Biotechnology (some definitions)

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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
• Separate strands by heating at 98C for 5
  minutes. Allows DNA to unwind.
• 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.
• Incubate, by cooling to 60C 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.

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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.

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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

• Cant 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

• 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
Buffer
Black background To make loading wells easier
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A gel as seen under UV light - some samples had 2
fragments of DNA, while others had none or one
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More

• Many samples can be run on one gel- but 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

• DNA cut with R.E. into small fragments
• Separated by gel electrophoresis
• Transferred from gel to nylon
• Gel soaked to denature DNA
• Gel put into long paper towel soaking in salt
  solution
• Nylon membrane placed onto gel, covered in
  blotting paper and towels

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• Blotting paper acts as a wick and draws salt
  solution up through gel
• Salt takes DNA with it and transfers it to nylon
  but in the same position that it was on the gel.
• 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 non-coding
  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 couriers 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 for testing to date through DNA Diagnostics
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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 individuals 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 loci- The 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. Different colors help with
interpretation
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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 mans analysis shows that the
DNA evidence is 25 million times more likely
that he is the biological father versus he is
not (odds 25 million1)
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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 wouldnt.
• 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 cloningClick and Clone