Download DNA Sequencing

Chapter 20
DNA Technology
and Genomics
PowerPoint Lectures for
Biology, Seventh Edition
Neil Campbell and Jane Reece
Lectures by Chris Romero
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Overview: Understanding and Manipulating Genomes
• 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
– DNA sequencing explained
– Sanger sequencing
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• DNA technology has launched a revolution in
the area of biotechnology
– The manipulation of organisms or their genetic
components to make useful products
• An example of DNA technology is the
microarray
– A measurement of gene expression of
thousands of different genes, all at once
– Also called a DNA chip see how it works
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microarray
Uses complementary probes (T,A,C,G)
Figure 20.1
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DNA Cloning
• 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 and their plasmids
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• Overview of gene cloning with a bacterial
plasmid, showing various uses of cloned genes
Bacterium
Cell containing gene
1 Gene inserted of interest
into plasmid
Plasmid
Bacterial
chromosome
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
Protein harvested
Copies of gene
Basic
research
on gene
Figure 20.2
DNA of
chromosome
4 Basic research and
various applications
Basic
research
on protein
Gene used to alter
Human growth
Gene for pest
Protein dissolves
resistance inserted bacteria for cleaning blood clots in heart hormone treats
up toxic waste
stunted growth
into plants
attack therapy
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Using Restriction Enzymes to Make Recombinant DNA
• Bacterial restriction enzymes
– Cut DNA molecules at a limited number of
specific DNA sequences, called restriction
sites
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• A restriction enzyme will usually make many cuts in
a DNA molecule restriction.exe
– Yielding a set of restriction fragments
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• Using a restriction enzyme and DNA ligase to
make recombinant DNA
Restriction site
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 5
3
1 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.
G AATT C
C TTAA G
G
G AATTC
CTTAA G
One possible combination
3 DNA ligase
Recombinant DNA molecule
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G
Fragment from different
DNA molecule cut by the
same restriction enzyme
seals the strands.
Figure 20.3
DNA ligase is an
enzyme that seals
the bonds
between
restriction
fragments
3
5
GAATTC
CTTAAG
Using Bacterial transformation to insert genes
• Transformation 1
• Transformation 2
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Cloning a Eukaryotic 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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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.
1 Isolate plasmid DNA and human DNA.
2 Cut both DNA samples with the same restriction
enzyme
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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lacZ gene
(lactose
Human
breakdown) cell
Bacterial cell
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid
Gene of
interest
Sticky
ends
Recombinant DNA plasmids
Human DNA
fragments
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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Identifying Clones Carrying a Gene of Interest
• A clone carrying the gene of interest
– Can be identified with a radioactively labeled
nucleic acid probe that has a sequence
complementary to the gene, a process called
nucleic acid hybridization
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• 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
Figure 20.5
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.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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
Phage
clones
(b) Phage library
• A genomic library made using bacteriophages
– Is stored as a collection of phage clones
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Complementary DNA (cDNA) library
Is made by cloning DNA made in vitro by reverse
transcription of all the mRNA produced by a particular
cell
The advantage of cDNA library is that it contains only the
coding region of a genome.
–
To prepare a cDNA library, the first step is to isolate the total
mRNA from the cell type of interest.
–
Because eukaryotic mRNAs consist of a poly-A tail, they can
easily be separated.
–
Then the enzyme reverse transcriptase is used to synthesize a
DNA strand complementary to each mRNA molecule.
–
After the single-stranded DNA molecules are converted into
double-stranded DNA molecules by DNA polymerase, they are
inserted into vectors and cloned.
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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
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Bacterial Expression Systems
• Several technical difficulties
– Hinder the expression of cloned eukaryotic
genes in bacterial (prokaryotic) 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
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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
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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
– Uses primers that bracket the desired
sequence
– Uses a heat-resistant DNA polymerase
– pcr.exe
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http://users.ugent.be/~avierstr/principles/pcrani.html
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• The PCR procedure
5
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 (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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3
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
Concept 20.2: Restriction fragment analysis
• RFLP detects DNA differences that affect
restriction sites, because they “cut” differently if
there are nucleotide variations
• Restriction fragment analysis
– Can rapidly provide useful comparative
information about DNA sequences
– Cut up the DNA region of interest using
restriction enzymes, run it through gel
electrophoresis
– gelelectrophoresis.exe
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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).
Cathode
1 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.
Power
source
Mixture
of DNA
molecules
of different sizes
Gel
Glass
plates
2 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
RESULTS
Figure 20.8
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.
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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Anode
Longer
molecules
Shorter
molecules
• Restriction fragment analysis
– Is useful for comparing two different DNA
molecules, such as two alleles for a gene
Normal  -globin allele
201 bp
175 bp
DdeI
DdeI
Large fragment
DdeI
DdeI
Sickle-cell mutant -globin allele
Large fragment
376 bp
DdeI
DdeI
DdeI
(a) DdeI restriction sites in normal and sickle-cell alleles of
-globin gene.
Normal
allele
Sickle-cell
allele
Large
fragment
376 bp
201 bp
175 bp
Figure 20.9a, b
(b) Electrophoresis of restriction fragments from normal and
sickle-cell alleles.
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• Specific DNA fragments can be identified by
Southern blotting
– Using labeled probes that hybridize to the DNA
immobilized on a “blot” of the gel
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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
1 Preparation of restriction fragments.
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 on homologous
chromosomes that result in restriction fragments of different
lengths
–
Polymorphisms are inherited differences found among the
individuals in a population.
• RFLPs have provided valuable information in many areas
of biology, including:
–
screening human DNA for the presence of potentially
deleterious genes
–
providing evidence to establish the innocence of, or a
probability of the guilt of, a crime suspect by DNA
"fingerprinting“
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RFLPs.html
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RFLPs
• Specific fragments
– Can be detected and analyzed by Southern
blotting
• The thousands of RFLPs present throughout
eukaryotic DNA
– Can serve as genetic markers
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Concept 20.3: Entire genomes can be mapped at the DNA level
• The Human Genome Project
– Sequenced the human genome
• 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
Cytogenetic map
Chromosome banding
pattern and location of
specific genes by
fluorescence in situ
hybridization (FISH)
Chromosome
bands
Genes located
by FISH
1 Genetic (linkage)
mapping
Ordering of genetic
markers such as RFLPs,
simple sequence DNA,
and other polymorphisms
(about 200 per chromosome)
2 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
3
Figure 20.11
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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Genetic
markers
Overlapping
fragments
…GACTTCATCGGTATCGAACT…
• The order of the markers and the relative
distances between them on such a map
– Are based on recombination frequencies
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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
– Gives the actual distance in base pairs
between markers
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DNA Sequencing
• Relatively short DNA fragments
– Can be sequenced by the dideoxy chaintermination method
– http://www.pbs.org/wgbh/nova/genome/sequencer.
html#
– cycseq.exe
– http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/
sequencing.html
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• Dideoxy chain-termination method for
sequencing DNA
DNA
(template strand)
5 C
T
G
A
C
T
T
C
G
A
C
A
A
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 (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.
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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3
5
3
C
T
G
A
C
T
T
C
G
A
C
A
A
Primer
3
T
G
T
T
Deoxyribonucleotides Dideoxyribonucleotides
(fluorescently tagged)
5
DNA
polymerase
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
P P P
P P P
G
OH
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)
ddC
T
G
T
T
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
• Linkage mapping, physical mapping, and DNA
sequencing
– Represent the overarching strategy of the
Human Genome Project
• An alternative approach to sequencing whole
genomes starts with the sequencing of random
DNA fragments
– Powerful computer programs would then
assemble the resulting very large number of
overlapping short sequences into a single
continuous sequence
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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
ACGATACTGGT
fragment
CGCCATCAGT
4 Order the
AGTCCGCTATACGA
sequences into one
overall sequence
with computer
software.
Figure 20.13
ACGATACTGGT
…ATCGCCATCAGTCCGCTATACGATACTGGTCAA…
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Concept 20.4: Genome sequences provide clues to important biological questions
• In genomics
– Scientists study whole sets of genes and their
interactions
– This has tapped into the need to store and analyze
massive amounts of data using computers,
technology: Bioinformatics
• Computer analysis of genome sequences
– Identifying Protein-Coding Genes in DNA
Sequences
• Helps researchers identify sequences that are
likely to encode proteins
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• Current estimates are that the human genome
contains about 25,000 genes
– But the number of human proteins is much
larger
Table 20.1
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Comparing Genomes of Different Species
• Comparative studies of genomes from related
and widely divergent species
– Are providing valuable information in many
fields of biology
• Comparison of the sequences of “new” genes
– With those of known genes in other species
may help identify new genes
– Some favored models: model_organisms.exe
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Determining Gene Function and location
• For a gene of interest (known or unknown
function)
– Can be identified using FISH (fluorescent in
situ hybridization) to “see” where a specific
section of RNA or DNA is in a cell
http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Baxter/Molecular
Tool.html
Human Papillomavirus DNA demonstrated by In Situ
Hybridization (pink) in epithelial cells identified by
indirect immunofluorescence using antibody against
cytokeratin (green)
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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
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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
– DNA chips dnachip.exe
– http://www.bio.davidson.edu/courses/genomics
/chip/chip.html
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• DNA microarray assay of gene expression levels
APPLICATION With this method, researchers can test thousands of genes 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 mRNA.
2 Make cDNA by reverse transcription, using fluores-cently labeled nucleotides.
Labeled cDNA molecules
(single strands)
3 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.
4 Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent
spot (yellow) represents a gene expressed in the tissue sample.
DNA
microarray
RESULT
The intensity of fluorescence at each spot is a measure 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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Size of an actual
DNA microarray
with all the genes
of yeast (6,400
spots)
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)
– Provide useful markers for studying human
genetic variation
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• 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
• Diagnosis of 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 diseasecausing mutation
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• 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
Figure 20.15
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Human Gene Therapy
• 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
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• Gene therapy using a retroviral vector
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
Figure 20.16
4 Inject engineered
cells into patient.
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Pharmaceutical Products
Applications of DNA technology include
•
Large-scale production of human hormones and other proteins with
therapeutic uses
•
Production of safer vaccines
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Forensic Evidence
• DNA “fingerprints” obtained
by analysis of tissue or
body fluids found at crime
scenes
Defendant’s
blood (D)
– Is a specific pattern of
bands of RFLP markers
on a gel
Figure 20.17
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defendant’s
clothes
4 g
D
– Can provide definitive
evidence that a suspect
is guilty or, more
specifically, not guilty
Blood from
Jeans
Victim’s
blood (V)
8 g
shirt
V
DNA fingerprinting
Can also be used in
establishing paternity
Figure: Electrophoresis of PCRamplified DNA fragments. (1)
Father. (2) Child. (3) Mother. The
child has inherited some, but not
all of the fingerprint of each of its
parents, giving it a new, unique
fingerprint.
http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Paternity_testing
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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
from the environment or degrade various types
of potentially toxic waste materials
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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
– Sometimes called “chimeras”
–
Fig 1. transgenic mouse lines expressing GFP known as “green
mice.”
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Animal Husbandry and “Pharm” Animals
• “Knockout” mice
http://www.nca-nl.org/English/Newsletters/Nb13/nl13txt.html
Since cancer is a multistage process, it is obvious that transgenic or knock out animals, which have
already undergone one step in the cancer process, may be a sensitive alternative for the
standard bioassay.
A number of mice models have been developed: either possessing an inactivated tumor suppressor
gene (p53), an activated oncogene (Tg.AC), over-expression of a (human) oncogene (rasH2)
or being deficient in nucleotide excision repair (Xpa, de Vries et al., 1995).
These mice models have several advantages:
•
the number of animals needed for one study is 120 instead of 400-500
•
the duration of the study is 6-9 instead of 24 months leading to less distress of the animals
and
•
the transgenic mouse model is considered more discriminating hence improving the accuracy
and reliability of human carcinogen identification.
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– Have been engineered to be pharmaceutical
“factories”
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
Bt corn (right)
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• 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
Figure 20.19
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.
2
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.
3
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.
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Agrobacterium tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
DNA with
the gene
of interest
Recombinant
Ti plasmid
Plant with
new trait
T DNA
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
– Centers on genetically modified (GMOs)
organisms used as food
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