DNA Technology and
Genomics
Chapter 15
Learning Objective 1
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How does a typical restriction enzyme cut
DNA molecules?
•
Give examples of the ways in which these
enzymes are used in recombinant DNA
technology
Recombinant DNA Technology
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Isolates and amplifies
•
•
•
specific sequences of DNA
incorporates them into vector DNA molecules
Resulting recombinant DNA
•
•
is propagated and amplified (cloned)
in organisms such as E. coli
Restriction Enzymes
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Recognize and cut DNA
•
•
at highly specific base sequences
May produce complementary, singlestranded sticky ends
Restriction Enzymes
Plus HindIII restriction enzyme
Sticky
ends
Fig. 15-1, p. 324
KEY CONCEPTS
•
Recombinant DNA techniques allow
scientists to clone many copies of specific
genes and gene products
Recombinant DNA Vectors
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Naturally occurring circular bacteria DNA
molecules (plasmids)
•
Bacterial viruses (bacteriophages)
Recombinant DNA Molecules
•
Construction
•
•
•
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ends of DNA fragment and vector
cut with same restriction enzyme
associate by complementary base pairing
DNA ligase
•
•
covalently links DNA strands
forms stable recombinant molecule
Plasmid from a
bacterium
DNA of interest from another organism
1 Plasmid and DNA from
another organism are cut
by the same restriction
enzyme (in this example, Clonable DNA fragment
Hin dIII). This produces
molecules with
complementary singlestranded ends.
two types of molecules
2 Mix
so their sticky ends pair.
DNA ligase then forms
covalent bonds at junctions,
linking fragments.
Recombinant
DNA
3 Transfer recombinant DNA molecule to
host cell, where it is copied and turned
on to produce gene product.
Fig. 15-2, p. 325
Plasmids
AatI
XbaI
HpaI
E. coli
origin of replication
PvuII
ClaI
SalI
BamHI
SmaI
Fig. 15-3a, p. 326
Main bacteria DNA
Bacterium
Plasmid
0.5 μ m
Fig. 15-3bc, p. 326
Learning Objective 2
•
What is the difference between a genomic
DNA library, a chromosome library, and a
complementary DNA (cDNA) library?
•
Why would one clone the same eukaryotic
gene from both a genomic DNA library and
a cDNA library?
Libraries (1)
•
Genomic DNA library
•
•
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thousands of DNA fragments
all DNA of an organism
Chromosome library
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all DNA fragments of a specific chromosome
Libraries (2)
•
Genomic DNA and chromosome libraries
•
•
DNA fragments stored in specific bacterial
strains
Provide information about genes and encoded
proteins
Chromosome
Library
Sites of cleavage
Fragment
1
Fragment
2
Human DNA
1
Produce recombinant
DNA
Gene for
resistance
to antibiotic
2
R
Fragment Fragment
3
4
Cut with a restriction enzyme
2
R
2
R
2
R
Transformation
3
Plate with antibioticcontaining medium
Bacteria with plasmid
live and multiply
4
Bacteria without
plasmid fail to grow
Fig. 15-4, p. 327
Sites of cleavage
Fragment
1
Fragment
2
Human DNA
1
Produce recombinant
DNA
Gene for
resistance
to antibiotic
2
Fragment Fragment
3
4
Cut with a restriction enzyme
2
R
R
2
R
Transformation
2
R
3
Plate with antibioticcontaining medium
Bacteria with plasmid
live and multiply
4
Bacteria without
plasmid fail to grow
Stepped Art
Fig. 15-4, p. 327
cDNA Library
•
Complementary DNA (cDNA)
•
•
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produced using reverse transcriptase
makes DNA copies of eukaryotic mRNA
Copies are incorporated into recombinant
DNA vectors
cDNA
Exon
Intron
DNA in a eukaryotic
chromosome
Pre-mRNA
Exon
Intron
Exon
Transcription
RNA processing (remove introns)
Mature mRNA
Formation of cDNA relies on RNA processing that occurs in the
nucleus to yield mature mRNA.
Fig. 15-6a, p. 328
Reverse transcriptase
1 mRNA
cDNA copy of mRNA
Degraded RNA
2
cDNA
3
DNA
polymerase
4
Double-stranded cDNA
Mature mRNA is extracted and purified.
Fig. 15-6b, p. 328
Introns (1)
•
Genes regions that do not code for protein
•
•
present in eukaryote genomic DNA and
chromosome libraries
Genes with introns
•
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can be amplified in bacteria
but protein is not properly expressed
Introns (2)
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Eukaryotic genes in cDNA libraries
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can be expressed in bacteria to produce
functional protein products
because introns have been removed from
mRNA molecules
Learning Objective 3
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What is the purpose of a genetic probe?
Genetic Probe
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Radioactive DNA or RNA sequence
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used to screen recombinant DNA molecules
in bacterial cells
to find specific colony with DNA of interest
Genetic Probe
Bacterial colonies
1
Radioactively
labeled nucleic
acid probe is
added
2
Transfer cells
from colonies to
nitrocellulose
filter
Filter with bacteria
from colonies; cells
are lysed and DNA
denatured
3 Some radioactive
nucleic acid probe
molecules become
hybridized to DNA of
some colonies
4 Exposed X-ray film;
dark spots identify
colonies with desired
DNA
Fig. 15-5, p. 328
Animation: Use of a Radioactive
Probe
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TO PLAY
Learning Objective 4
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How does the polymerase chain reaction
amplify DNA in vitro?
Polymerase Chain Reaction (PCR)
•
Automated in vitro technique
•
•
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targets a particular DNA sequence by specific
primers
clones it using heat-resistant DNA
polymerase
Used to analyze tiny DNA samples
•
from crime scenes, archaeological remains
Fig. 15-7, p. 329
Learning Objective 5
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What is the difference between DNA,
RNA, and protein blotting?
Southern Blot
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Detects DNA fragments
•
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separates using gel electrophoresis
transfer to nitrocellulose or nylon membrane
Probe is hybridized
•
•
by complementary base pairing to DNA bound
to membrane
bands of DNA identified by autoradiography
or chemical luminescence
Gel
Electrophoresis
Fig. 15-8a, p. 330
DNA
Cut with restriction enzyme
100 base pairs
200 base pairs
Mixture placed in
well
300 base pairs
Standards of
known size
+ –
Origin
Direction of
movement
300 base pairs
200 base pairs
100 base pairs
Gel
Fig. 15-8a, p. 330
Fig. 15-8b, p. 330
Southern
Blot
5
Load DNA
2 fragments on gel
for electrophoresis.
1 Digest DNA with
restriction enzymes.
–
DNA
+
DNA fragments
Buffer solution
Agarose gel
Fig. 15-9, p. 332
Buffer solution moves
DNA fragments are in 4 upward, transferring DNA
5 same location as those fragments to a DNAbinding filter.
on gel.
3 Separate DNA by
electrophoresis.
Longer
DNA
fragments
Weight
Absorbent
paper
6
7
Nitrocellulose
filter
Gel
Wick
Buffer
Shorter
DNA
fragments
Fig. 15-9, p. 332
Place filter and
radioactively
6 labeled probe
together in sealed
bag so it can
hybridize.
Wash filter to remove
excess probe and then
expose filter to X-ray film;
7
resulting autoradiograph
shows hybridized DNA
fragments.
Radioactive
probe solution
Fig. 15-9, p. 332
RNA and Proteins
•
Northern Blot
•
•
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RNA molecules separated by electrophoresis
transferred to a membrane
Western Blot
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Proteins or polypeptides previously separated
by gel electrophoresis
Learning Objective 6
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What is the chain termination method of
DNA sequencing?
DNA Sequencing
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Yields information about gene structure
•
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and amino acid sequence of encoded proteins
Geneticists compare DNA sequences
•
with other sequences stored in databases
Automated DNA Sequencing
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Based on chain termination method
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•
•
•
uses dideoxynucleotides
tagged with colored fluorescent dyes
terminates elongation during DNA replication
Gel electrophoresis
•
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separates resulting fragments
laser identifies nucleotide sequence
Dideoxynucleotide
Dideoxyadenosine
triphosphate
(ddATP)
Fig. 15-10, p. 333
Chain Termination Method
Single-strand DNA fragment to be sequenced
+ddATP
+ddCTP
+ddGTP
+ddTTP
Fig. 15-11a, p. 334
Radioactive
primer
+ddATP
Direction of
synthesis
Reaction products from mixture
containing dideoxyATP
Fig. 15-11b, p. 334
Larger
fragments
Smaller
fragments
Fig. 15-11c, p. 334
A
C
G T
Fig. 15-11d, p. 334
Automated DNA Sequence
KEY CONCEPTS
•
Biologists study DNA using gel
electrophoresis, DNA blotting, automated
sequencing, and other methods
Animation: Automated DNA
Sequencing
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TO PLAY
Learning Objective 7
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What are the three main areas of interest
in genomics?
Genomics (1)
Field of biology that studies the entire DNA
sequence of an organism’s genome
1. Structural genomics
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mapping and sequencing genomes
Genomics (2)
2. Functional genomics
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functions of genes and nongene sequences in
genomes
3. Comparative genomics
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comparing genomes of different species
understanding evolutionary relationships
KEY CONCEPTS
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Genomics is an emerging field that
comprises the structure, function, and
evolution of genomes
Learning Objective 8
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What does a DNA microarray do?
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Give an example of its research and
medical potential
DNA Microarrays (1)
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Used in diagnostic tests
•
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different DNA molecules placed on glass chip
Enable researchers to compare
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many genes in normal and diseased cells
DNA Microarrays (2)
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Cancer and other diseases exhibit altered
patterns of gene expression
•
DNA microarrays identify disease-causing
genes (or the proteins they code for)
DNA
Microarray
1 Prepare microarray. Each microdot
contains multiple copies of a specific
single-stranded cDNA.
Treated cell
Mature mRNA
Untreated (control) cell
2 Prepare cDNA from
two cell populations
(treated and control).
Reverse
transcriptase
Mature mRNA
cDNA copy of
mRNA
3 Tag each cDNA with
different fluorescent
dye.
cDNA
mRNA (discard)
cDNA
Reverse
transcriptase
cDNA copy of
mRNA
mRNA (discard)
Fig. 15-13, p. 336
4 Hybridize two cDNA
populations to array.
Laser 1
5 Scan array to identify
Laser 2
fluorescence where
hybridization has
occurred.
Emissions
6 Computer analysis
produces color-coded
readout.
Gene in treated cell that
increased activity, compared
to control
Gene in treated cell that
decreased activity, compared
to control
Gene that was active in both
treated and untreated cells
Gene that was inactive in
both treated and untreated
cells
Fig. 15-13, p. 336
Learning Objective 9
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What are pharmacogenetics and
proteomics?
Pharmacogenetics
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Science of gene-based medicine
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analyzes individual’s genetic makeup
customizes drugs to match
Proteomics
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Study of all proteins encoded by genome
•
•
Try to identify all proteins made by a cell
Harder than sequencing the human genome
Learning Objective 10
•
Describe at least one important application
of recombinant DNA technology in each of
the following fields: medicine and
pharmacology, DNA fingerprinting, and
transgenic organisms
Genetically Altered Bacteria
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Produce important human protein products
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insulin
growth hormone
tissue plasminogen activator (TPA)
tissue growth factor-beta (TGF- β)
clotting factor VIII
Dornase Alpha (DNase)
DNA Fingerprinting
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Analysis of individual’s DNA
•
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based on short tandem repeats (STRs)
(molecular markers, highly polymorphic)
Applications in
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•
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law enforcement
disputed parentage
tracking tainted foods
1
2
3
From 4
blood at
crime
scene
5
6
7
Fig. 15-14, p. 339
Animation: DNA Fingerprinting
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TO PLAY
Transgenic Organisms
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Foreign DNA
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Transgenic livestock
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incorporated into genetic material
produce foreign proteins in milk
Transgenic plants
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have great potential in agriculture
Fig. 15-15, p. 340
Fig. 15-16, p. 341
Fig. 15-17, p. 342
Fig. 15-17a, p. 342
Fig. 15-17b, p. 342
KEY CONCEPTS
•
DNA technology and genomics have wide
applications, from medical to forensic to
agricultural
Learning Objective 11
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Describe at least two safety issues
associated with recombinant DNA
technology
•
How are these issues being addressed?
Safety Concerns
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Genetically engineered organisms
•
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Scientists have specific safety guidelines for
using recombinant DNA technology
Introduction of transgenic plants and
animals into the natural environment
•
may spread in an uncontrolled manner
Genetically Engineered Plant
Animation: Gene Transfer Using a Ti
Plasmid
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TO PLAY
Animation: Base-pairing of DNA
Fragments
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TO PLAY
Video: Cloned Pooch
CLICK
TO PLAY
•
From ABC News, Biology in the Headlines, 2005 DVD.