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Chapter 20
DNA Technology
and Genomics
Biology, Seventh Edition
Neil Campbell and Jane Reece
Lectures by Chris Romero
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Understanding and Manipulating Genomes
• DNA technology has launched a revolution in the
area of Biotechnology which is the manipulation
of organisms or their components to make useful
products such as insulin, blood clotting factors,
and lots of other proteins.
• This manipulation can be accomplished by DNA
Technology and this would be central to the field of
Genetic engineering.
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• DNA technology is now applied in areas ranging
from agriculture to criminal low with the most
important achievements in basic research.
• For instance the level of expression of thousands of
different genes can now be measured at the same
time using what is known as DNA microarray
(Figure 20.1).
• One of the great achievements of modern science
has been completing the sequence of the entire
human genome by the year 2003.
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DNA microarray revealing expression of 2400 genes
Figure 20.1
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• 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 have developed methods for
preparing well-defined, gene-sized pieces of
DNA in multiple identical copies, a process
called gene cloning.
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DNA Cloning and Its Applications: A Preview
• Most methods for cloning pieces of DNA in the
laboratory
– Share certain general features, such as the
use of bacteria (e.g Escherichia coli) and their
plasmids.
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Overview of Gene Cloning
•
Isolation of plasmid DNA from bacteria and DNA carrying
a gene of interest from another
•
A piece of DNA containing the gene is inserted into a
plasmid (cloning vector) producing a recombinant DNA
plasmid
•
The recombinant plasmid is returned to a bacterial cell
•
The cell is then grown in culture forming a clone of cells.
With the foreign DNA replicating with the rest of the
bacterial chromosome.
•
Identification of the bacterial clone that carries the gene
of interest
•
Once the clone is identified, then a large scale production
of the product of the gene is initiated in a bioreactor.
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Overview of gene cloning with a bacterial plasmid, showing various uses of cloned genes
Bacterium
1 Gene inserted
into plasmid
Plasmid
Bacterial
chromosome Recombinant
DNA (plasmid)
2
Gene of
interest
Plasmid put into
bacterial cell
Recombinate
bacterium
3
Gene of
interest
Copies of gene
Basic
research
on gene
Figure 20.2
Gene for pest
resistance inserted
into plants
4
Basic
research and
various
applications
Gene used to alter
bacteria for cleaning
up toxic waste
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Cell containing gene
of interest
3
DNA of
chromosome
Host cell grown in culture,
to form a clone of cells
containing the “cloned”
gene of interest
Protein expressed
by gene of interest
Protein harvested
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
– Revolutionize the recombinant DNA Technology
– Cut DNA molecules at a limited number of specific
DNA sequences, called restriction sites
– Naturally these enzymes protect bacteria from
intruding DNA, but how can they spare the bacterial
own DNA?
– It adds a methyl group to adenine or cytosine within
the sequences recognized by these enzymes.
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How can these enzymes be used for creating a recombinant DNA?
• 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
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Restriction enzymes… Cont.
• Most restriction sites are symmetrical; the same 5’→ 3’
sequence of four to eight nucleotides is found on both
strands.
• Restriction enzymes cut covalent phosphodiester
bonds of both strands and always in a very
reproducible way producing restriction fragments with
at least one single stranded end called sticky end.
• Sticky ends will form H-bonded bp with complementary
single stranded DNA from another DNA of anther
organism that was cut using the same enzyme.
• An enzyme called the DNA ligase will seal these
fragments together by forming phosphdiester bonds.
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Using a restriction enzyme and DNA ligase to make recombinant DNA
Restriction site
DNA
1
5
3
3
5
GAATTC
CTTAAG
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow
G
G
Sticky end
2
DNA fragment from
another source is added.
Base pairing of sticky
ends produces various
combinations.
GA A T TC
CT TA A G
3
Figure 20.3
G
Fragment from different
DNA molecule cut by the
same restriction enzyme
G AATTC
CTTAA G
One possible combination
DNA ligase
seals the strands.
Recombinant DNA molecule
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G
Cloning a Eukraryotic Gene in a Bacterial Plasmid
• In gene cloning, the original plasmid is called a
cloning vector
– Defined as a DNA molecule that can carry
foreign DNA into a cell and replicate there.
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Procedure for cloning a eukaryotic gene in a bacterial plasmid
•
Isolation of vector and gene-source DNA; we begin
by preparing two kinds of DNA;
–
–
Bacterial plasmid to be used as a vector, form E. coli
that carry two useful genes;
•
ampR (conferes resistance to ampicillin)
•
and lac Z that encodes the enzyme βgalactosidase that hydrolysis the sugar lactose.
•
In addition this plasmid has a single recognition
sequence that falls within the lac Z gene.
DNA containing the gene of interest let us say from
humans.
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Insertion of DNA into the vector
–
Both DNA and the vector should be digested by the
same restriction enzyme.
•
In this case the enzyme cuts the plasmid at its
single cutting site thus disrupting the lacZ gene.
•
The enzyme also cuts the human DNA generating
many thousands of fragments that have sticky
ends.
•
One of these fragments carries the gene of interest
–
Fragments of human DNA are mixed with clipped
plasmid vectors. The sticky ends of both types pair with
each other
–
Use the DNA ligase to join the DNA molecules by
covalent bonds
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Introduction of cloning vector into cells
•
In this step bacterial cells take up the
recombinant plasmids by transformation. Now
the bacteria that are lacZ- (negative) will be
a mutation that are not able to hydrolyse
lactose are most probably recombinant and
have a foreign DNA.
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Most critical step in cloning; identifying clones that accepted foreign DNA
•
We plate out the transformed bacteria on sold nutrient agar
medium containing ampicillin and a sugar called X-gal
–
Each reproducing bacteria forms a clone that a pears as a
colony on the medium.
–
In this process most of the human genes will be cloned
–
The antibiotic makes us sure that only those bacteria have the
ampR gene will grow while the X-gal makes us identify only the
bacteria that contain the foreign DNA, how?
•
X-gal is hydrolysed by β-galactosidase to produce colonies
with blue color
•
Colonies with blue color means that the lac Z gene was
NOT disrupted, therefore, they did not include the foreign
DNA, while the colonies appear whitish in color, means the
lac Z is not working, i.e it was disrupted by the insert,
therefore, this colony contains the human DNA insert.
(Figure 20. 4)
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Producing Clones of Cells
APPLICATION 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.
TECHNIQUE
In this example, a human gene is inserted into a plasmid from E. coli. The plasmid
contains the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin.
It also contains the lacZ gene, which encodes -galactosidase. This enzyme hydrolyzes
a molecular mimic of lactose (X-gal) to form a blue product. Only three plasmids and
three human DNA fragments are shown, but millions of copies of the plasmid and a
mixture of millions of different human DNA fragments would be present in the samples.
Bacterial cell
1 Isolate plasmid DNA and human DNA.
lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
2 Cut both DNA samples with the same restriction
enzyme
ampR gene
(ampicillin
resistance)
Bacterial
plasmid
Gene of
interest
Sticky
ends
3 Mix the DNAs; they join by base pairing.
The products are recombinant plasmids and
many nonrecombinant plasmids.
Figure 20.4
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Human DNA
fragments
Recombinant DNA plasmids
4 Introduce the DNA into bacterial cells that have a
mutation in their own lacZ gene.
Recombinant
bacteria
5 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
RESULTS
Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form
a colony.
Colonies with nonrecombinant plasmids will be blue, because they can
hydrolyze X-gal. Colonies with recombinant plasmids, in which lacZ is disrupted,
will be white, because they cannot hydrolyze X-gal. By screening the white
colonies with a nucleic acid probe (see Figure 20.5), researchers can identify
clones of bacterial cells carrying the gene of interest.
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How can we identify the bacteria that contain the gene of interest
•
We can look either for the gene itself or we
can look for the protein product of the gene
•
All methods of detecting the gene are based
on the base pairing between the gene and
other a complementary sequence on another
nucleic acid molecule in a process called
DNA
•
Hybridization using a nucleic acid probe
(Figure 20.5).
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Identifying Clones Carrying a Gene of Interest
• The probe is traced by labeling it with a radio label or a
fluorescence tag following the procedure;
–
Bacterial colonies on agar are pressed against special filter thus
transferring the colonies to the filter
–
Filter is treated to break open the cells and denature their DNA,
with the resulting single stranded DNA molecules stick to the filter
–
A solution of probe molecules is incubated with the filter. The
probe DNA hybridizes with any complementary DNA in the filter
–
The filter is laid on a photographic film allowing any radioactive
areas to expose the film
–
The developed film an autoradiograph, is compared with the
mastered culture plate to determine which colonies carry the gene
of interest.
–
Once the clone carrying the gene of interest is identified, cells can
be grown in liquid medium in a large tank to produce large
amounts of the gene or its products such as a protein.
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Nucleic acid probe hybridization
APPLICATION
TECHNIQUE
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.
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.
Colonies
containing
gene of
interest
Master plate
Master plate
Probe
DNA
Solution
containing
probe
Radioactive
single-stranded
DNA
Gene of
interest
Single-stranded
DNA from cell
Filter
Film
Filter lifted and
flipped over
Hybridization
on filter
1 A special filter paper is
pressed against the
master plate,
transferring cells to
the bottom side of the
filter.
RESULTS
Figure 20.5
2 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.
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Storing Cloned Genes in DNA Libraries
• A genomic library made using bacteria
– Is the collection of recombinant vector clones
produced by cloning DNA fragments derived from an
entire genome
Foreign genome
cut up with
restriction
enzyme
or
Recombinant
plasmids
Bacterial
clones
Figure 20.6
(a) Plasmid library
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Recombinant
phage DNA
(b) Phage library
Phage
clones
• The gene cloning procedure outlined in Figure
20-4 is called the shotgun approach as no
single gene is targeted, instead thousands of
different recombinant plasmids are actually
produced and a clone of each ends up in a
white colony.
• The complete set of these clones is called
genomic library, Figure 20-6a. Such library
can be used later for exploring some other
genes or for genome mapping.
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A genomic library made using bacteriophages
– In addition to plasmids, certain bacteriophages
are used as cloning vectors for using even
larger genomic libraries. A genomic library
made using phage is stored as collections of
phage clones (Figure 20-6b).
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A complementary DNA (cDNA) library
– Another type of genomic libraries is called cDNA
library.
– In this, researchers isolate mRNA from the cell that
was transcribed from a number of genes.
– Thus the cDNA library is made of a set of genes that
were transcribed in the starting cells and the new DNA
strand that is produced is called complementary DNA
or cDNA.
– This cDNA represents only part of the genome
– This is advantageous for;
• Studying the genes responsible for specialized functions of
a particular type of cells such as brain or liver cells.
• In addition by making cDNA library from cells of same type at
different stages of life of an organism, researchers can trace
changes in patterns of gene expression.
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Cloning and Expressing Eukaryotic Genes
• As an alternative to screening a DNA library for
a particular nucleotide sequence
– The clones can sometimes be screened for a
desired gene based on detection of its
encoded protein, How?
• Activity of the protein can be measured if for
example it is an enzyme
• Detection of the protein by antibodies.
– Once the protein is identified, it can be
produced in large amounts.
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Bacterial Expression Systems
• Several technical difficulties
– Hinder the expression of cloned eukaryotic genes in
bacterial host cells
• To overcome such problems
– Scientists usually employ an expression vector, a
cloning vector that contains a highly active prokaryotic
promoter that leads to the expression of the eukaryotic
protein.
– The other problem is the presence of introns in the
eukaryotic gene and the un-ability of the prokaryotes to
process RNA so a cDNA of the gene which contains
only the exons should be used in which the bacterial
vector will be able to express.
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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 and the
incompatibility of prokaryotic/eukaryotic system
– Scientists has developed the YAC which combines the
essentials of a eukaryotic chromosome, origin of
replication, a centromere and two telomeres with
foreign DNA.
– YAC can carry a longer DNA segment than the
bacterial plasmid thus the chance to clone the entire
gene is much more than that in plasmids
– Posttranslational modification is another advantage of
using mammalian vector over the plasmid
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How to introduce DNA into cells?
• There are different methods for this purpose
– Electroporation; application of a brief
electrical pulse applied to a solution contains
cells will open membrane holes so that DNA
can inter.
– DNA can be injected directly into a single
eukaryotic cell with the aid of microscope
– In plants, the soil microbe, Agrobacterium can
be used to infect the plant and introduce DNA
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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 and proves to be very useful when DNA is
present in very few copies.
• Items needed for this procedure;
– The DNA to be amplified
– Two short nucleotide primers that determines the DNA
sequence that is amplified
– Nucleotides ( dATP, dCTP, dGTP and dTTP)
– Heat resistant DNA polymerase
– PCR cycler
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Characteristics and uses of PCR
• PCR is so specific to the point that starting material need not be
purified
• It is fast to the point that in about 3-4 hours you can do 40 cycles
and of course amplifying your DNA millions of times
• It can be used to amplify specific gene prior to cloning
• Only minute amounts of DNA are needed to do the amplification
It can be used for;
–
ancient DNA form 40,000 years old frozen woolly mammoth;
–
DNA from tiny amounts of blood, tissue or semen found at a crime
scene.
–
DNA from single embryonic cells fro rapid prenatal diagnosis of
genetic disorders and lots of other applications.
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The PCR procedure
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.
3
Genomic DNA
5
3
5
1 Denaturation:
Heat briefly
to separate
DNA strands
5
3
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.
During each PCR cycle, the target DNA
RESULTS
sequence is doubled. By the end of the third cycle, onefourth of the molecules correspond exactly to the target
sequence, with both strands of the correct length (see
white boxes above). After 20 or so cycles, the target
sequence molecules outnumber all others by a billionfold
or more.
Figure 20.7
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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
Concept 20.2: Restriction fragment analysis
• detects DNA differences that affect restriction
sites
• Can rapidly provide useful comparative
information about DNA sequences
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Gel Electrophoresis and Southern Blotting
• Gel electrophoresis separates DNA restriction fragments of
different lengths
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
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.
charged DNA
molecules move toward the positive electrode, with shorter
Power
source
Gel
Glass
plates
When the current is turned on, the negatively
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
Cathode
Mixture
of DNA
molecules
of different sizes
Anode
Longer
molecules
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
Shorter
molecules
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.
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Figure 20.8
Restriction fragment analysis
• Is useful for comparing two different DNA molecules, such
Normal  -globin allele
as two alleles for a gene
201 bp
175 bp
DdeI
DdeI
Large fragment
DdeI
DdeI
Sickle-cell mutant -globin allele
Large fragment
376 bp
DdeI
(a)
DdeI
DdeI
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)
Figure 20.9a, b
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Electrophoresis of restriction fragments from
normal and sickle-cell alleles.
Southern blotting
an overview
• Identifies Specific DNA fragments in the genome.
• the gel will be overlaid with a nylon or
nitrocellulose membrane and several layers of
filter papers.
• The setting will be submerged with an alkaline
solution that will pull the bands out of the gel and
transfer them to the membrane meanwhile
denaturing the DNA by the action of the alkaline
solution
• After certain time, the filter papers pulled out and
the bands are exposed to a solution containing
radioactive probe which is single stranded DNA
that is complementary to the gene of interest
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• Rinse a way unattached probe and expose membrane to
an X-ray film. In this case the radioactive probe that
hybridized with the gene of interest will expose the film to
form an image corresponding to DNA bands that
hybridized to the probe.
• From looking at the band pattern, it appears that band
patters of sample I and II and III are different from each
other as shown in Figure 20.10.
• This process is called southern blotting after E. M
Southern who developed the method in 1975.
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Southern blotting of DNA fragments
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
Preparation
of restriction fragments.
1
2 Gel electrophoresis.
Figure 20.10
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3 Blotting.
Paper
towels
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).
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Restriction Fragment Length Differences as
Genetic Markers
• Restriction fragment length polymorphisms (RFLPs)
– Are differences in DNA sequences in non-coding
sequences on homologous chromosomes that
result in restriction fragments of different lengths
– They are scattered abundantly through out
genome and serve as a genetic markers for
particular locations (locus) in the genome.
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RFLPs
• Can be detected and analyzed by Southern
blotting (Figure 20.10).
• The thousands of RFLPs present throughout
eukaryotic DNA
– Can serve as genetic markers i.e a measure of
closeness of two loci in a chromosome.
– Because they are inherited in a mendelian
fashion, they can serve as genetic markers for
making linkage maps
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GENOME MAPPING
• Concept 20.3: Entire genomes can be mapped
at the DNA level
• The Human Genome Project
– Sequenced the human genome from 1990-2003
• Scientists have also sequenced genomes of
other organisms
– Providing important insights of general biological
significance
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Genetic (Linkage) Mapping: Relative Ordering of Markers
• The initial stage in mapping a large genome
– Is to construct a linkage map of several
thousand genetic markers spaced throughout
each of the chromosomes
– The order of the markers and the relative
distances between them on such a map are based
on recombination frequencies
– These markers can be genes or RFLPs or
short repetitive sequences (microsatellites).
Based on these, researchers finished the
human genetic map with 5000 markers.
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Three stage approach to mapping the entire genome
Cytogenetic map
Chromosome banding
pattern and location of
specific genes by
fluorescence in situ
hybridization (FISH)
1
2
3
Figure 20.11
Chromosome
bands
Genes located
by FISH
Genetic (linkage)
mapping
Ordering of genetic
markers such as RFLPs,
simple sequence DNA,
and other polymorphisms
(about 200 per chromosome)
Genetic
markers
Physical mapping
Ordering of large overlapping fragments
cloned in YAC and BAC
vectors, followed by
ordering of smaller
fragments cloned in
phage and plasmid
vectors
DNA sequencing
Determination of
nucleotide sequence of
each small fragment and
assembly of the partial
sequences into the complete genome sequence
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Overlapping
fragments
GACTTCATCGGTATCGAACT…
…
Physical Mapping: Ordering DNA Fragments
• A physical map gives the actual distance in base pairs
between markers
– Is constructed by cutting a DNA molecule into many short
fragments and then determine the original order of the
fragments in the DNA
– The key is to make fragments that overlap and then use
probes to identify the overlaps
– Researchers carry out several rounds of DNA cutting,
cloning and physical mapping
– Supplies of DNA fragments used for physical mapping
are prepared by cloning.
– First cloning vector is often YAC or BAC for long DNA
pieces and then smaller plasmids are used for ordering
shorter DNA pieces.
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DNA Sequencing
• Relatively short DNA fragments can be sequenced by
the dideoxy chain-termination method as following;
– Incubate DNA template strand (single) with DNA
polymerase, 4 dioxynucleotides and the 4 flouresent
labelled didioxyribonucleotides
– Synthesis of the new strands starts at the 3’ end and
continues until a ddN is incorporated instead of the
regular dioxyribonucleotide which prevents further
elongation.
–
Eventually a set of labeled strands of various length is
produced with the color of the tag representing the last
nucleotide in sequence.
–
Labeled strands are separated by capillary
electrophoresis with shorter strands moving faster.
–
The color of the tag will be detected by a fluorescence
detector.
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Dideoxy chain-termination method for sequencing DNA
DNA
Primer
(template strand) T
3
APPLICATION
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.
TECHNIQUE
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. 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.
RESULTS
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
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G
T
T
5
C
T
G
A
C
T
T
C
G
A
C
A
A
3
5
3
C
T
G
A
C
T
T
C
G
A
C
A
A
Dideoxyribonucleotides
Deoxyribonucleotides (fluorescently tagged)
5
DNA
polymerase
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
P P P
P P P
G
OH
DNA (template
strand)
ddG
C
T
G
T
T
ddC
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
Detector
G
A
C
T
G
A
A
G
C
H
Labeled strands
Direction
of movement
of strands
Laser
G
3
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
• Linkage mapping, physical mapping, and DNA
sequencing
– Represent the overarching (umbrella) strategy
of the Human Genome Project
• An alternative approach to sequencing whole
genomes starts with the sequencing of random
DNA fragments (approach of Craig Venter
from Celera Genomics)
– Powerful computer programs would then
assemble the resulting very large number of
overlapping short sequences into a single
continuous sequence (Figure 20-13).
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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.
Figure 20.13
ACGATACTGGT
AGTCCGCTATACGA
…ATCGCCATCAGTCCGCTATACGATACTGGTCAA…
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• Concept 20.4: Genome sequences provide
clues to important biological questions such as;
– Genome organization,
– control of gene expression,
– growth and development;
– and evolution.
In genomics
– Scientists study whole sets of genes and their
interactions
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Identifying Protein-Coding Genes in DNA Sequences
• How can we determine a gene and recognize
its function?
• Computer analysis of genome sequences
– Helps researchers identify sequences that are
likely to encode proteins.
– Certain softwares looks for shorts sequences
that are similar to known genes
– Thousands of such sequences are called
Expressed Sequence Tags are catalogued in
computer data base.
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• Current estimates are that the human genome contains
about 25,000 genes which is only 1 and ½ the number of
genes in the fruit fly.
• So What makes humans are more complex than flies or worms?
–
Gene expression is regulated in more subtle and complicated way
than in the flies or worms. The long none coding sequences play
a role in this regulation.
–
Other explanation is that typically one human gene specifies at
least 2 or 3 different polypeptides by using different combinations
of exons.
– In addition it appears that our polypeptides tend to be
more complex than those of invertebrates due to
posttranslational modification.
– Possible interaction between gene products.
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Table 20.1
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Comparison of the sequences of “new” genes
with those of known genes in other species may
help identify new genes
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Determining Gene Function For a gene of unknown function
• Experimental inactivation of the gene and
observation of the resulting phenotypic effects
can provide clues to its function.
– In vitro mutagenesis is one application of this
approach.
• a mutation is introduced into a cloned gene
• The gene is returned to a cell
• If the mutation alter or destroy the gene,
then the phenotype of the cell may help
reveal the function of the missing protein.
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Silencing gene expression
– A simple method to silence gene expression is
by exploiting the phenomenon of RNA
interference (RNAi). However, this approach
has only limited success in humans while
prove to be very efficient in silencing nematode
and fruit flies genes.
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Studying Expression of Interacting Groups of Genes
•
DNA microarray assays allow researchers to
compare patterns of gene expression
–
•
In different tissues, at different times, or
under different conditions
A basic principal to study the gene
expression is done as following;
–
Isolate mRNA made in particular cells
–
Use these mRNA as a template for making
cDNA library using reverse transcription
–
Compare this cDNA with other collections of
DNA by hybridization
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DNA microarray method of studying gene expressions (current method)
•
Tiny amounts of a large number of single
stranded DNA fragments representing different
genes are fixed to a glass slide in a tightly
spaced array (DNA chip).
•
Ideally these fragments represents all the genes
of an organism
•
The fragments are then tested for hybridization
with various samples of cDNA molecules which
has been labeled with fluorescent dyes.
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DNA microarray assay of gene expression levels
With this method, researchers can test thousands of genes
APPLICATION
simultaneously to determine which ones are expressed in a particular tissue,
under different environmental conditions in various disease states, or at
different developmental stages. They can also look for coordinated gene
expression.
Tissue sample
TECHNIQUE
mRNA molecules
1 Isolate
2
3
4
mRNA.
Make cDNA by reverse transcription, using fluores-cently
labeled nucleotides.
Apply the cDNA mixture to a microarray, a microscope slide on which
copies of single-stranded 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 fluorescence.
Each fluorescent spot (yellow) represents a gene expressed
in the tissue sample.
The intensity of fluorescence at each spot is a measure
RESULT
of the expression of the gene represented by that spot in the tissue
sample. Commonly, two different samples are tested together by labeling
the cDNAs prepared from each sample with a differently colored
fluorescence label. The resulting color at a spot reveals the relative levels
of expression of a particular gene in the two samples, which may be from
different tissues or the same tissue under different conditions.
Figure 20.14
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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
– Are providing valuable information in many fields of
biology particularly evolution relations
– The more closely related the genomes of two species the
more closely related in their evolution.
– Comparison of genomes of bacteria, archaea and
eukarya strongly support that these are the three
domains of life.
– Similarities between genomes of disparate species led
some researchers to consider the fruit fly as “little people
with wings”.
– There is 80% similarity between human and mouse
genes can be used in studying human genetic diseases.
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Future Directions in Genomics
• The success in genomics have encouraged
scientist to proceed to the area of proteomics
which the study of full protein sets encoded by
genomes. However, proteomics are challenging as
the numbers of proteins far exceed number of
genes and there are extremely varied in their
structure, chemical and physical properties.
• Application of bioinformatics which is the
application of computer science and mathematics
to genetic and other biological information will play
a crucial role in dealing with the enormous data
obtained.
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Single nucleotide polymorphisms (SNPs)
– Is a single base pair variation in a genome. In
the human genome SNPs occurs at about 1 in
1000 pairs that is if you could compare your
personal DNA sequence with that of a person
setting beside you and with that of some one in
Antarctica you would find them to be 99.9%
identical.
– Provide useful markers for studying human
genetic variation, differences between human
populations and the migratory routs of human
population throughout histroy.
– SNPs and other polymorphisms will be helpful
in identifying disease genes that affect human
health in more subtle way.
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PARACTICAL APPLICATIONS OF DNA TECHNOLOGY
• Concept 20.5: The practical applications of
DNA technology affect our lives in many ways
• Numerous fields are benefiting from DNA
technology and genetic engineering
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Medical Applications
• One obvious benefit of DNA technology
– Is the identification of human genes whose
mutation plays a role in genetic diseases
– In addition, DNA technology also plays an
important role in studying none genetic
diseases such as AIDS and Arthritis.
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Diagnosis of Diseases
•
Medical scientists can now diagnose hundreds of
human genetic disorders
–
PCR, RT-PCR and DNA hybridization has opened a
door for the diagnosis of infectious agents such as
HIV, and other elusive organisms.
–
Hundreds of human genetic disorders were
diagnosed using DNA technology
–
It is possible to diagnose such diseases before onset
or even before birth.
–
It is also possible to diagnose symptomless carriers
of potentially harmful recessive alleles.
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Diagnosis of Diseases… cont.
– It is possible to find an abnormal allele of a
gene that has not been cloned if a closely
linked RFLP marker has been found
particularly if they are located close to each
other so that no crossing over happens while
the gamete formation. i.e they will be inherited
together. Figure 20-15.
– Genes have been cloned for many human
diseases including hemophilia, cystic fibrosis,
Duchenne muscular dystrophy, huntingtons,
sickle cell disease.
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This diagram depicts homologous segments of DNA from a family in
which some members have a genetic disease. In this family
different versions of RFLP marker are found in unaffected family
members and in those who exhibit the disease. If a family
member has inherited the version of the RFLP marker with two
restriction sites near the gene ( rather than one) there a high
probability that the individual has also inherited the disease
causing allele.
RFLP marker
DNA
Restriction
sites
Disease-causing
allele
Normal allele
Figure 20.15
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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 (reteroviral) for delivery
of the normal allele into somatic cells
– For gene therapy of somatic cells to be
permanent, the cells that receive the normal
allele must be one that multiply for life such as
the bone marrow cells.
– Unfortunately, so far the inserted genes were
found to be effective only for short time.
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Gene therapy using a retroviral vector
Cloned gene (normal
allele, absent from
patient’s cells)
1 Insert RNA version of normal allele
into retrovirus.
Viral RNA
2
2 Let retrovirus infect bone marrow cells
Retrovirus
capsid
that have been removed from the
patient and cultured.
3 Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
Figure 20.16
4 Inject engineered
cells into patient.
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• Several trials were conducted to treat 10 children with
SCID with some success. Nine of them showed some
success however, two of them developed leukemia as the
reterovirus inserted the genes near a gene that is involved
in the proliferation and development of blood cells that led
to leukemia.
• There are some other questions need to be asked too
about gene therapy such as what guarantees that the
gene will be placed in the right place and produce the
exact protein and does not disrupt other genes.
• Further, there is some ethical issues regarding
tampering with human genome in a way that leads to
the practice of eugenics, a deliberate effort to control
human gene make up.
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Pharmaceutical Products
– Large-scale production of human hormones and
other proteins with therapeutic uses such as
insulin, Tissue plasminogen factor, human
growth hormone.
– Production of safer vaccines (Recombinant
proteins) than the traditional vaccines which are
either heat killed pathogens or viable but
attenuated pathogens
– Production of genetically engineered proteins
that either block or mimic receptors on cell
membranes.
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Forensic Evidence
• 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 with high degree of certainty as
the DNA of every person is unique except for
identical twins.
– Traditional methods, such as ELISA and blood
typing needs fresh blood and cannot give a
definitive answer that a person did it, but can
exclude some body.
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• RFLP of samples obtained at the crime scene
will be analyzed by southern blotting that
allows researchers to detect similarities and
differences in DNA and requires only tinny
amounts (1000 blood cells).
• Radioactive probes marks the electrophoresis
bands that contain certain RFLP markers. 5
markers usually are enough, in other words,
only a small portion of DNA should be enough.
• Figure 20-17 shows a real example of samples
collected from crime scenes.
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A DNA fingerprint from crime scene samples
Defendant’s
blood (D)
Blood from
defendant’s
clothes
4 g
D
Jeans
Figure 20.17
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Victim’s
blood (V)
8 g
shirt
V
DNA fingerprinting for establishing paternity
– Another use for the DNA finger printing is
comparing DNA from a child, a mother and
purported father which can for sure settle a
question of paternity.
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Satellite DNA or short repetitive sequences
• is another new technique that is increasingly used
as a DNA fingerprint. The most useful satellite
sequences for forensic purposes are the
microsatellites ranging from 10-100 bp long, have
repeating units of only few bp and highly variable
from person to person.
• Example; one person might have the unit ACA
repeated 65 times at one locus of a gene and 118
times at another locus, and so on. Now it is highly
unlikely that another person will have the exact
same arrangement. Such polymorphic genetic loci
are called simple tandem repeats (STRs).
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• PCR is usually used to amplify certain STRs or
other markers before electrophoresis
particularly when the amount of DNA recovered
is very minute.
• What is the probability that two people have
identical DNA fingerprints? It is 1in 100,000 to
1in a billion. That depends on the number of
markers compared and on the frequency of
these markers in the population.
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Environmental Cleanup
• Genetic engineering can be used to modify the
metabolism of microorganisms so that they can be
used to extract minerals (cu, S, Ni, lead) from the
environment or degrade various types of
potentially toxic waste materials such as
chlorinated hydrocarbons that are used as
pesticides while other bacteria able to degrade oil
spills at the sea.
• Likewise, sewage treatment plants rely heavily on
microbes to degrade many organic compounds
into non-toxic form before these compounds are
released to the environment.
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Agricultural Applications
• DNA technology
– Is being used to improve agricultural productivity
and food quality
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Animal Husbandry and “Pharm” Animals
Transgenic animals contain genes from other organisms.
Goals of creating transgenic animals (examples)
–
Producing sheep with better quality wool
–
Producing pigs with leaner meat
–
Producing cows that mature faster than the normal ones
–
Engineering a transgenic animal that produces a
pharmaceutical drug that gets secreted in the milk such
as a hormone or an anti-clotting factor… etc. and are
used to produce vaccines, hormones and pharmaceutical
proteins.
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How to make transgenic animals?
–
Remove egg cells from a female and fertilize
them in vitro
–
At the same time clone the desired gene from
another organism
–
Inject the cloned DNA directly into the nuclei of
the eggs so that some of the cells integrate the
inserted DNA into their genomes
–
The engineered eggs are then surgically
implanted into surrogate mothers
–
If the pregnancy happened, the result might be
a transgenic animal
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Pharm Animals
• These animals contain genes for a human blood
protein which they secrete in their milk
Figure 20.18
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Genetic Engineering in Plants
• Agricultural scientists
– Have already endowed a number of crop
plants with genes for desirable traits such as
delayed ripening and resistance to spoilage
and disease. In addition, engineered plants are
now produces human proteins for therapeutic
purposes or antibodies for diagnosis.
– Plants are easily engineered than animals
because a single plant cell can give rise to a
whole plant, thus genetic manipulation can be
done on only one single cell and let grow to a
full grown tree.
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Ti plasmid from soil bacteria Agrobacterium tumefaciens.
–
Is the most commonly used vector for introducing new genes into plant
cells
Genes conferring useful traits, such as pest
APPLICATION 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.
Example !
TECHNIQUE
1
2
3
RESULTS
Agrobacterium tumefaciens
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.
Site where
restriction
enzyme cuts
Ti
plasmid
T DNA
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.
Transformed cells carrying the transgene of interest can
regenerate complete plants that exhibit the new trait
conferred by the transgene.
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Plant with
new trait
Figure 20.19
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.
•
There should be certain safety measures to control
such potential hazard;
–
Protecting researchers from infection by engineered
microbes and to prevent microbes from accidentally
leaving the laboratory.
–
Strains to be used for Recombinant DNA technology
should be genetically crippled to insure that they can
not survive outside the lab.
–
Certain obvious dangerous experiments have to be
banned.
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• Today, most public concern about possible hazards
– Centers on genetically modified organisms
(GMOs) used as food however, GM food must be
included in the food label thus enabling the
consumer to decide whether to buy them or not.
– In year 2000, 130 countries signed an agreement
to label the bulk food shipments if they have
GMOs or not.
– Why the big fuzz about GM Foods and GMOs?
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• One argument is what if a wild plant acquired a
herbicidal resistance gene form a house plant and
spread this resistance to wide areas producing super
weeds?! AS of yet nothing like this happen.
• For humans some think that some genetically
modified proteins might be allergic.
• Completing the sequence of the entire human genome
poses some ethical issue to who should be allowed to
know somebody’s genetic make up or should these
information be used as a criteria for job selection…..
etc.
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End of Chapter 20
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