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Chapter 20
DNA Technology
and Genomics
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
• DNA technology has revolutionized
biotechnology, the manipulation of
organisms or their genetic components to
make useful products
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
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
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
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
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
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
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
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.
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
• 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
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
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
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
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
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
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
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
• Go to video
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
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
DNA Sequencing
• Relatively short DNA fragments can be
sequenced by the dideoxy chaintermination method
• Inclusion of special dideoxyribonucleotides
in the reaction mix ensures that fragments
of various lengths will be synthesized
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
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
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
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
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
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
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
Concept 20.5: 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
• 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
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
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
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
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
Agricultural Applications
• DNA technology is being used to improve
agricultural productivity and food quality
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
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
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