Genetics & Evolution Series: Extra Set
Copyright © 2005
Version: 2.0
What is Gene Technology?
Gene technology is a broad field which includes analysis of DNA
as well as genetic engineering and other forms of genetic
modification.
Genetic engineering refers the artificial manipulation of genes:
adding or subtracting genes, or changing the way genes work.
Organisms with artificially altered DNA are referred to as
genetically modified organisms (GMOs).
Gene technologies have great
potential to benefit humanity through:
increasing crop production
increasing livestock production
preventing and fighting disease
reducing pollution and waste
producing new products
detecting and preventing crime
Why Gene Technology?
Despite potential benefits, gene technology is highly controversial.
Some people feel very strongly that safety concerns associated
with the technology have not been adequately addressed.
Environmentally friendly
Could improve the
sustainability of crop and
livestock production
Could potentially benefit
the health of many
More predictable and
directed than
selective breeding
Who owns and regulates
the GMOs?
Third world economies are
at risk of exploitation
Biological risks have not
been adequately addressed
Animal ethics issues
The costs of errors
Photos courtesy of GreenPeace
The Beginning of GE
Genetic engineering (GE) was made possible
by the discovery of new techniques and tools in
the 1970s and 1980s.
It builds on traditional methods of genetic
manipulation, including selective breeding programs
and the deliberate introduction of novel traits by
exposing organisms (particularly plants) to mutagens.
Methods were developed to insert ‘foreign’ DNA
into cells using vectors. New recombinant DNA
technology involved ‘recombining’ DNA from
different individuals and even different species.
Organisms such as bacteria, viruses, and
yeasts are used to propagate recombinant
genes and/or transfer genes to target cells (cells
that receive the new DNA).
The bacterium Escherichia
coli (above) and the yeast
Saccharomyces cerevisiae
(below): favorite organisms
of gene research
Producing GMOs
GMOs may be created by modifying
their DNA in one of three ways:
Adding a Foreign Gene
A foreign gene is added which will enable the GMO
to carry out a new genetic program. Organisms
altered in this way are referred to as transgenic.
Alter an Existing Gene
An existing gene already present in the organism
may be altered to make it express at a higher level
(e.g. growth hormone) or in a different way (in tissue
that would not normally express it). This method is
also used for gene therapy.
Delete or ‘Turn Off’ a Gene
An existing gene may be deleted or deactivated
to prevent the expression of a trait (e.g. the
deactivation of the ripening gene in tomatoes).
Host DNA
Existing gene altered
Host DNA
Host DNA
Restriction Enzymes
Restriction enzymes are one of the essential tools of genetic
engineering. Purified forms of these naturally occurring bacterial
enzymes are used as “molecular scalpels”, allowing genetic
engineers to cut up DNA in a controlled way.
Restriction enzymes are used to cut DNA molecules at very precise
sequences of 4 to 8 base pairs called recognition sites (see below).
By using a ‘tool kit’ of over 400 restriction enzymes recognizing about 100
recognition sites, genetic engineers are able to isolate and sequence
DNA, and manipulate individual genes derived from any type of organism.
Recognition Site
Recognition Site
cut
The restriction enzyme
EcoRI cuts here
GAATTC
GAATTC
DNA
CTTAAG
CTTAAG
cut
cut
Specific Recognition Sites
Restriction enzymes are named according to the bacterial species
they were first isolated from, followed by a number to distinguish
different enzymes isolated from the same organism.
e.g. BamHI was isolated from the bacteria Bacillus amyloliquefaciens strain H.
A restriction enzyme cuts the double-stranded DNA molecule at its
specific recognition site:
Enzyme
Source
Recognition Sites
EcoRI
Escherichia coli RY13
GAATTC
BamHI
Bacillus amyloliquefaciens H
GGATCC
HaeIII
Haemophilus aegyptius
GGCC
HindIII
Haemophilus influenzae Rd
AAGCTT
Hpal
Haemophilus parainfluenzae
GTTAAC
HpaII
Haemophilus parainfluenzae
CCGG
MboI
Moraxella bovis
GATC
NotI
Norcardia otitidis-caviarum
GCGGCCGC
TaqI
Thermus aquaticus
TCGA
Sticky Ends
A restriction enzyme cuts the double-stranded
DNA molecule at its specific recognition site
It is possible to use
restriction enzymes
that cut leaving an
overhang; a so-called
“sticky end”.
Fragment
GAAT T C
GAAT T C
C T TAA G
C T TAA G
DNA cut in such a way Restriction enzyme: EcoRI
produces ends which
may only be joined to A A T T C
G
other sticky ends
with a complementary
G
C T TAA
DNA from
base sequence.
another source
See steps 1-3
opposite:
Restriction
enzyme: EcoRI
Sticky end
The cuts produce a
DNA fragment with
two “sticky” ends
The two different fragments cut
by the same restriction enzyme
have identical sticky ends and
are able to join together
When two fragments of DNA cut by the same restriction
enzyme come together, they can join by base-pairing
Blunt Ends
Recognition Site
It is possible to use
restriction enzymes that
cut leaving no overhang;
a so-called “blunt end”.
DNA cut in such a way is
able to be joined to any
other blunt end fragment,
but tends to be nonspecific because there
are no sticky ends as
recognition sites.
A special group of
enzymes can join
the pieces together
Recognition Site
C C CG G G
C C CG G G
G G GC C C
G G GC C C
DNA
Restriction enzyme
cut
cut
cuts here
The cut by this type of restriction
enzyme leaves no overhang
CCC
GGG
GGG
CCC
GGG
CCC
CCC
GGG
DNA from another source
Ligation
DNA fragments produced using restriction enzymes may be
reassembled by a process called ligation.
Pieces of DNA are joined together using an enzyme called DNA ligase.
DNA of different origins produced in this way is called recombinant DNA
because it is DNA that has been recombined from different sources.
Steps 1-3 are involved in creating a recombinant DNA plasmid:
Two pieces of DNA
are cut using the
same restriction
enzyme.
Plasmid
DNA
fragment
This other end of the
foreign DNA is attracted
to the remaining sticky
end of the plasmid.
The two different DNA
fragments are attracted to each
other by weak hydrogen bonds.
AAT T C
G
G Foreign DNA fragment
C T TAA
Annealing
When the two matching “sticky ends” come together, they join by
base pairing. This process is called annealing.
This can allow DNA fragments from a different source, perhaps a
plasmid, to be joined to the DNA fragment.
The joined fragments will usually form either a linear molecule or a
circular one, as shown here for a plasmid.
Detail of Restriction Site
Plasmid
DNA
fragment
Restriction sites on the
fragments are attracted
by base pairing only
Gap in DNA
molecule’s
‘backbone’
Foreign
DNA
fragment
Recombinant DNA Plasmid
The fragments of DNA are joined together by the enzyme DNA
ligase, producing a molecule of recombinant DNA.
These combined techniques of using restriction enzymes and
ligation are the basic tools of genetic engineering.
DNA ligase
Detail of Restriction Site
Recombinant
Plasmid DNA
Fragments linked
permanently by
DNA ligase
No break in
DNA molecule
The fragments are able to
join together under the
influence of DNA ligase.
DNA Amplification
Using the technique called polymerase chain reaction (PCR),
researchers are able to create vast quantities of DNA identical to
trace samples. This process is also known as DNA amplification.
Many procedures in DNA technology
require substantial amounts of DNA to
work with, for example;
A crime scene
(body tissue samples)
DNA sequencing
DNA profiling/fingerprinting
Gene cloning
A single viral particle
(from an infection)
Transformation
Making artificial genes
Samples from some sources,
including those shown here,
may be difficult to obtain in
any quantity.
Fragments of DNA from
a long extinct animal
PCR Equipment
Amplification of DNA can be carried out with simple-to-use PCR
machines called thermal cyclers (shown below).
Thermal cyclers are in common use in the biology departments of
universities as well as other kinds of research and analytical laboratories.
Steps in the PCR Process
The laboratory process
called the polymerase
chain reaction or PCR
involves the following
steps 1-3 each cycle:
Separate Strands
Separate the target DNA strands
by heating at 98°C for 5
minutes
Add Reaction Mix
Add primers (short RNA strands
that provide a starting sequence
for DNA replication), nucleotides
(A, T, G and C) and DNA
polymerase enzyme.
Incubate
Cool to 60°C and incubate for a few
minutes. During this time, primers
attach to single-stranded DNA. DNA
polymerase synthesizes
complementary strands.
Repeat for about 25
cycles
Repeat cycle of heating
and cooling until enough
copies of the target DNA
have been produced.
Polymerase Chain Reaction
Although only three
cycles of replication
are shown here,
following cycles
replicate DNA at an
exponential rate
and can make
literally billions of
copies in only a few
hours.
Original
DNASample
Cycle 1
Cycle 2
The process of PCR
is detailed in the
following slide
sequence
of steps 1-5.
Cycle 3
PCR
cycles
No. of target
DNA strands
1
2
2
4
3
8
4
16
5
32
6
64
7
128
8
256
9
512
10
1024
11
2048
12
4096
13
8192
14
16 384
15
32 768
16
65 536
17
131 072
18
262 144
19
524 288
20
1 048 576
21
2 097 152
22
4 194 304
23
8 388 608
24
16 777 216
25
33 554 432
The Process of PCR 1
A DNA sample called the
target DNA is obtained
DNA is denatured (DNA strands
are separated) by heating the
sample for 5 minutes at 98C
Primers (short strands of mRNA)
are annealed (bonded) to the DNA
Primer annealed
The Process of PCR 2
Nucleotides
The sample is cooled to 60°C.
A thermally stable DNA
polymerase enzyme binds to
the primers on each side of the
exposed DNA strand.
This enzyme synthesizes a
complementary strand of DNA
using free nucleotides.
After one cycle, there are now
two copies of the original sample.
Nucleotides
Gel Electrophoresis
A technique known as gel
electrophoresis can be used
to separate large molecules
(including nucleic acids or
proteins) on the basis of their
size, electric charge, and other
physical properties.
Cathode
Sample
Wells into which samples
to be analyzed are placed.
Buffer
Plastic Frame
To prepare DNA for
electrophoresis, the DNA is
often cut up into smaller
pieces. Called a restriction
digest, and it produces a
range of DNA of different
lengths.
To carry out electrophoresis,
the DNA samples are placed
in wells and covered with a
buffer solution that gradually
dissolves them into solution.
Anode
DNA fragments, shown symbolically
above, move towards the positive
terminal (smaller fragments move
faster than longer ones).
Gel
Buffer solution
Analyzing DNA
By applying an electric field to the solution, the molecules move towards
one or other electrode depending on the charge on the molecule itself. DNA
is negatively charged because the phosphates have a negative charge.
Molecules of different
sizes (molecular weights)
become separated
(spread out) on the
gel surface.
These can be visualized
by applying dyes or
radio-labeled probes.
Wells: Holes created
in the gel with a comb.
-ve terminal
DNA solutions:
Mixtures of
different sizes of
DNA fragments
are loaded into
each well.
DNA fragments:
The gel matrix
acts as a seive
for the DNA
molecules.
Large fragments
Small fragments
+ve terminal
Tray: Contains the set gel.
DNA markers:
A mixture of DNA
molecules with
known molecular
weights. They are
used to estimate
the sizes of the
DNA fragments in
the sample lanes.
DNA Profiling
DNA profiling (DNA fingerprinting) is a technique for genetic analysis,
which identifies the variations found in the DNA of every individual.
The profile refers to the distinctive
pattern of DNA restriction fragments
or PCR products which is used to
identify an individual.
There are different methods of DNA
profiling, each with benefits and drawbacks.
DNA profiling does not determine a base
sequence for a sample but merely sorts
variations in base sequences.
Only one in a billion (i.e. a thousand
million) persons is likely to have an
identical DNA profile, making it a useful
tool for forensic investigations and
paternity analysis.
Visualizing the Profile
DNA fragments (PCR product after endonuclease digestion) visualized
under UV light after staining with ethidium bromide and migration in an
agarose electrophoresis gel.
DNA Profiling Methods
DNA profiling begins by extracting DNA from the cells in
a sample of blood, saliva, semen, or other fluid or tissue.
Two methods are commonly used. Both are based on
the analysis of short repetitive sequences in the DNA.
Profiling using probes (RFLP analysis) was the first profiling
technique to be developed. Restriction enzymes are applied to
a DNA sample and the DNA fragments are separated on a gel.
Radioactive probes are used to label DNA fragments with
complementary sequences.
Profiling using PCR is newer technique which uses highly
polymorphic regions of DNA that have short repeated
sequences of DNA. These sequences are amplified using PCR
and then separated on a gel.
This technique is suitable when there is very little DNA
available or the sample is old.
Uses of DNA Profiling
DNA profiling can be used for investigating:
the presence of a particular gene,
such as cystic fibrosis) in a family.
genetic relatedness of different organisms
e.g. checking on pedigree in stock
breeding programs.
e.g. checking that captive populations of
endangered species are not inbred.
DNA Profiling Using Probes
The most commonly used manual method of DNA profiling is called
the Southern blot. It uses the older technology of DNA probes and
another type of repeat sequence to that used in profiling with PCR.
The repeat sequences are called
minisatellites or variable tandem
repeats (VNTRs) and comprise
repeating units of a few tens
of nucleotides long.
Equivalent sequences in different
people have the same core sequence
of 10-15 bases (to which a DNA
probe is attached), but thereafter the
patterns vary considerable in
length from one person to the next.
Southern Blotting Method
Steps 1-7 show the DNA profiling procedure called the Southern blot.
Gene of
interest
Cut Up DNA
The DNA is cut up into fragments
using restriction enzymes,
yielding thousands of fragments
of all different sizes.
Extract DNA From Sample
A sample collected from the tissue of a
living or dead organism is treated with
chemicals and enzymes to extract the
DNA, which is separated and purified.
Buffer solution
Separate Fragments
+ve
terminal
-ve
terminal
The fragments are separated by
length, using electrophoresis.
DNA, which is negatively charged,
moves toward the positive terminal.
The smaller fragments travel faster
than longer ones.
Gel
DNA fragments
Southern Blotting Method
Transfer DNA Fragments to Filter Sheet
DNA molecules are split into single strands using alkaline
chemicals. The DNA is transferred onto a nitrocellulose
filter sheet by pressing it against the gel.
Paper towels
Filter sheet
Gel
The salt solution passes
through the gel, carrying the
DNA fragments onto the
surface of the filter sheet.
Sponge
Remove Filter Sheet
The gel with filter sheet still attached
is removed and separated. The DNA
fragments that have now moved to
the filter sheet are in exactly the
same position as on the gel.
Filter
Tray containing
salt solution
DNA transferred
to filter
Gel
Southern Blotting Method
Attach Radioactively Labeled Probes
The filter sheet is immersed in a bath with radioactive
probes; synthetic complementary DNA.
Many thousands of these segments bind to the sample
DNA fragments where they are localized as bands.
Radioactive
probes
Bands
Developed X-ray film
Create Autoradiograph
The filter sheet is exposed to X-ray film. The
radioactive probes attached to the sorted
fragments show up as dark bands on the film.
The spacing of these bands is the DNA
fingerprint, and can be used as evidence.
DNA fingerprint
Forensic DNA Evidence
DNA fingerprints from tissue
samples can be used as
evidence in the same way
traditional fingerprinting is used.
Which DNA fingerprint from the
three suspects matches that of
the tissue sample submitted as
evidence?
Why would the DNA from the
victim be included in this test?
How DNA Probes Work
Artificially constructed DNA probes work by binding to a
specific sequence on DNA that is of interest to the investigator.
A DNA probe is a small fragment of nucleic
acid (either cloned or artificially synthesized),
that is labeled with an enzyme, a radioactive
tag, or a fluorescent dye tag.
Fluorescent dye tag: Shows up
as fluorescent bands when gel is
exposed to ultraviolet light source.
or
Radioactive tag: Shows up
as a dark band when the gel is
exposed to photographic film.
Under appropriate conditions, the probe
will bind to a complementary DNA
sequence by base pairing, identifying
the presence and location of the target
DNA sequence for further analysis.
Target DNA strand (such as a tandem
repeat) with a complementary sequence
that is being searched for by the probe.
What Gene Probes Do
Gene probes may be used
to search for:
the presence of a specific allele of a
gene (e.g. cystic fibrosis gene).
the approximate location of a gene on
a chromosome
(i.e. which chromosome and what
position on its p or q arm it binds to).
the ‘genetic fingerprint’ of a person to
tell them apart from others
(e.g. paternity testing, forensic
identification of suspects, the
identification of bodies from plane
crashes and exhumed graves).
DNA Profiling Using PCR
Microsatellites or short tandem repeats (STRs) are found
throughout the genome within genes and between genes, particularly
near telomeres and centromeres.
Microsatellites consist of a
variable number of tandem
repeats of a 2 to 6 base pair
sequence. In the example shown
it is a two base sequence (CA)
that is repeated.
Microsatellite
containing 4
repeat units
Homologous
pair of
chromosomes
The human genome contains
about 100 000 separate blocks
of tandem repeats of the dinucleotide:
CA. One such block at a known
location on a chromosome is shown
in the diagram opposite.
telomeres
centromeres
Microsatellite
containing 7
repeat units
Flanking regions to which PCR
primers can be attached
STRs Used in DNA Profiling
Extract DNA from
sample
DNA from
Individual ‘A’:
DNA from
Individual ‘B’:
DNA from
Individual ‘C’:
A sample is collected from the
tissue of three individuals from
which the DNA is extracted,
separated and purified.
Microsatellite
Amplify microsatellite
using PCR
Specific primers (arrowed and
orange) attach to the flanking
regions (green) either side of
the microsatellite (yellow).
They are used to make large
quantities of the microsatellite
and flanking regions sequence
only (no other part of the DNA
is amplified).
DNA
Microsatellites from
Individual ‘A’
Microsatellites from
Individual ‘B’
Microsatellites from
Individual ‘C’
PCR and Electrophoresis
7
2
6
4
5
3
The results of PCR are
many fragments
A
B
C
Largest fragments
7
Visualize fragments on a gel
The fragments are separated by length
using gel electrophoresis. DNA, which
is negatively charged, moves towards the
positive terminal. The smaller fragments
travel faster than the larger ones.
6
5
4
3
2
Smallest fragments
Manual DNA Sequencing
T C G A
DNA sequencing techniques are used to
determine the nucleotide (base) sequence
of DNA.
Two manual methods are in current use:
the Maxim-Gilbert procedure and
the Sanger procedure (the most common
method).
Both of these methods use electrophoresis.
Autoradiography is used to analyze the DNA
sequence (see opposite for a typical
autoradiograph).
Each of the dark shadows (blobs) contains
millions of DNA fragments.
Sanger Method
The Sanger method of manual DNA sequencing is illustrated
over the next few screens. It is based on the premature
termination of DNA synthesis resulting from the inclusion of
specially modified nucleotides called dideoxynucleotides.
These modified nucleotides are missing a crucial oxygen atom.
Because they lack the oxygen atom, when they are incorporated into
normally growing DNA strands, the next nucleotide cannot be added,
and synthesis of that DNA fragment stops.
How long each fragment will be depends on what position one of the
chemically altered nucleotides is incorporated into the sequence.
T
Thymine
C
Cytosine
G
Guanine
A
Adenine
Chemically altered so
that they prevent
further synthesis of the
complementary DNA
Forming Fragments 1
The DNA sample being analyzed consists of many millions of individual
molecules, each being used as a template to make fragments.
Each template molecule itself will produce thousands of complementary DNA
fragments of varying lengths.
In the sample DNA below, the guanine reaction can produce two fragments of
as illustrated below:
different lengths: T G and T G A C C A G
The sample DNA being analyzed
is used repeatedly as a template
to produce complementary
fragments of different lengths
‘Unknown’ DNA sequence
3’
Complementary DNA
strands of varying lengths
will form opposite the
sequence to be analyzed
5’
5’
Radioactive primer is
attached to each DNA
fragment (this is what
causes the blob on the film)
Synthesis in
this direction
Synthesis of this particular
fragment stops at the 7th base
because a modified guanine was
added which stops further growth
of the complementary DNA strand
Forming Fragments 2
DNA synthesis is initiated from a primer which is radio-labeled
(contains a radioactive isotope that will appear on a photographic film
called an autoradiograph).
Four separate reactions are
run, each containing a modified
nucleotide mixed with its normal
counterpart, as well as the 3
other normal nucleotides.
When a modified
nucleotide is added to the
growing complementary
DNA, synthesis stops at
that point.
The reaction for creating
thymine fragments is
shown opposite:
Radioactive primer
attached to each
fragment
1
The nucleotides for a
sequencing reaction for
thymine includes
normal nucleotides
Modified thymine is
added at random to each
synthesizing fragment
which stops the DNA
growing any longer
9
Four Sequencing Reactions
Using the same DNA sample to be sequenced (A C T G G T C T A G), a separate
sequencing reaction is carried out for each of the 4 bases: T, C, G and A
Each reaction yields a series of different sized fragments:
Thymine Reaction
1% modified TT
is added to cause
termination at random
thymine sites
Cytosine Reaction
C
1% modified C
is added to cause
termination at random
cytosine sites
Guanine Reaction
1% modified GG
is added to cause
termination at random
guanine sites
Adenine Reaction
1% modified AA
is added to cause
termination at random
adenine sites
1
2
3
4
5
6
7
8
9
10
Running the Gel
Electrophoresis Gel A jelly-like
material that allows DNA fragments
to move through it when an electric
charge is applied. It is usually made
of a material called acrylamide.
Largest DNA
fragments
DNA Samples The four reactions
containing DNA fragments are placed
in separate wells at the top of the gel.
-ve
Direction of movement of
radio-labeled nucleotides
The fragments from
the four reactions are
separated by
electrophoresis
and analyzed by
autoradiography
to determine the
DNA sequence:
Smallest DNA
fragments
Radio-labeled DNA Fragments
Attracted to the positive terminal, millions of DNA
fragments of similar size and sequence move as
a dark shadow down the gel. Larger pieces move
more slowly and therefore do not travel as far.
+ve
Positive Terminal Attracts
the fragments of DNA which
are negatively charged.
Automated DNA Sequencing
The process of DNA sequencing can be
automated using gel electrophoresis
machines that can sequence up to 600
bases at a time (see opposite). The
throughput of DNA samples is greatly
improved over the manual method.
Computer software automatically interprets
the data coming from the machine to
produce a base sequence.
One automated method uses nucleotides that are
labelled with fluorescent dyes; a different color for each
of the four types of bases (see next series of slides).
Another advantage of this automated method is that the
entire base sequence for the sample can be determined
from a single lane on the gel (not four lanes as in the
manual method).
Preparing a DNA Sample
Step 1: A DNA sample arrives for automated DNA
sequencing
Purified DNA samples
may contain linear DNA
or plasmids.
The sample should contain
about 1 x 1011 DNA
molecules.
The sample is checked to
ensure that there is enough
DNA present in the sample
to work with.
Adding Primer and Mix
Step 2: The primer and reaction mix are added
A DNA primer is added to
the sample which provides
a starting sequence for
synthesis.
Also added is the
sequencing reaction mix
containing the polymerase
enzyme and free
nucleotides, some which
are labeled with dye.
Creating Tagged Fragments
Step 3: Creation of dye-labeled fragments
(also see the next slide for greater detail)
A PCR machine creates fragments of DNA complementary to the
original template DNA.
Each fragment is tagged with a fluorescent dye-labeled nucleotide.
Running for 25 cycles,
it creates 25 x 1011
single-stranded DNA
molecules.
Creating Tagged Fragments
The key ingredients for
preparing a DNA sample
for automatic sequencing
are shown (a)-(c) opposite
(this process is also shown
in the previous slide):
(a) Original DNA template (the sample)
(b) Many normal unlabeled nucleotides:
A
T
G
C
(c) Terminal nucleotides labeled with fluorescent dye (a
different color for each of the 4 bases). These
nucleotides have had their structure altered so they act
as terminators to stop further synthesis of the strand:
Two examples of synthesized DNA fragments are shown
below. One is relatively short, the other is longer:
Normal
nucleotides
Terminal nucleotide
labeled with dye
Precipitation & Centrifugation
Step 4: Centrifugation to create a DNA pellet
The sample is chemically precipitated and centrifuged to settle
the DNA fragments as a solid pellet at the bottom of the tube.
Unused nucleotides,
still in the liquid, are
discarded.
Preparing the Pellet
Step 5: The DNA pellet is
washed, and buffer added
The pellet is washed with
ethanol, dried, and a gel
loading buffer is added.
All that remains now are single
stranded DNA molecules with
one dye-labeled nucleotide at
the end of each molecule.
Loading the Acrylamide Gel
Step 6: Acrylamide gel is
loaded (also see the next
two slides for greater detail)
The DNA sequencer is
prepared by placing the
acrylamide gel (sandwiched
between two sheets of glass)
into position.
A 36 channel ‘comb’ for
receiving the samples is
placed at the top of the gel.
Samples
placed here
Gel
Laser
Loading the Samples
Step 7: Loading DNA samples onto the gel
Different samples can be placed in each of the 36 “wells”
(funnel shaped receptacles) above the gel.
A control DNA sample of known sequence is applied to the first lane
of the sequencer. If there are problems with the control sequence
then results for all other lanes are considered invalid.
Running the Gel
The fragments from
the four reactions
are separated by
electrophoresis
and analyzed by
autoradiography to
determine the DNA
sequence:
Prepared samples
are placed in ‘wells’
Negative terminal
repels DNA fragments
Comb with 36 lanes into
which different samples
can be placed
DNA fragments with dyelabeled nucleotides move
down the gel over a 10
hour period
The smallest fragments
move fastest down the gel
and reach the argon laser
first, with larger fragments
arriving later
2400 volts
50mA
Acrylamide gel
See next slide for detail
Argon laser excites
fluorescent dye labels
on nucleotides
Lenses collect the
emitted light and focus it
into a spectrograph. An
attached digital camera
detects the light.
Positive terminal
attracts DNA fragments
Running the Gel
Green
TGGCATACT
Red
TGGCATAC
Blue
TGGCATA
Green
TGGCAT
Red
TGGCA
Green
TGGC
Gel through which
the DNA
fragments travel
Blue
TGG
Yellow
TG
T
Large fragments
travel slowly
Green
TGGCATACTA
Fragments travel in this direction
Detail from the
previous slide: DNA
fragments of different
sizes are drawn down
through the gel,
separating into distinct
bands of color as they
are illuminated by the
laser:
Yellow
T G G CATAC TAA
Yellow
Red
Small fragments
travel quickly
Green
Red
Yellow
Red
Blue
Laser scans across the gel to detect
the passing of each colored dye
Running the DNA Sequencer
Step 8: Running the DNA sequencer
Powerful computer software controls the activity of the DNA sequencer.
The gel is left to run
for up to 10 hours.
During this time an
argon laser is
constantly scanning
across the bottom of
the gel to detect the
passing of dye-labeled
nucleotides attached
to DNA fragments.
Analyzing the Data
Step 8: Running the DNA
sequencer
The data from the digital
camera is collected by
computer software.
The computer software records
the strength of the radiolabeled glow for each fragment
on the gel as a peak on a
graph sequence.
Each base (A T C G) is given a
different color on the graph.
Source: Four Peaks software http://mekentosj.com/4peaks/
Collecting the Data
Step 10: Computer analysis of results
The data from the digital camera is collected by computer software (e.g.
provided by Four Peaks software, a freeware program available for
download). A typical screen display is shown below:
Download Four Peaks software at: http://mekentosj.com/4peaks/
DNA Chips
DNA chips are also called gene
chips or microarrays.
They provide a tool to quickly
compare a known DNA
sequence with an unknown DNA
sequence.
This will allow determination of:
the genes present in a sample.
the code of an unsequenced
string of DNA.
the activity level (expression)
of the genes present.
DNA Chips
A microarray (DNA chip) consists
of DNA probes fixed to a small
solid support such as a glass slide
or a nylon filter.
The DNA probes are single
stranded DNA molecules,
each representing a gene.
Each spot on the microarray has
thousands to millions of copies
of a different DNA probe.
DNA chip
(microarray)
Segment of
a DNA chip
DNA Chips
Microarrays rely on nucleic acid hybridization in which a known
DNA fragment is used as a probe to find complimentary sequences.
DNA fragments of known genes are fixed to a solid support in an
orderly pattern as a series of dots.
The fragments are tested for
hybridization with samples of
labeled complementary DNA
(cDNA) molecules.
Computer analysis reveals
which genes are active in
different tissues, in different
stages of development or in
tissues in different stages of
health.
Part of one
DNA strand
Spot on the microarray
containing copies of a
single DNA molecule
How DNA Chips Work
DNA chip with
DNA probes
The following steps 1-5 show how
DNA chips work:
1. A microarray (DNA chip) contains DNA
probes. Each tiny spot on the microarray
has many copies of a different single
stranded DNA probe.
2. RNA is extracted from cells.
3. Reverse transcription in the presence
of a labeled nucleotide produces more
stable cDNA molecules, each with a
fluorescent tag.
RNA
Note: Molecules of cDNA representing
more than one tissue, or the same tissue
under different conditions, can be tested
together using a different colored label
for each.
Labeled cDNA molecules
How DNA Chips Work
4. The labeled cDNAs are applied to the
chip. The tagged cDNA will bind with
any complementary probe. Such binding
indicates that the gene represented by
the chip DNA was expressed, or active,
in the sample.
Labeled cDNA molecules
binding to DNA probes
5. After hybridization, the color of the spot
indicates the relative amount of mRNA
in the samples. The microarray is
scanned and a computer quantifies the
amount of gene activity in the sample
and generates a color-coded read-out.
Labeled cDNA molecules
binding to DNA probes
Gene Cloning
Gene cloning is a process of making large quantities of a
desired piece of DNA once it has been isolated.
Biologists obtain genes for cloning from two main sources:
DNA isolated directly from an organism.
Complementary DNA made in the laboratory from messenger RNA
(mRNA) templates (see the slides Making an Artificial Gene).
Cloning allows for an unlimited number of copies of a gene to
be produced for analysis or for production of a protein product.
Methods have been developed to insert a DNA fragment of
interest (e.g. a segment of human DNA) into the DNA of a
vector, resulting in a recombinant DNA molecule or
molecular clone.
Large quantities of the desired gene can be obtained if the
recombinant DNA is allowed to replicate in an appropriate host.
Vectors for Gene Cloning
A vector is a self-replicating DNA molecule (e.g. plasmid or viral
DNA) used to transmit a gene from one organism into another.
All vectors must have the following properties:
be able to replicate inside their host organism.
have one or more sites at which a restriction enzyme can cut.
have some kind of genetic marker that allows them to be easily identified.
Organisms such as bacteria, viruses and yeasts have DNA which
behaves in this way.
Using
Plasmids
Plasmid vectors,
found in bacteria,
are prepared for
cloning following
steps 1-6:
Preparation of the Clone
Human cell
DNA in
chromosome
Escherichia coli
bacterial cell
Human gene
Sticky
end
Plasmid
Sticky
end
Restriction enzyme
recognition sequence
A gene of interest (DNA
fragment) is isolated from
human tissue cells
Chromosome
Plasmid
vector
Tetracyclineresistance gene
Human DNA and plasmid are
treated with the same restriction
enzyme to produce identical
sticky ends
Gene
disrupted
Sticky
ends
Restriction enzyme cuts the plasmid
DNA at its single recognition
sequence, disrupting the tetracycline
resistance gene
Mix the DNAs together and add
the enzyme DNA ligase to bond
the sticky ends
Human gene
Recombinant plasmid is introduced into a
bacterial cell by simply adding the DNA to
a bacterial culture where some bacteria
take up the plasmid from solution
Ampicillinresistance
gene
An appropriate
plasmid vector is
isolated from a
bacterial cell
Recombinant
DNA
molecule
Cloning
the Gene
Bacteria containing the
recombinant plasmid are
spread onto an agar plate
containing ampicillin.
Bacterium
Human gene
The actual gene cloning
process (making multiple
copies of the human gene)
occurs when the bacterium
with the recombinant plasmid
is allowed to reproduce.
Recombinant plasmid
All colonies look identical but
only some have the plasmid
with the human gene.
Dish A
Filter
paper
Dish B
Agar containing ampicillin allows
only bacterial colonies with the
appropriate plasmid to grow.
Filter paper is pressed against the
agar plate thereby transferring
colonies of bacteria to the paper.
Colonies of bacteria
that carry the
recombinant plasmid
can be identified by
differential response
to antibiotics (e.g.
resistant to ampicillin
but sensitive to
tetracycline).
The filter paper is pressed against agar that
contains tetracycline. Those colonies that
grow do not have the human gene
disrupting the tetracycline resistance gene.
Colonies with the human gene
can be located according to their
position on the original dish A.
Using
Viruses
A gene is isolated from
human tissue cells
An appropriate bacteriophage vector is
selected that is capable of infecting the
target cell
Human cell
Protein capsule
enclosing DNA
Bacteriophage or
‘phage’
Some bacteriophages are
convenient for
cloning large
fragments of DNA
(15 to 20 thousand
base range).
Viral vectors are
prepared for
cloning following
steps 1-8:
DNA in
chromosome
Restriction
enzyme
cleavage site
Restriction enzyme
cleavage site
Human gene
Viral DNA
Human and the viral DNA are
cut with the same restriction
enzyme
Mix the DNAs together and the sticky
ends will be attracted to each other. Add
the enzyme DNA ligase to bond the
sticky ends.
Recombinant DNA
Gene Cloning Using Viruses
The recombinant DNA is
packaged into phage particles by
being mixed with phage proteins.
Recombinant DNA
Phage
proteins
Phage injecting
DNA into cell
The assembled phages are
then used to infect a bacterial
host cell.
The viral genes and enzymes
cause the replication of the
recombinant DNA within the
bacterial host cell.
The bacterial host cell
succumbs to the viral infection.
The cell ruptures (lysis) and
thousands of phages, each with
recombinant DNA, are released
to infect neighboring bacteria.
Viral clones containing human gene
Making an Artificial Gene
One problem with cloning DNA directly from an organism’s cell is
that it often contains long non-coding regions called introns.
These introns can be of enormous length and cause problems
when the gene as a whole is inserted into plasmids or viral DNA
vectors for cloning:
Plasmids tend to lose large inserts of foreign DNA.
Viruses cannot fit the very long DNA into their protein coats (capsids).
To avoid this problem, it is possible to make an artificial gene that
lacks introns.
This is possible by using an enzyme called reverse transcriptase
which is able to reverse the process of transcription by making
DNA molecules out of their mRNA products.
The important feature of this process is that mRNA has already
had the introns removed. By using mRNA as the template to
recreate the gene, the DNA will also lack the troublesome intron
regions.
Transcription
Using an enzyme
called reverse
transcriptase, it is
possible to create an
artificial gene that
lacks introns.
Steps 1-3 in the
diagram opposite
illustrate the normal
process of
transcription in the
cell, and steps 4-6 on
the following slide
show how scientists
are able to make the
artificial gene.
Double stranded DNA of a gene
Double stranded molecule
from a eukaryotic organism
of genomic DNA
(e.g. human) containing introns.
Intron
DNA
Exon
Intron Intron
Exon Exon
As a normal part of the cell
process of gene expression,
transcription creates a
primary RNA molecule.
Intron
Exon
Intron
Exon
Exon
Transcription
Primary RNA
The introns are removed
by splicing enzymes to
form a mature mRNA (now
excluding the introns) that
codes for a single protein.
Exons are
spliced together
mRNA
Introns are
removed
Introns
Reverse Transcription
The mRNA is extracted
from the cell and purified
mRNA
Reverse transcriptase is added
which synthesizes a single
stranded DNA molecule
complementary to the mRNA
mRNA
DNA
The second DNA strand is
made by using the first as a
template, and adding the
enzyme DNA polymerase
DNA
DNA
Reverse
Transcription
DNA strand being synthesized
by reverse transcriptase
Completed artificial gene consisting
of a double stranded molecule of
complementary DNA (cDNA)
The Nature of Transformation
Transformation, using genetic engineering
techniques, is concerned with the movement of genes
from one species to another.
An organism that develops from a cell into which foreign DNA
has been introduced is called a transgenic organism.
Recombinant DNA technology allows direct modification of
an organism’s genome, allowing traits to be introduced that
are not even present in the species naturally.
Insertion of DNA can be between species or between taxa
that are not even closely related.
Example: In January 2001, for the first time scientists
modified the DNA of a primate. They inserted a fluorescent
gene from a jellyfish into the DNA of an unfertilized primate
egg. This resulted in the birth of “ANDi” (“inserted DNA”
backwards), the first transgenic primate (a Rhesus monkey).
Applications of Transformation
Dolly and her
surrogate
mother
The applications associated with
transformation are numerous, e.g.
Improvement of crop yields
Production of herbicide resistant plants
Enhancement of desirable traits in livestock
Treatment of human defects through gene therapy.
Cloning technology can be used to propagate
transgenic organisms so that introduced genes
quickly become inherited.
Populations of transgenic organisms may be
rapidly obtained by various methods:
Bacteria: Grown in a fermenter
Plants: Micropropagation (tissue culture) techniques
Animals: Cloning using embryo splitting and
nuclear transfer techniques.
Photos courtesy of the Roslin Institute, Edinburgh
Dolly and
her progeny
Dolly
Transformation Methods
The process of introducing new DNA into an
organism is called transformation.
There are a number of methods employed to achieve
a transfer of genetic material, and they include:
Protoplast fusion
Ballistic DNA injection
(see Helios gene gun opposite)
Liposomes
Viral vectors
Pronuclear injection
(Microinjection)
Plasmid vectors
Protoplast Fusion
Protoplast fusion requires the
cell walls of plants to be removed
by digesting enzymes.
Enzymes
digest cell
walls
polyethylene glycol
stimulates fusion
The resulting protoplasts (cells that
have lost their cell walls) are then
treated with polyethylene glycol
which causes them to fuse.
In the new hybrid cell, the DNA derived
from the two "parent" cells may undergo
natural recombination (they may merge).
Cell
membrane
DNA from 2
different cells
Ballistic DNA Injection
This remarkable way of introducing foreign DNA into cells literally
shoots it directly into the plant or animal tissue using a "gene gun".
Microscopic particles of gold or tungsten are coated with DNA
and propelled by a burst of helium into the tissue.
Some of the cells express the introduced DNA as if it were their own.
barrel spacer
Gold palettes
coated with DNA
Cartridge holder:
takes up to 12 DNA
gold coated cartridges
Compressed
helium gun
Nucleus
Target plant
or animal cell
Liposomes
Liposomes are small spherical vesicles made of a single membrane.
They can be made commercially to precise specifications.
When coated with appropriate surface molecules, they are attracted to
specific cell types in the body.
DNA carried by the
liposome can enter the
cell by endocytosis or
fusion.
They can be used to
deliver genes to these
cells to correct defective
or missing genes.
Liposome
membrane
Surface molecule
Gene carried
inside liposome
DNA
Liposome fusing
with cell membrane
Animal
cell
Nucleus
Viral Vectors
Some viruses are well suited for gene therapy. They can
accommodate up to 7500 bases of inserted DNA in their protein
capsule.
When viruses infect and reproduce inside the target cells, they are also
spreading the recombinant gene.
Viral vectors have
already been used in
several clinical trials of
gene therapy for
different diseases.
A problem with this method
involves the host’s immune
system reacting to and killing
the virus.
Retrovirus with
normal human gene
Normal gene
introduced into
human cell by virus
Cell transplanted
into body to
correct genetic
disease
Pronuclear Injection
DNA can be introduced directly into an animal cell
(usually an egg cell) by microinjection.
This technique requires the use of a glass micropipette with a
diameter that is much smaller than the cell itself. The sharp tip
of the micropipette is used to puncture the cell membrane.
The DNA is then injected through it and into the nucleus.
Micropipette
injects gene
Egg cell
Blunt holding pipette
Pieces of DNA
forming the gene
Egg nucleus
Transgenic Mice
Steps 1-4 show the
successful trial
experiment using
pronuclear injection to
produce the world’s
first transgenic animal.
Two eggs are removed from a single female
mouse and are fertilized artificially in a test tube.
One fertilized egg
is left unaltered.
Micropipette injects rat
growth hormone gene
into a fertilized egg.
Normal egg is cultured to
an embryo, then implanted
in a surrogate mother.
Transformed egg is cultured to
an embryo, then implanted in a
surrogate mother.
A rat growth hormone
was introduced as a
plasmid into the
fertilized egg of
a mouse.
Weight: 29 g
Weight: 44 g
The mice above are siblings, but the mouse on
the right was transformed by the introduction of
a rat growth hormone gene.
Plasmid Vectors
Plasmids are accessory chromosomes occurring naturally in bacteria.
In nature, plasmids are usually transferred between closely related microbes
by cell-to-cell contact (a process called conjugation).
Simple chemical treatments
can make mammalian cells,
yeast cells and some
bacterial cells that do not
naturally transfer DNA,
able to take up external DNA.
The bacterium
Agrobacterium tumefaciens
(opposite and the next slide)
can insert part of its plasmid
directly into plant cells.
Bacterium:
Agrobacterium
tumefaciens
Plasmid
Plant infected by
bacterium with
foreign gene
Plasmid with
foreign gene
The Ti Plasmid
The Ti plasmid is
from a soil bacteria
that causes tumors
(galls) in plants.
It can be successfully
transferred to plant
cells where a segment
of its DNA can be
integrated into the
plant’s chromosome.
Ti plasmid isolated from bacteria:
Agrobacterium tumefaciens
Site where restriction
enzyme cuts the plasmid
DNA containing gene for
disease resistance is
added to Ti plasmid
Recombinant
plasmid
Ti plasmid
Restriction enzyme
and DNA ligase splice
the gene of interest
into the plasmid
Introduce plasmid
into plant cells
Part of the plasmid
containing the gene
of interest integrates
into the plant’s
chromosomal DNA
Transformed plant
cells are grown by
tissue culture