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Genetics & Evolution Series:
Version: 2.0
Set 9
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.