Download Definition of a Gene - Kaikoura High School

Biotechnology
(some definitions)
• Biotechnology is the development of products using a
living organisms to meet a human need or demand. Note
that this includes traditional processes such as wine and
cheese production as well as more modern technologies.
• Genetic engineering is a technology used to alter the
genetic material of living cells in order to make them
capable of producing new substances or performing new
functions.
• Cloning is the production of exact copies (clones) of
particular genes or cells.
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• If you are going to handle genes, first you
need to find them (probes), cut them out of
a chromosome (restriction enzymes), glue
them (ligation) back into another
chromosome (plasmid) then put them into a
cell (bacteria, virus, yeast) that can make
copies (simple replication or protein
synthesis).
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Tools of Biotech
Collecting the DNA
- break open the cells to release DNA
- remove unwanted debris
-remove unwanted proteins
- precipitate out DNA
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Restriction Enzymes
• Restriction enzymes are proteins produced by bacteria to
restrict invasion by foreign DNA (such as viruses).
• Restriction enzymes recognise and cut at specific locations
along the DNA molecule called recognition sites.
• A restriction site is a 4- or 6- base-pair sequence that is a
palindrome, ie. The “top” strand read from 5’ to 3’ is the
same as the “bottom” strand read from 5’ to 3’.
• For example:
5’ GAATTC 3’
3’ CTTAAG 5’
is the recognition site for the restriction enzyme EcoRI
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Restriction Enzyme Action
• EcoRI makes one cut between the G and the A in each of
the DNA strands. The hydrogen bonds holding the bases
together then break.
5’ G
AATTC 3’
3’ CTTAA
G 5’
• The single-strands of exposed bases on the cut DNA are
called “sticky ends”. Ends cut with the same restriction
enzyme can be joined together.
• Some restriction enzymes cut the DNA strands directly
across from one another producing a “blunt end”.
• Hundreds of restriction enzymes have been discovered and
are now used.
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DNA Ligase
• DNA ligase joins together
Okazaki fragments on the
lagging strand during DNA
replication.
• Genetic engineers use
DNA ligase to join
together fragments of DNA
(usually from different
sources) that have been cut
using the same restriction
enzyme.
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Ligation
• Reassembling of DNA strands once they have
been cut. e.g. two different pieces of DNA that
have been cut with the same restriction enzyme
have sticky ends that ‘match’.
• These are annealed (bonded) together to get a new
piece of DNA in either:
Liner
or plasmid form
• This is then a piece of recombinant DNA. DNA
ligase is used to make the pieces join.
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PCR – polymerase chain reaction
• This is the method by which a small piece of DNA
can be quickly copied many times over. It is
faster than cloning, you only need a small piece of
sample and the sample can be old.
• DNA polymerases are the enzymes that copy
DNA – to do this they need a template strand and
a primer.
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PCR
(Polymerase Chain Reaction)
• A PCR cycle consists of 3 steps:
1. Separate strands by heating at 98°C for 5 minutes. Allows
DNA to unwind.
2. Cooled and then Add primers (which are short DNA
strands that provide a starting sequence for DNA
replication), nucleotides (A, T, G & C) and DNA
polymerase.
3. Incubate, by cooling to 60°C for a few minutes. The
primers attach to the single-stranded DNA and DNA
polymerase synthesises complementary strands.
• Automated DNA sequencing uses thermophilic enzymes
so step two is required only for the first cycle. Each cycle
takes approx. 5min so many cycles can occur quickly.
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PCR animation
• AnimationPolymerase Chain Reaction
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Applications of PCR
• Used by police when have small piece of tissue to
identify criminals
• Anthropologists and archaeologists to check
ancient fossils
• Gene checking – i.e. to see if carry cystic fibrosis.
• Identify viral genes earlier and quicker than
normal methods
• Identify genetic disorders in prenatal cells
• Detect cancer cells
• Identify unknown skeletons
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Pros and cons
• Advantages are:
•
•
•
•
Only need a small piece of tissue
Tissue can be old
Fast
Can be automated
• Disadvantages;
– Need to be extremely careful of cross
contamination.
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Cloning
Cloning can be an entire organism or a
single cell many times over.
 A vector is any vehicle that carries DNA
into a host cell – most modern clones are
vectors
• Transformation is when external genetic
material is assimilated by a cell.
• Once engineered DNA needs to be put back
in a cell to function.

Natural vectors (application gene
cloning)
• Plasmids from bacteria are used as vectors
These are small rings of DNA separate from
the bacteria chromosome. They can easily
be removed from the bacteria and cut like
other DNA. The two pieces of DNA are
joined together and put back into the
bacteria.
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



The bacteria divides and so copies the
foreign gene.
These are used in many ways: to make lots of
copies of the gene
: or to make bacteria that have a new
function
: to make the protein the gene codes for in
large quantities.
Other vectors
• Viruses: in a virus DNA is a string in a protein
coat. New DNA spliced into virus DNA then
returned to virus coat. Then infects host cell and
replicates (normally bacteria). Some can carry
DNA into animals and an advantage is they are
normally host specific so only invade certain cells
(cystic fibrosis).
• Yeast: if protein to be made is too complicated for
prokaryote cell then need a eukaryote cell. Yeast
is rare as has plasmids so can be used like bacteria.
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Other vectors
• Can’t always use natural vectors –
especially with plant and animal cells.
• Electroporation: an electric current is used
to force DNA over a cell membrane.
• DNA gun: DNA of interest is coated onto
microscopic pellets (gold or tungsten) and
fired into cells.
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Host cells in gene cloning
• Usually bacteria as easy to insert genes and
replicate quickly. But because prokaryote
and eukaryote cells have different enzymes
for transcription and translation the prok.
does not always read the eukaryote gene
correctly, so need to use a eukaryote cell.
This is difficult and not many eukaryote
cells will take up engineered DNA.
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What is DNA cloning?
• When DNA is
extracted from an
organism, all its
genes are obtained
• In gene (DNA)
cloning a particular
gene is copied
(cloned)
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Whole organisms are cloned
too, but differently
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Why Clone DNA?
• A particular gene can be isolated and its
nucleotide sequence determined
• Control sequences of DNA can be identified &
analyzed
• Protein/enzyme/RNA function can be
investigated
• Mutations can be identified, e.g. gene defects
related to specific diseases
• Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect
resistance, etc.
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DNA Synthesis
•
•
1.
2.
Scientists can now synthesise short
one-sided pieces of DNA, called oligonucleotides. These
are made by machines from a computer program.
These oligonucleotides are used as:
Primers for the Polymerase Chain Reaction (PCR).
They do this by providing an attachment point for DNA
polymerase to synthesise new strands.
Gene probes.
These are oligonucleotides that hybridise with specific
DNA sequences. A radioactive marker or fluorescent
dye is attached to the probe so it is visible.
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Separating DNA – Gel
Electrophoresis
• This method depends on the fact that
restriction enzymes produce DNA fragments
of different lengths and DNA has a negative
charge due to the phosphate groups.
• When DNA is exposed to an electrical field,
the particles migrate toward the positive
electrode
• Smaller pieces of DNA can travel further in a
given time than larger pieces
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Gel Electrophoresis – the method
• The gel is made from Agarose - a
polysaccharide made from seaweed.
Agarose is dissolved in buffer and
heated, then cools to a gelatinous solid.
• Some gels are made with acrylamide if
sharper bands are required
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• The gel chamber is set up, the ‘comb’ is
inserted – this leaves little holes when
the gel sets.
• The agarose may have a DNA ‘dye’
added (or it may be stained later). The
agarose is poured onto the gel block
and cooled, then flooded with a buffer
solution.
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• Buffer - the gel slab
is submerged
(submarine gel) in
buffer after
hardening
• The buffer provides
ions in solution to
ensure electrical
conductivity.
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• The comb is
removed, leaving
little ‘wells’
• The DNA samples
are mixed with a
dense loading dye
so they sink into
their wells and can
be seen
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• The power source is turned on and the
gel is run. The time of the run depends
upon the amount of current and % gel,
and requires experimentation
• At the end of the run the gel is removed
(it is actually quite stiff)
• The gel is then visualized - UV light
causes the bands of DNA to fluoresce
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A gel being run
Positive electrode
Comb
Agarose block
DNA loaded in
wells in the agarose
Black background
To make loading wells easier
Buffer
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A gel as seen under UV light - some samples had 2 fragments
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of DNA, while others had none or one
More……
• Many samples can
be run on one gelbut it is important to
keep track
• Most gels have one
lane as a ‘DNA
ladder’ - DNA
fragments of known
size are used for
comparison
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Still more….
• The DNA band of interest can be cut out
of the gel, isolated and purified and then
have full biological activity.
• Or DNA can be removed from the gel by
Southern Blotting
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Southern Blotting Summary
(Developed by Ed Southern of Edinburgh University).
• The method uses gel electrophoresis and
hybridisation to find a gene of interest.
• Since probes cannot work on a gel, the DNA is
transferred to a nylon membrane.
• A radioactive probe is then added and hybridises
with a specific DNA sequence.
• A sheet of photographic film is placed over the
membrane and developed to show the position of
the probe.
• More probes can be used to identify other regions
of DNA, since each probe is specific to a
particular DNA sequence.
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Southern Blotting Method
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Method
1. DNA cut with R.E. into small fragments
2. Separated by gel electrophoresis
3. Transferred from gel to nylon
a) Gel soaked to denature DNA
b) Gel put into long paper towel soaking in salt
solution
c) Nylon membrane placed onto gel, covered in
blotting paper and towels
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d) Blotting paper acts as a wick and draws salt
solution up through gel
e) Salt takes DNA with it and transfers it to
nylon but in the same position that it was on
the gel.
4. Radioactive probe added that sticks only to
the genes of interest and X-ray film can be
developed.
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DNA Sequencing
• This uses gel electrophoresis to find out the order of
the nucleotides A, C, T and G on a DNA strand. If
you know the order you can then work out the amino
acid order of the protein.
• Known as the Sanger method after discoverer.
• Dideoxynucleotides are used to stop synthesis of a
complementary DNA strand at the point they are
incorporated.
• By using dideoxynucleotide versions of A, C, T and G
mixed in with normal versions, it is possible to stop
synthesis at every nucleotide.
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• Since the resulting complementary
strands are of different lengths, gel
electrophoresis can be used to separate
them.
• Large modern laboratories use
fluorescent dyes and the gels are read
by a computer to sequence the DNA.
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Why
• Understanding a particular DNA sequence
can shed light on a genetic condition and
offer hope for the eventual development of
treatment
• DNA technology is also extended to
environmental, agricultural and forensic
applications
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DNA Fingerprinting/profiling
• Used to form a genetic
fingerprint to identify
person, animal or plant.
Remember that some of
the DNA in humans is
common to all organisms,
some common to all
humans but the unique
parts (VNTR and STR)
can be used for
identification as they are
only in one individual.
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What is Analyzed in the DNA?
• DNA profiling depends on regions of noncoding DNA that show great variability
between individuals (are polymorphic which
means many forms)
• Modern profiling uses Short Tandem
Repeats, STRs
• These are short sequences of DNA, usually
2-5 base pairs (bp) long, that repeat, or
‘stutter’ many times
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New Technology
• STR analysis has largely replaced the original
RFLP analysis (DNA Fingerprinting)
developed in 1985 by Dr Alec Jeffreys
• RFLP analysis requires good amounts of
non-degraded DNA but STR analysis can be
done on less than one billionth of a gram (a
nanogram) of DNA (as in a single flake of
dandruff)
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DNA Fingerprinting & DNA Profiling
- same or different?
• DNA fingerprinting, as developed by Sir
Alec Jeffries, produces patterns unique
to an individual. It requires good DNA
samples and takes 1 - 2 weeks.
• DNA profiling produces patterns of
inheritance for individual loci, and then
uses laws of probability to predict the
likelihood of a match. It uses minute
amounts of DNA and can be processed
within 24 hours
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Why Test?
• Parentage - e.g. disputes over who is the
father of a child & is thus responsible for child
support
• Determining whether twins are identical or
fraternal
• Estate cases (these may involve obtaining
pathology samples of deceased individuals)
• Immigration - establishing that individuals are
the true children/parents/siblings in cases of
family reunification
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Why Test? ctd
• Bone marrow transplant monitoring - to check
that the transplanted marrow is still present
• Determination of maternal cell contamination
in chronic villus sampling (used to investigate
the possibility that a fetus has a severe
inherited disease)- is the tissue sample really
fetal?
• Etc.
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The Steps, II
• DNA samples are
collected- in the case of
parentage testing, from
the mother, child and
putative (possible)
father(s)
• They are usually blood,
but a buccal (cheek
cell) swab is acceptable
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The Steps, III
• If the samples need
transport they must
be sent in leak
proof containers for
the courier’s safety.
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The Steps, IV
• The samples are
processed, and
DNA is extracted
from each
• Primers for each
locus are added.
Each primer is
labeled with a
fluorescent marker
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The Steps, IV, ctd
• DNA Diagnostics
currently uses an
AmpFlSTR Identifiler TM
PCR Amplification Kit
which targets 15 STR
regions plus a sex
specific region.
• Kits allow
standardization and
accuracy, as DNA
samples are added to a
pre-made mix
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The Steps, V
• The DNA and
fluorescent primers are
run through the
polymerase chain
reaction (PCR) to
amplify the targeted
STR regions on the
DNA
• The samples are
audited continually to
ensure accuracy
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The Steps, VI
• The amplified DNA in a sample is separated
by electrophoresis in a genetic analyzer
• The analyzer has a gel-filled capillary tube
through which the DNA travels (this replaces
the gel slab of earlier days)
• DNA fragments move through the gel tube by
size, smallest first
• A laser reads the fluorescent marked DNA
loci
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An ABI Prism 310 Genetic Analyser
Capillary tube
Sample tray
Note-other models of this Analyzer have more capillary tubes and can
process more samples at a time, but this model is sufficient for the demand
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for testing to date through DNA Diagnostics
Analyzing the Read-out
• Digital output from the
Analyzer is read and
interpreted by genotyping
software
• Each STR region read has
two peaks, for the regions
(loci) on an individual’s
maternal and paternal
chromosomes with that
locus. note - if both regions
are the same length, there is
one peak
• Data is shown both
graphically and numerically
A sample showing 4 lociThe top line is a ‘ladder’
for comparison
Locus D19S433 = 14,15
Locus vWA = 15,16
Locus TPOX= 8,8
Locus D18S51= 13,16
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A sample print -out for one person, showing all loci tested.
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Different colors help with interpretation
Whose STR?
• A child will inherit one of the STRs at each locus from
its mother, and since usually in parentage tests these
are determined, then by elimination the other STRs at
each locus come from its father
• The father can donate either of his two STRs at each
locus
• If a child has STRs different from those of the
putative father, then that man can be eliminated as a
possible father
• If a child has a particular STR that is the same as the
putative father, it is necessary to examine possible
matches with other STR loci and examine probability
in Parentage Analysis
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Parentage Analysis
• For each STR tested, the data obtained is
used to calculate a paternity index (the
probability of the evidence given that a
particular man is the father versus he is
not the father)
• This is based on the frequency in the
population of the alleles at that locus
• In New Zealand there are databases for
European, Maori/Cook Islander, Asian
and Tongan/Samoan. International
databases are used for other ethnicities
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Analysis II
• Each STR site index is an independent
event, so using probability law that says
“the probability that two independent
events may happen together is the
product of their individual probabilities”,
an overall paternity index is calculated
by multiplying together the indices for
each locus
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Parentage Analysis II, ctd
Paternity index
The index in this man’s analysis shows that the DNA
evidence is 25 million times more likely that he is the
biological father versus he is not (odds 25 million:1)
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Cost?
• A standard Paternity/Maternity test for two or
three people costs $1125 including GST in
2003, payable in advance
• If more than three persons are tested at one
time, each additional person tested costs
$250 + GST.
• These costs include blood collection and
transport
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Transgensis
• A transgenic organism is one that has had
its genetic makeup altered by having a gene
from another species transferred into it. As
a result it can make a protein it normally
wouldn’t.
• Many examples in microorganisims (human
insulin by bacteria, human growth
hormones, hep. B vaccine in yeast)
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• In animals the gene is placed into the
nucleus of a fertilised egg before it starts to
divide (examples in milking cows to make
proteins and vaccines, salmon – make them
grow faster, mice and pigs).
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Technical limitations
• Not all species will take on strange genes
• Regulation of expression of gene – sometimes this
is in a different place on the DNA
• Physiological problems – growth hormones can
make animals grow faster but have other problems
like heart, liver, diabetes.
• Cost effectiveness – costs a lot to develop and
implement.
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Salmon as an example
• Pros: fast growth, better returns for fish,
flavour enhanced = better product, breed all
year round not limited season.
• Cons: environmental spread of new gene if
escape and cross breed with wild, public
acceptance of G.E food, health and safety –
can it affect humans who eat the G.E
product?
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Xenotransplantation
• The transplantation of tissues and organs
between different species – mainly the
transplant of animal tissue into humans.
• Issues
• Totally random mouse cloning:Click and
Clone
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