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DNA Technology
and Genomics
Understanding and Manipulating Genomes
• DNA technology has launched a revolution in the area
of biotechnology - The manipulation of organisms or
their genetic components to make useful products
• One of the greatest achievements of modern science
has been the sequencing of the human genome,
which was largely completed by 2003
• DNA sequencing accomplishments have all depended
on advances in DNA technology, starting with the
invention of methods for making recombinant DNA
Recombinant DNA
• Formed by joining DNA from 2 different individuals into
a single molecule.
• Various natural mechanisms can combine DNA from 2
individuals of the same species
• Scientists have also developed techniques to combine
DNA from any 2 individuals.
Recombinant DNA
•
Two key enzymes are used to make artificially recombined
DNA.
•
•
Restriction enzymes (also called restriction endonucleases) cut DNA
into fragments – so called “molecular scissors”

Each one recognizes and cuts DNA only where a specific sequence
of base pairs occurs.

Many do not cut straight through both strands, but make a jagged
cut leaving unpaired bases at both ends. Because these unpaired
bases can pair with complimentary bases, they are called “sticky
ends”.
DNA ligase is used to join DNA fragments together. This is the
“molecular glue”
Restriction Enzymes and Recombinant DNA
• Bacterial restriction enzymes cut DNA molecules at a limited
number of specific DNA sequences, called restriction sites
• A restriction enzyme will usually make many cuts in a DNA
molecule yielding a set of restriction fragments
• The most useful restriction enzymes cut DNA in a staggered
way producing fragments with “sticky ends” that can bond with
complementary “sticky ends” of other fragments
• DNA ligase is an enzyme that seals the bonds between
restriction fragments
Procedure for Recombining DNA
Restriction site
•
Isolate DNA from 2 different sources
DNA 5
3
•
Cut the DNA from both sources into
fragments using the same restriction
enzyme.
1 Restriction enzyme cuts
3
5
GAATTC
CTTAAG
the sugar-phosphate
backbones at each arrow
G
•
•
G
Mix the DNA fragments together.
Since they were cut with the same
restriction enzyme, fragments from
different sources will have the same
“sticky ends” and can pair up.
Use the enzyme DNA ligase to join
the paired fragments together
Sticky end
2 DNA fragment from
another source is added.
Base pairing of sticky
ends produces various
combinations.
G AATT C
C TTAA G
G
G
Fragment from different
DNA molecule cut by the
same restriction enzyme
G AATTC
CTTAA G
One possible combination
3 DNA ligase
seals the strands.
Recombinant DNA molecule
Recombinant Plasmids
• Recombinant DNA technology can be used to
create recombinant plasmids (or other agents such
as viruses) used to insert foreign genes into
recipient cells.
• Plasmids (or other recombinant agents) used to
insert foreign DNA into recipient cells are called
vectors
• Recombinant plasmids can then be used to
produce multiple copies of the DNA fragment
Copying DNA Fragments
• Why produce multiple copies of a DNA fragment?
•
Study its structure and function
•
Compare the fragment with DNA from other sources
•
If it codes for a useful protein, to produce large
quantities of the protein
Two Basic Copy Methods
• Cloning – insert the gene into a living host cell and use the
cell to replicate the gene along with its own DNA whenever it
divides
• Polymerase Chain Reaction (PCR) – create the conditions
needed for replication inside a test tube that contains a copy
of the gene
DNA Cloning
•
DNA cloning permits
production of multiple copies
of a specific gene or other
DNA segment
Bacterium
Bacterial
chromosome
• Most methods for cloning
pieces of DNA in the
laboratory involve
•
•
•
Use of bacteria and
their plasmids
Restriction Enzymes
In gene cloning, the original
plasmid is called a cloning
vector a DNA molecule that
can carry foreign DNA into a
cell and replicate there
Cell containing gene
1 Gene inserted of interest
into plasmid
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
2 Plasmid put into
bacterial cell
Recombinate
bacterium
3 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
DNA of
chromosome
Protein harvested
4 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
Gene Cloning
Stage 1: DNA from two sources
is isolated and cleaved with the
same restriction endonuclease.
Animal cell
Restriction
endonuclease
cut sites
E. coli
Restriction
Gene of
site
interest
ampR
gene
DNA
Stage 2: The two
types of DNA can
pair at their sticky
ends when mixed
together; DNA
ligase joins the
segments.
Sticky
ends
Plasmid
lacZ'
gene
Reclosed plasmid with
functional lacZ' gene
Recombinant plasmids with
nonfunctional lacZ' genes
Gene Cloning
Stage 3: Plasmids are inserted into
bacterial cells by transformation;
bacterial cells reproduce and form
clones.
Clone 1
Clone 2
Clones with recombinant plasmids
Clone 3
Can be screened for
the gene of interest
Amplifying DNA in Vitro
• Polymerase chain reaction (PCR)
•
Can produce many copies of a specific target segment
of DNA
•
Uses primers that bracket the desired sequence
•
Uses a heat-resistant DNA polymerase
Polymerase Chain Reaction (PCR)
1. Denaturation – a solution containing RNA primers and the DNA
fragment to be amplified is heated so that the DNA dissociates
into single strands.
2. Annealing of primers – the solution is cooled, and the primers
bind to complementary sequences on the DNA flanking the
gene to be amplified.
3. Primer extension – DNA polymerase then copies the remainder
of each strand, beginning at the primer.
4. Repeat steps 1 – 3 many times, each time doubling the number
of copies, until a sufficient number of copies are produced.
The Polymerase Chain Reaction (PCR)
5
3
Target
sequence
APPLICATION With PCR, any specific segment—the target
sequence—within a DNA sample can be copied many times
(amplified) completely in vitro.
TECHNIQUE The starting materials for PCR are doublestranded DNA containing the target nucleotide sequence to be
copied, a heat-resistant DNA polymerase, all four nucleotides,
and two short, single-stranded DNA molecules that serve as
primers. One primer is complementary to one strand at one end
of the target sequence; the second is complementary to the
other strand at the other end of the sequence.
RESULTS
During each PCR cycle, the target DNA
sequence is doubled. By the end of the third cycle, one-fourth
of the molecules correspond exactly to the target sequence,
with both strands of the correct length. After 20 or so cycles,
the target sequence molecules outnumber all others by a
billionfold or more.
3
Genomic DNA
1 Denaturation:
5
5
3
3
5
Heat briefly
to separate
DNA strands
2 Annealing:
Cycle 1
yields
2
molecules
Cool to allow
primers to
hydrogen-bond.
Primers
3 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
New
nucleotides
Identifying Clones Carrying a Gene of Interest
• Cloning is used to prepare many copies of a gene of
interest for use in sequencing the gene, in producing
its encoded protein, in gene therapy, or in basic
research
• A clone carrying the gene of interest can be identified
with a radioactively labeled nucleic acid probe
• The probe has a sequence complementary to the
gene
• The process is called nucleic acid probe hybridization
Using a Genetic Probe to Screen for the Gene of
Interest
5. A comparison with the original
plate identifies the colony
containing the gene.
1. Colonies of bacteria, each grown
from cells taken from a white colony.
2. A replica of the plate
is made by pressing
a filter against the
colonies. Some cells
from each colony
adhere to the filter.
Filter
Film
3. The filter is washed with a solution
that denatures the DNA and contains
the radioactively labeled probe. The
probe contains nucleotide sequences
complementary to the gene of interest
and binds to cells containing the gene.
4. Only those colonies
containing the gene will
retain the probe and emit
radioactivity on film placed
over the filter.
Nucleic Acid Probe Hybridization
APPLICATION Hybridization with a complementary nucleic acid probe detects a specific DNA within a mixture of DNA molecules.
In this example, a collection of bacterial clones (colonies) are screened to identify those carrying a plasmid with a
gene of interest.
TECHNIQUE
Cells from each colony known to contain recombinant plasmids (white colonies in Figure 20.4, stap 5) are
transferred to separate locations on a new agar plate and allowed to grow into visible colonies. This
collection of bacterial colonies is the master plate.
Master plate
Master plate
Colonies
containing
gene of
interest
Probe
DNA
Solution
containing
probe
Radioactive
single-stranded
DNA
Gene of
interest
Single-stranded
DNA from cell
Filter
Film
Filter lifted and
flipped over
1 A special filter paper is
pressed against the
master plate,
transferring cells to
the bottom side of the
filter.
RESULTS
2
Hybridization
on filter
The filter is treated to break
open the cells and denature
their DNA; the resulting singlestranded DNA molecules are
treated so that they stick to
the filter.
3 The filter is laid under
photographic film,
allowing any
radioactive areas to
expose the film
(autoradiography).
4 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.
Colonies of cells containing the gene of interest have been identified by nucleic acid hybridization. Cells from
colonies tagged with the probe can be grown in large tanks of liquid growth medium. Large amounts of the DNA
containing the gene of interest can be isolated from these cultures. By using probes with different nucleotide
sequences, the collection of bacterial clones can be screened for different genes.
DNA Library
•
A DNA library is a collection of DNA fragments
representing all the DNA of an organism.
•
Two Types:
•
Genomic libraries
•
cDNA libraries
Genomic Library
•
The simplest kind, the entire genome of an organism is fragmented.
•
The fragments are then inserted into a vector, such as a plasmid or phage, and
introduced into a host:
DNA fragments from source DNA
DNA inserted
into plasmid
vector
DNA inserted
into phage
vector
Storing Cloned Genes in DNA Libraries
•
A genomic library made using bacteria is a collection of recombinant vector
clones produced by cloning DNA fragments derived from an entire genome
•
A genomic library made using bacteriophages is stored as a collection of
phage clones
Foreign genome
cut up with
restriction
enzyme
or
Recombinant
plasmids
Bacterial
clones
(a) Plasmid library
Recombinant
phage DNA
Phage
clones
(b) Phage library
cDNA 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
• Includes only the DNA fragments that code for
proteins, introns not included.
• To produce a cDNA library, scientists first isolate the
mature mRNA from an organism.
• An enzyme called reverse transcriptase is then used
to make a complementary DNA copy of each mature
mRNA molecule:
Intron (noncoding region)
Exon (coding region)
Eukaryotic DNA
Transcription
Primary RNA
transcript
Introns are cut out
and coding regions are
spliced together
Mature mRNA transcript
Isolation of mRNA
Addition of reverse
transcriptase
mRNA-cDNA hybrid
Addition of mRNAdegrading enzymes
DNA polymerase
Double-stranded cDNA
gene without introns
Restriction Fragment Length Polymorphisms
(RFLP)
• Different DNA sequences on homologous
chromosomes, result in restriction fragments of
different lengths
• Specific fragments can be detected and analyzed by
Southern blotting
• The thousands of RFLPs present throughout
eukaryotic DNA can serve as genetic markers
Restriction Fragment Analysis
• Detects DNA differences
that affect restriction sites
• Can rapidly provide useful
comparative information
about DNA sequences
•
For example comparing
two alleles for a gene
• Gel electrophoresis
separates DNA restriction
fragments of different
lengths
Normal  -globin allele
175 bp
201 bp
DdeI
DdeI
Large fragment
DdeI
DdeI
Sickle-cell mutant -globin allele
376 bp
Large fragment
DdeI
DdeI
DdeI
(a) DdeI restriction sites in normal and sickle-cell alleles of
-globin gene.
Normal Sickle-cell
allele
allele
Large
fragment
376 bp
201 bp
175 bp
(b) Electrophoresis of restriction fragments from
normal and sickle-cell alleles.
Gel Electrophoresis and Southern Blotting
APPLICATION
Gel electrophoresis is used for separating nucleic acids or proteins that
differ in size, electrical charge, or other physical properties. DNA
molecules are separated by gel electrophoresis in restriction fragment
analysis of both cloned genes (see Figure 20.9) and genomic DNA (see
Figure 20.10).
1
2
Cathode
Each sample, a mixture of DNA molecules, is placed in a separate
well near one end of a thin slab of gel. The gel is supported by
glass plates, bathed in an aqueous solution, and has electrodes
attached to each end.
When the current is turned on, the negatively charged DNA
molecules move toward the positive electrode, with shorter
molecules moving faster than longer ones. Bands are shown here
in blue, but on an actual gel, DNA bands are not visible until a
DNA-binding dye is added. The shortest molecules, having
traveled farthest, end up in bands at the bottom of the gel.
TECHNIQUE Gel electrophoresis separates macromolecules on the basis of their rate
of movement through a gel in an electric field. How far a DNA molecule
travels while the current is on is inversely proportional to its length. A
mixture of DNA molecules, usually fragments produced by restriction
enzyme digestion, is separated into “bands”; each band contains
thousands of molecules of the same length.
RESULTS
After the current is turned off, a DNA-binding dye is added. This dye
fluoresces pink in ultraviolet light, revealing the separated bands to which it
binds. In this actual gel, the pink bands correspond to DNA fragments of
different lengths separated by electrophoresis. If all the samples were initially
cut with the same restriction enzyme, then the different band patterns
indicate that they came from different sources.
Power
source
Mixture
of DNA
molecules
of different sizes
Gel
Glass
plates
Anode
Longer
molecules
Shorter
molecules
Southern Blotting
• Specific DNA fragments can be identified by Southern blotting
• Uses labeled probes that hybridize to the DNA immobilized on a
“blot” of the gel
APPLICATION Researchers can detect specific nucleotide sequences within a DNA sample with this
method. In particular, Southern blotting is useful for comparing the restriction fragments
produced from different samples of genomic DNA.
TECHNIQUE In this example, we compare genomic DNA samples from three individuals: a homozygote
for the normal -globin allele (I), a homozygote for the mutant sickle-cell allele (II), and a
heterozygote (III).
DNA + restriction enzyme
Restriction
fragments
I
II
III
Nitrocellulose
paper (blot)
Heavy
weight
Gel
Sponge
I Normal
-globin
allele
Alkaline
solution
II Sickle-cell III Heterozygote
allele
1 Preparation of restriction fragments.
2 Gel electrophoresis.
3 Blotting.
Paper
towels
Stack of paper towels
Nitrocellulose paper
Sponge
Gel
Buffer
2. The gel is covered with a sheet of
nitrocellulose and placed in a tray of buffer
on top of a sponge. Alkaline chemicals in the
buffer denature the DNA into single strands.
The buffer wicks its way up through the gel
and nitrocellulose into a stack of paper
towels placed on top of the nitrocellulose.
Nitrocellulose paper now
contains nucleic acid "print"
Gel
3. Pattern on gel is copied faithfully, or
“blotted,” onto the nitrocellulose.
Southern Blotting – Labeled Probes
Radioactively
labeled probe
for -globin
gene is added
to solution in
a plastic bag
I
II
III
Paper blot
1 Hybridization with radioactive probe.
RESULTS
Probe hydrogenbonds to fragments
containing normal
or mutant -globin
Fragment from
sickle-cell
-globin allele
Fragment from
normal -globin
allele
I
II
III
Film over
paper blot
2 Autoradiography.
Because the band patterns for the three samples are clearly different, this method can be used to
identify heterozygous carriers of the sickle-cell allele (III), as well as those with the disease, who have
two mutant alleles (II), and unaffected individuals, who have two normal alleles (I). The band patterns
for samples I and II resemble those observed for the purified normal and mutant alleles, respectively,
seen in Figure 20.9b. The band pattern for the sample from the heterozygote (III) is a combination
of the patterns for the two homozygotes (I and II).
Film
Size
markers
Hybridized
nucleic acids
5. Photographic film is laid over the paper and is
exposed only in areas that contain radioactivity
(autoradiography). Nitrocellulose is examined
for radioactive bands, indicating hybridization
of the original nucleic acids with the
radioactively labeled ones.
Restriction
endonuclease
cutting sites
RFLP Analysis
Larger
fragments
Smaller
fragments
–
+
–
+
–
+
Single base-pair
change
Sequence duplication
Three different
DNA duplexes
Cut DNA
Gel electrophoresis of
restriction fragments
Genomes
• The complete set of genetic instructions for an
organism is referred to as its genome.
• Genomics – the science of mapping and studying the
genomes of living organisms
• Involves
• Genetic (Linkage) maps
• Physical maps
• DNA Sequencing
Genetic maps
•
Genetic maps are linkage maps show the relative location of genes on
a chromosome, determined by recombination frequencies.
•
Distances on genetic maps are measured in centimorgans (cM). One
cM represents 0.01% recombination frequency.
I
Y
II
X
IV
III
Mutant phenotypes
Short
aristae
Black
body
0
Long aristae
(appendages
on head)
Cinnabar
eyes
Vestigial
wings
48.5 57.5 67.0
Gray
body
Red
eyes
Brown
eyes
104.5
Normal
wings
Wild-type phenotypes
Red
eyes
Physical maps
• Show the distance between DNA landmarks, such as
the recognition sites for restriction enzymes.
• Distances between landmarks on a physical map are
measured in base-pairs (1000 base-pairs equals 1
kilobase kb).
• Created by cutting genomic DNA with different
restriction enzymes, and then determining the size of
the pieces and how they fit together, into a continuous
segment of the genome
• Such a map shows the distance between the different
recognition sites of the restriction enzymes:
2. The fragments produced by
enzyme A only, by enzyme B only,
and by enzymes A and B
simultaneously are run out sideby-side on a gel, which separates
them according to size, smaller
fragments running faster.
DNA
Molecular
weight
marker
14kb
10kb
6kb
2kb
3. The fragments are arranged so
that the smaller ones produced by
the simultaneous cut can be
grouped to generate the larger
ones produced by the individual
enzymes
4. A physical map is constructed.
enzyme B
1. Multiple copies of a segment of
DNA are cut with restriction
enzymes.
14kb
9kb
8kb
9kb
5kb
2kb
5kb
3kb
2kb
2kb
8kb
9kb
A
A
5kb
14kb
B
2kb 3kb 5kb
9kb
A B
A
A B
A
2kb 5kb 10kb
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
19kb
DNA Sequencing
• Ultimate map - organism’s DNA base-pair sequence of
the entire genome.
• Sequencing an entire genome, which may contain
billions of base pairs
• DNA must be cut into small fragments and automated
sequencers are used to determine the base-pair
sequence of DNA
• Only accurate on fragments of DNA up to about 500
base-pairs in length.
DNA Sequencing
•
Starts with the
sequencing of random
DNA fragments
•
Powerful computer
programs then assemble
the resulting very large
number of overlapping
short sequences into a
single continuous
sequence
1
Cut the DNA from
many copies of an
entire chromosome
into overlapping fragments short enough
for sequencing.
2 Clone the fragments
in plasmid or phage
vectors
3 Sequence each
fragment
ACGATACTGGT
CGCCATCAGT
4 Order the
sequences into one
overall sequence
with computer
software.
ACGATACTGGT
AGTCCGCTATACGA
…ATCGCCATCAGTCCGCTATACGATACTGGTCAA…
DNA Sequencing
•
Short DNA fragments can be sequenced by the dideoxy chain-termination method
DNA
(template strand)
C
5 T
APPLICATION
TECHNIQUE
RESULTS
The sequence of nucleotides in any cloned DNA
fragment up to about 800 base pairs in length can
be determined rapidly with specialized machines
that carry out sequencing reactions and separate
the labeled reaction products by length.
This method synthesizes a nested set of DNA strands
complementary to the original DNA fragment. Each
strand starts with the same primer and ends with a
dideoxyribonucleotide (ddNTP), a modified nucleotide.
Incorporation of a ddNTP terminates a growing DNA
strand because it lacks a 3’—OH group, the site for
attachment of the next nucleotide (see Figure 16.12).
In the set of strands synthesized, each nucleotide
position along the original sequence is represented by
strands ending at that point with the complementary
ddNT. Because each type of ddNTP is tagged with a
distinct fluorescent label, the identity of the ending
nucleotides of the new strands, and ultimately the
entire original sequence, can be determined.
The color of the fluorescent tag on each strand indicates
the identity of the nucleotide at its end. The results can
be printed out as a spectrogram, and the sequence,
which is complementary to the template strand, can then
be read from bottom to top. (Notice that the sequence
here begins after the primer.)
Figure 20.12
G
A
C
T
T
C
G
A
C
3 A
A
C
5 T
G
A
C
T
T
C
G
A
C
A
3
A
Primer
3
T
G
T
T
Deoxyribonucleotides Dideoxyribonucleotides
(fluorescently tagged)
5
DNA
polymerase
dATP
ddATP
dCTP
dTTP
ddCTP
ddTTP
dGTP
ddGTP
P P P
P P P
G
OH
ddC
T
G
T
T
ddG
C
T
G
T
T
ddA
G
C
T
G
T
T
ddA
A
G
C
T
G
T
T
ddG
A
A
G
C
T
G
T
T
ddT
G
A
A
G
C
T
G
T
T
Direction
of movement
of strands
Laser
Detector
G
A
C
T
G
A
A
G
C
H
Labeled strands
DNA (template
strand)
G
ddC
T
G
A
A
G
C
T
G
T
T
ddA
C
T
G
A
A
G
C
T
G
T
T
ddG
A
C
T
G
A
A
G
C
T
G
T
T
3
Human Genome Project
• Project to sequence the entire human genome is
called the human genome project
• Sequencing the human genome is a major step
towards improving the diagnosis and treatment of
disease in humans - practical applications of this
information will be enormous.
• Project has already led to some important findings:
•
Human genome is surprisingly similar to other
genomes
•
Only about 1% of the human genome codes for
proteins
Human Genome
•
Eukaryotes tend to have many more genes than prokaryotes.
•
However -complexity of an organism is not directly related to the
number of genes
•
For example, the human genome has around 30,000 genes, about
the same number as in mice and far fewer than in rice.
•
Current estimates are that the human genome contains about
25,000 genes, but the number of human proteins is much larger
Human Genome
• Humans have only about 25,000 protein-encoding genes, these
genes can code for at least 87,000 different proteins.
• How? Alternative splicing of exons can produce several different
mature mRNAs from a single gene
Alternative Splicing of Exons
DNA
Introns
Unmodified
RNA
transcript
Processed RNAin brain
Processed RNAin muscle
Exons
Genomics
• Genome sequences provide clues to important biological
questions
• In genomics scientists study whole sets of genes and their
interactions
• Computer analysis of genome sequences helps researchers
identify sequences that are likely to encode proteins
• Comparison of the sequences of “new” genes with those of
known genes in other species may help identify new genes
• For a gene of unknown function experimental inactivation of the
gene and observation of the resulting phenotypic effects can
provide clues to its function
DNA Micro-arrays
•
A DNA microarray, is a small square of glass that is covered with
thousands of short pieces of single-stranded DNA arranged in a grid.
•
The short chains of DNA nucleotides, which are attached to the glass
surface, are called oligonucleotides.
•
Can be used to screen a genome for the presence of thousands of
specific alleles at once.
•
To screen a person’s genome, a DNA sample is obtained, cut into
fragments, heated to separate the strands, and then flooded over the
chip.
•
Specific alleles can be determined based on which oligonucleotides
bind to the chip
DNA Technology
• The practical applications of DNA technology affect
our lives in many ways
• Numerous fields are benefiting from DNA technology
and genetic engineering
• One obvious benefit of DNA technology is the
identification of human genes whose mutation plays a
role in genetic diseases
Future Directions in Genomics
• Genomics is the study of entire genomes
• Proteomics is the systematic study of all the
proteins encoded by a genome
• Single nucleotide polymorphisms (SNPs) single
base-pair variations that provide useful markers
for studying human genetic variation
Practical Applications Of DNA
• Numerous fields are benefiting from DNA technology
and genetic engineering
• One obvious benefit of DNA technology is the
identification of human genes whose mutation plays a
role in genetic diseases
• Medical scientists can now diagnose hundreds of
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
Diagnosis of Diseases
• Hundreds of human genetic disorders are diagnosed
•
using PCR and primers corresponding to cloned disease genes
•
sequencing the amplified product to look for the disease-causing
mutation
• Even when a disease gene has not yet been cloned the
presence of an abnormal allele can be diagnosed with
reasonable accuracy if a closely linked RFLP marker has been
found
RFLP marker
DNA
Restriction
sites
Disease-causing
allele
Normal allele
Human Gene Therapy
• Is the alteration of an
afflicted individual’s
genes
• Holds great potential for
treating disorders
traceable to a single
defective gene
• Uses various vectors for
delivery of genes into
cells
Cloned gene
(normal
allele,
absent
from
patient’s
cells)
Retrovirus
capsid
1 Insert RNA version of normal allele
into retrovirus.
Viral RNA
2 Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
3 Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
4 Inject engineered
cells into patient.
Pharmaceutical Products
•
Large-scale production of human hormones and other proteins with
therapeutic uses
•
Production of safer vaccines
2. Surface protein
gene is isolated.
1. DNA is extracted.
Gene specifying
herpes simplex
surface protein
Herpes simplex virus
Human immune
response
Harmless vaccinia
(cowpox) virus
6. Antibodies
directed against
herpes simplex
viral coat are
made.
5. Harmless
engineered virus
(the vaccine) with
surface like herpes
simplex is injected
into the human
body.
3. Vaccinia DNA
is extracted and
cleaved.
4. Fragment
containing surface
gene combines
with cleaved
vaccinia DNA.
Construction of Vaccine
for Herpes Simplex
Forensic Evidence
• A DNA fingerprint is a specific
pattern of bands of RFLP
markers on a gel
•
DNA “fingerprints” obtained by
analysis of tissue or body fluids
found at crime scenes
•
Can provide definitive evidence
that a suspect is guilty or not
• DNA fingerprinting can also be
used in establishing paternity
Defendant’s
blood (D)
Blood from
defendant’s
clothes
4 g
D
Jeans
Victim’s
blood (V)
8 g
shirt
V
Manipulation of Microganisms
• Genetic engineering can be used to modify the
metabolism of microorganisms
•
Can be used to extract minerals from the environment
•
Degrade various types of potentially toxic waste
materials
•
Improve agricultural productivity and food quality
Animal Husbandry and “Pharm” Animals
• Transgenic animals contain genes from other organisms
• Have been engineered to be pharmaceutical “factories”
Escherichia coli
Plasmid
1. Plasmid is removed
and cut open with
restriction endonuclease.
2. Cow
somatotropin
gene is isolated
from cow cell.
Gene
of interest
5. Bacteria producing
bovine somatotropin
are grown in
fermentation tanks.
3. Somatotropin gene is
inserted into bacterial
plasmid.
4. Plasmid is
reintroduced into
bacterium.
Cow DNA
Production of Bovine
Somatotropin
6. Somatotropin is
removed from
bacteria and purified.
7. Bovine
somatotropin
is administered to
cow to enhance
milk production.
Genetic Engineering in Plants
•
Agricultural scientists have already 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
APPLICATION
Genes conferring useful traits, such as pest resistance, herbicide
resistance, delayed ripening, and increased nutritional value, can be
transferred from one plant variety or species to another using the Ti
plasmid as a vector.
TECHNIQUE
1
2
3
The Ti plasmid is isolated from the bacterium Agrobacterium
tumefaciens. The segment of the plasmid that integrates into
the genome of host cells is called T DNA.
Isolated plasmids and foreign DNA containing a gene of
interest are incubated with a restriction enzyme that cuts in
the middle of T DNA. After base pairing occurs between
the sticky ends of the plasmids and foreign DNA
fragments, DNA ligase is added. Some of the resulting
stable recombinant plasmids contain the gene of interest.
Agrobacterium tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
DNA with
the gene
of interest
Recombinant
Ti plasmid
Recombinant plasmids can be introduced into cultured plant
cells by electroporation. Or plasmids can be returned to
Agrobacterium, which is then applied as a liquid suspension
to the leaves of susceptible plants, infecting them. Once a
plasmid is taken into a plant cell, its T DNA integrates into
the cell‘s chromosomal DNA.
RESULTS
Transformed cells carrying the transgene of interest can regenerate
complete plants that exhibit the new trait conferred by the
transgene.
Plant with
new trait
T DNA
Transgenic Rice
Beans
Ferritin gene
is transferred
into rice from
beans.
Fe
Ferritin protein
increases iron
content of rice.
Aspergillus fungus
Wild rice
Phytase gene is
transferred into
rice from a
fungus.
Metallothionin
gene is
transferred into
rice from wild
rice.
Pt
Rice
chromosome
Phytate, which
inhibits iron
reabsorption,
is destroyed by the
phytase enzyme.
S
Metallothionin
protein supplies
extra sulfur to
increase iron
uptake.
Daffodil
Enzymes for
-carotene
synthesis are
transferred into
rice from daffodils.
A1 A2 A3 A4
-carotene, a
precursor to
vitamin A, is
synthesized.
Safety and Ethical Questions Raised by DNA
Technology
• The potential benefits of genetic engineering
•
Must be carefully weighed against the potential
hazards of creating products or developing procedures
that are harmful to humans or the environment
• Today, most public concern about possible hazards is
focused on genetically modified (GM) organisms used
as food