Genetics & Evolution Series: Set 9 Version: 2.0. Gene technology is a broad field which includes...
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Transcript of Genetics & Evolution Series: Set 9 Version: 2.0. Gene technology is a broad field which includes...
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
What is Gene Technology?
Why Gene Technology?
Photos courtesy of GreenPeace
Who owns and regulatesthe GMOs?
Third world economies areat risk of exploitation
Biological risks have notbeen adequately addressed
Animal ethics issues
The costs of errors
Environmentally friendly
Could improve thesustainability of crop andlivestock production
Could potentially benefitthe health of many
More predictable anddirected thanselective breeding
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.
The Beginning of GE
The bacterium Escherichia coli (above) and the yeast Saccharomyces cerevisiae (below): favorite organisms
of gene research
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).
Producing GMOsGMOs may be created by modifying their DNA in one of three ways:
Adding a Foreign GeneA 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.
Host DNA
Delete or ‘Turn Off’ a GeneAn 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
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.
Existing gene altered
Host DNA
Restriction Enzymes
Recognition SiteRecognition Site
GAATTC
CTTAAG
DNA
CTTAAG
GAATTC
cutThe restriction enzyme
EcoRI cuts here
cut cut
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.
Specific Recognition SitesRestriction 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
It is possible to use restriction enzymes that cut leaving an overhang; a so-called “sticky end”.
DNA cut in such a way produces ends which may only be joined to other sticky ends with a complementary base sequence.
See steps 1-3 opposite:
C T T A A
A A T T C G
G
Sticky Ends
FragmentRestriction
enzyme: EcoRI
Sticky endRestriction enzyme: EcoRI
DNA from
another source
A restriction enzyme cuts the double-stranded
DNA molecule at its specific recognition site
The two different fragments cut
by the same restriction enzyme
have identical sticky ends and
are able to join together
The cuts produce a
DNA fragment with
two “sticky” ends
When two fragments of DNA cut by the same restriction
enzyme come together, they can join by base-pairing
C T T A A
A A T T C
G
G A A T T C
C T T A AG
G
C T T A A
A A T T C G
G
C C C
G G G
G G G
C C C
C C C
G G G
G G G
C C C
C C C
G G G
G G G
C C C
Blunt Ends
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 non-specific because there are no sticky ends as recognition sites.
Restriction enzyme
cuts here
Recognition Site Recognition Site
DNA from another source
The cut by this type of restriction
enzyme leaves no overhang
cutcut
C C C
G G G
G G G
C C C
C C C
G G G
G G G
C C C
G G G
G G G
C C C
C C C
DNA
A special group of
enzymes can join
the pieces together
LigationDNA 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:
Plasmid DNA fragment
Two pieces of DNA are cut using the same restriction enzyme.
Foreign DNA fragment
A A T T C G
C T T A AG
The two different DNA fragments are attracted to each other by weak hydrogen bonds.
This other end of the foreign DNA is attracted
to the remaining sticky end of the plasmid.
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.
Annealing
Detail of Restriction Site
Restriction sites on the fragments are attracted by base pairing only
Gap in DNA molecule’s ‘backbone’
Foreign
DNA
fragment
A A T TC
A A T T C
G
G
C A
Plasmid
DNA
fragment
G
G
T T A
AATTC
DNA ligase
The fragments are able to
join together under the
influence of DNA ligase.
Recombinant DNA Plasmid
GA A T T
C
G A A T T C
C T T A A G
GAATT
C
Recombinant Plasmid DNA
Detail of Restriction Site
Fragments linked
permanently by
DNA ligase
No break in
DNA molecule
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 Amplification
A crime scene(body tissue samples)
Fragments of DNA from
a long extinct animal
A single viral particle
(from an infection)
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;
DNA sequencing
DNA profiling/fingerprinting
Gene cloning
Transformation
Making artificial genes
Samples from some sources,including those shown here,may be difficult to obtain inany quantity.
PCR EquipmentAmplification 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 StrandsSeparate the target DNA strands by heating at 98°C for 5 minutes
Add Reaction MixAdd primers (short RNA strands that provide a starting sequence
for DNA replication), nucleotides (A, T, G and C) and DNA
polymerase enzyme.
IncubateCool 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.
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.
The process of PCR is detailed in the following slide sequence of steps 1-5.
Polymerase Chain ReactionPCR
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
Cycle 1
Cycle 2
Cycle 3
Original DNASample
The Process of PCR 1
Primer annealed
A DNA sample called the
target DNA is obtained
DNA is denatured (DNA strands
are separated) by heating the
sample for 5 minutes at 98C
Primers (short strands of mRNA)
are annealed (bonded) to the DNA
The Process of PCR 2Nucleotides
Nucleotides
After one cycle, there are now two copies of the original sample.
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.
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.
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.
Gel ElectrophoresisWells into which samples to be analyzed are placed.
Buffer solution
Cathode
Anode Gel
Plastic Frame
Buffer
Sample
DNA fragments, shown symbolically above, move towards the positive terminal (smaller fragments move faster than longer ones).
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.
Analyzing DNA
-ve terminal
+ve terminal
Small fragments
Large fragments
Tray: Contains the set gel.
DNA solutions: Mixtures of different sizes of DNA fragments are loaded into each well.
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 fragments:
The gel matrix acts as a seive for the DNA molecules.
Wells: Holes created in the gel with a comb.
DNA ProfilingDNA 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.
DNA fragments (PCR product after endonuclease digestion) visualized under UV light after staining with ethidium bromide and migration in an agarose electrophoresis gel.
Visualizing the Profile
DNA Profiling MethodsDNA 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.