heat to
primers to form
separate
DNA strands hydrogen bonds
with ends of
target sequences
5
3
5
3
Primer
3
5
DNA
polymerase adds
nucleotides
to the 3 end
of each primer
5
3
New DNA
Cycle 3
yields 8 molecules
Cycle 1
yields 2 molecules
Genomic
DNA
3
3
5
5
3
Target
sequence
5
3
3
5
5
3
2 Cool to allow
1 Heat to
primers to form
separate
DNA strands hydrogen bonds
with ends of
target sequences
5
5
3
5
3
Primer
5
DNA
polymerase adds
nucleotides
to the 3 end
of each primer
5
3
New DNA
Cycle 2
yields 4 molecules
Cycle 3
yields 8 molecules
12.13 Gel electrophoresis sorts DNA molecules
by size
 Gel electrophoresis separates DNA molecules
based on size
– DNA sample is placed at one end of a porous gel
– Current is applied and DNA molecules move from the
negative electrode toward the positive electrode
– Shorter DNA fragments move through the gel pores
more quickly and travel farther through the gel
– DNA fragments appear as bands, visualized through
staining or detecting radioactivity or fluorescence
– Each band is a collection of DNA molecules of the
same length
Video: Biotechnology Lab
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Mixture of DNA
fragments of
different sizes
Power
source
Longer
(slower)
molecules
Gel
Shorter
(faster)
molecules
Completed gel
12.14 STR analysis is commonly used for DNA
profiling
 Short tandem repeats (STRs) are genetic
markers used in DNA profiling
– STRs are short DNA sequences that are repeated
many times in a row at the same location
– The number of repeating units can differ between
individuals
– STR analysis compares the lengths of STR
sequences at specific regions of the genome
– Current standard for DNA profiling is to analyze 13
different STR sites
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STR site 1
STR site 2
Crime scene DNA
Number of short tandem Number of short tandem
repeats match
repeats do not match
Suspect’s DNA
Crime scene
DNA
Suspect’s
DNA
12.15 CONNECTION: DNA profiling has
provided evidence in many forensic
investigations
 Forensics
– Evidence to show guilt or innocence
 Establishing family relationships
– Paternity analysis
 Identification of human remains
– After tragedies such as the September 11, 2001, attack on the
World Trade Center
 Species identification
– Evidence for sale of products from endangered species
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12.16 RFLPs can be used to detect differences in
DNA sequences
 Single nucleotide polymorphism (SNP) is a
variation at one base pair within a coding or
noncoding sequence
 Restriction fragment length polymorphism
(RFLP) is a variation in the size of DNA fragments
due to a SNP that alters a restriction site
– RFLP analysis involves comparison of sizes of
restriction fragments by gel electrophoresis
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Restriction
enzymes added
DNA sample 1
DNA sample 2
w
Cut
z
x
Cut
Cut
y
y
Longer
fragments
z
x
Shorter
fragments
w
y
y
GENOMICS
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12.17 Genomics is the scientific study of whole
genomes
 Genomics is the study of an organism’s complete
set of genes and their interactions
– Initial studies focused on prokaryotic genomes
– Many eukaryotic genomes have since been
investigated
 Evolutionary relationships can be elucidated
– Genomic studies showed a 96% similarity in DNA
sequences between chimpanzees and humans
– Functions of human disease-causing genes have
been determined by comparisons to similar genes in
yeast
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12.18 CONNECTION: The Human Genome
Project revealed that most of the human
genome does not consist of genes
 Goals of the Human Genome Project (HGP)
– To determine the nucleotide sequence all DNA in the
human genome
– To identify the location and sequence of every human
gene
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12.18 CONNECTION: The Human Genome
Project revealed that most of the human
genome does not consist of genes
 Results of the Human Genome Project
– Humans have 21,000 genes in 3.2 billion nucleotide
pairs
– Only 1.5% of the DNA codes for proteins, tRNAs, or
rRNAs
– The remaining 88.5% of the DNA contains
– Control regions such as promoters and enhancers
– Unique noncoding DNA
– Repetitive DNA
– Found in centromeres and telomeres
– Found dispersed throughout the genome, related to
transposable elements that can move or be
copied from one location to another
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Exons (regions of genes coding for protein
or giving rise to rRNA or tRNA) (1.5%)
Repetitive
DNA that
includes
transposable
elements
and related
sequences
(44%)
Introns and
regulatory
sequences
(24%)
Unique
noncoding
DNA (15%)
Repetitive
DNA
unrelated to
transposable
elements
(15%)
12.19 The whole-genome shotgun method of
sequencing a genome can provide a wealth
of data quickly
 Three stages of the Human Genome Project
– A low-resolution linkage map was developed using
RFLP analysis of 5,000 genetic markers
– A physical map was constructed from nucleotide
distances between the linkage-map markers
– DNA sequences for the mapped fragments were
determined
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12.19 The whole-genome shotgun method of
sequencing a genome can provide a wealth
of data quickly
 Whole-genome shotgun method
–
Restriction enzymes were used to produce fragments
that were cloned and sequenced
–
Computer analysis assembled the sequence by
aligning overlapping regions
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Chromosome
Chop up with
restriction enzyme
DNA fragments
Sequence
fragments
Align
fragments
Reassemble
full sequence
12.20 Proteomics is the scientific study of the full
set of proteins encoded by a genome
 Proteomics
– Studies the proteome, the complete set of proteins
specified by a genome
– Investigates protein functions and interactions
 The human proteome may contain 100,000
proteins
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12.21 EVOLUTION CONNECTION: Genomes
hold clues to the evolutionary divergence of
humans and chimps
 Comparisons of human and chimp genomes
– Differ by 1.2% in single-base substitutions
– Differ by 2.7% in insertions and deletions of larger
DNA sequences
– Human genome shows greater incidence of
duplications
– Genes showing rapid evolution in humans
– Genes for defense against malaria and tuberculosis
– Gene regulating brain size
– FOXP2 gene involved with speech and vocalization
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Bacterial
clone
Cut
Bacterium
DNA
fragments
Cut
Recombinant
DNA
plasmids
Recombinant
bacteria
Plasmids
Genomic library
Mixture of DNA
fragments
A “band” is a
collection of DNA
fragments of one
particular length
Longer
fragments
move slower
Shorter
fragments
move faster
DNA attracted to +
pole due to PO4– groups
Power
source
DNA
amplified
via
(a)
Bacterial
plasmids
DNA
sample
treated with
treated with
(b)
DNA
fragments
sorted by size via
(c)
Recombinant plasmids
are inserted
into bacteria
Add
(d)
Particular
DNA
sequence
highlighted
are copied via
(e)
(f)
Collection
is called a
You should now be able to
1. Distinguish between terms in the following
groups: restriction enzyme—DNA ligase; GM
organism—transgenic organism; SNP—RFLP;
genomics—proteomics
2. Define the following terms: cDNA, gel
electrophoresis, gene cloning, genomic library,
“pharm” animal, plasmid, probe, recombinant
DNA, repetitive DNA, reverse transcriptase, STR,
Taq polymerase, vector, whole-genome shotgun
method
3. Describe how genes are cloned
Copyright © 2009 Pearson Education, Inc.
You should now be able to
4. Describe how a probe is used to identify a gene of
interest
5. Describe how gene therapy has been attempted
and identify challenges to the effectiveness of this
treatment approach
6. Distinguish between the use of prokaryotic and
eukaryotic cells in producing recombinant DNA
products
7. Identify advantages to producing pharmaceuticals
with recombinant DNA technology
Copyright © 2009 Pearson Education, Inc.
You should now be able to
8. Describe the basis for DNA profiling and explain
how it is used to provide evidence in forensic
investigations
9. Explain how PCR provides copies of a specific
DNA sequence
10. Identify ethical concerns related to the use of
recombinant DNA technology
11. Describe how comparative information from
genome projects has led to a better
understanding of human biology
Copyright © 2009 Pearson Education, Inc.