DNA Technology and Genomics

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DNA Technology and Genomics
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Why?

• Understand the way in which plants and animals,
  including humans, develop, function and evolve
• Investigate the molecular basis of disease
• Develop products for medicine and crops for
  agriculture
• Solve crimes and paternity disputes
• Investigate endangered species for conservation
  management

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Studying DNA

• A number of methods have been developed that can
  be used to identify the DNA (genetic) profile of
  an individual
• These methods can also be employed to measure
  genetic differences between individuals in a
  population
• Techniques for working with DNA can be broken
  down into four major categories
• Copying DNA
• Cutting and pasting DNA
• Measuring DNA length
• Probing DNA

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Extracting DNA

• Break open (lyse) the cells or virus containing
  the DNA of interest. This is often done by
  sonicating or bead beating the sample. Vortexing
  with phenol (sometimes heated) is often effective
  for breaking down protienacious cellular walls or
  viral capsids. The addition of a detergent such
  as SDS is often necessary to remove lipid
  membranes.
• DNA associated proteins, as well as other
  cellular proteins, may be degraded with the
  addition of a protease. Precipitation of the
  protein is aided by the addition of a salt such
  as ammonium or sodium acetate. When the sample is
  vortexed with phenol-chloroform and centrifuged
  the proteins will remain in the organic phase and
  can be drawn off carefully. The DNA will be found
  at the interface between the two phases.
• DNA is the precipitated by mixing with cold
  ethanol or isopropanol and then centrifuging. The
  DNA is insoluble in the alcohol and will come out
  of solution, and the alcohol serves as a wash to
  remove the salt previously added.
• Wash the resultant DNA pellet with cold alcohol
  again and centrifuge for retrieval of the pellet.
  
• After pouring the alcohol off the pellet and
  drying, the DNA can be re-suspended in a buffer
  such as Tris or TE.

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Why we need so many copies

• Biologists needed to find a way to read DNA
  codes.
• How do you read base pairs that are angstroms in
  size?
• It is not possible to directly look at it due to
  DNAs small size.
• Need to use chemical techniques to detect what
  you are looking for.
• To read something so small, you need a lot of it,
  so that you can actually detect the chemistry.
• Need a way to make many copies of the base pairs,
  and a method for reading the pairs.

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Polymerase Chain Reaction (PCR)

• Polymerase Chain Reaction (PCR)
• Used to massively replicate DNA sequences.
• Exploits enzymes and process of replication that
  normally occurs in cells.
• How it works
• Separate the two strands with low heat
• Add some base pairs, primer sequences, and DNA
  Polymerase
• Creates double stranded DNA from a single strand.
• Primer sequences create a seed from which double
  stranded DNA grows.
• Now you have two copies.
• Repeat. Amount of DNA grows exponentially.
• 1?2?4?8?16?32?64?128?256

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Polymerase Chain Reaction

• Problem Modern instrumentation cannot easily
  detect single molecules of DNA, making
  amplification a prerequisite for further analysis
• Solution PCR doubles the number of DNA fragments
  at every iteration

1 2 4 8
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Polymerase Chain Reaction

• Polymerase Chain Reaction (PCR) is broken down
  into three separate steps which are repeated
  until enough DNA is obtained (usually between 25
  and 40 cycles)
• Step 1 Denaturation
• Temperature is raised (94oC) in order to separate
  dsDNA into single strands
• Step 2 Annealing
• Temperature decreased (50-60oC) in order for
  primers to anneal and provide a starting point
  for DNA polymerase
• Step 3 Extension
• Temperature increased (72oC) which allows Taq
  polymerase to extend DNA
• Taq polymerase is a heat resistant DNA polymerase
  isolated from the bacterium Thermus aquaticus
  which is found in hot springs and hydrothermal
  vents

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Denaturation
Raise temperature to 94oC to separate the duplex
form of DNA into single strands
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Design primers

• To perform PCR, a 10-20bp sequence on either side
  of the sequence to be amplified must be known
  because DNA polymerase requires a primer to
  synthesize a new strand of DNA

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Annealing

• Anneal primers at 50-65oC

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Annealing

• Anneal primers at 50-65oC

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Extension

• Extend primers raise temp to 72oC, allowing Taq
  pol to attach at each priming site and extend a
  new DNA strand

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Extension

• Extend primers raise temp to 72oC, allowing Taq
  pol to attach at each priming site and extend a
  new DNA strand

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Repeat

• Repeat the Denature, Anneal, Extension steps at
  their respective temperatures

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Polymerase Chain Reaction
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RT-PCR Variation of PCR

• PCR reaction amplifies DNA from a single copy in
  the absence of cells
• RT-PCR is a variant of PCR in which DNA is first
  reverse transcribed (copied in reverse) from RNA
  extracted from cells prior to being amplified by
  PCR as per usual protocol
• Reverse transcription process involves the use of
  a reverse transcriptase enzyme and various other
  reagents including either a primer for a specific
  gene or an oligo-dT primer (string of Ts that
  will bind to poly-A tail of RNA.
• Enzyme, reagent mix, primer and RNA are usually
  incubated at 37oC for 1 hour before the RT enzyme
  is inactivated at 72oC for approximately 15
  minutes. The resulting cDNA is then used as the
  template for a PCR reaction.

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RT-PCR
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Cloning DNA to achieve copies

• Use restriction enzymes and DNA ligase to insert
  the fragment of interest into the genome of
  another organism (e.g. bacteria) in order for it
  to multiply.
• The resulting DNA is referred to as recombinant
  DNA as the genes from two different organisms are
  combined.
• Once you have a large quantity of bacteria, you
  will be able to isolate a large quantity of the
  gene of interest

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Restriction Enzymes

• Discovered in the early 1970s
• Used as a defense mechanism by bacteria to break
  down the DNA of attacking viruses.
• They cut the DNA into small fragments.
• Can also be used to cut the DNA of organisms.
• This allows the DNA sequence to be in a more
  manageable bite-size pieces.
• It is then possible using standard purification
  techniques to single out certain fragments and
  duplicate them to macroscopic quantities.

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Restriction Enzymes

• Definition
• A restriction enzyme is a bacterial enzyme that
  recognises a short sequence of bases in a DNA
  molecule and cuts the DNA at this recognition
  site.
• The position where a cutting enzyme can snip is
  its recognition sequence and is where a
  particular order of nucleotides occurs.
• Some restriction enzymes cut the two strands of a
  DNA molecule at points directly opposite each
  other to produce cut ends that are blunt.
• Other cutting enzymes cut one strand at one
  point, but cut the second strand at a point that
  is not directly opposite.
• The overhanging cut ends made by these cutting
  enzymes are called sticky. These sticky ends
  are complementary.

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Blunt and Sticky Ends
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Pasting DNA

• Once separated, DNA fragments from different
  sources can be joined (ligated) together.
• Sticky ended fragments will initially join by
  hybridization or complementary base pairing.
• Bonds within the single strands of DNA are then
  repaired by DNA ligase (this is similar to the
  action of DNA ligase in linking of Ozaki
  fragments on the lagging strand during DNA
  replication)

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Gel Electrophoresis

• A copolymer of mannose and galactose, agaraose,
  when melted and recooled, forms a gel with pores
  sizes dependent upon the concentration of
  agarose.
• The phosphate backbone of DNA is highly
  negatively charged, therefore DNA will migrate in
  an electric field.

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Gel Electrophoresis

• The size of DNA fragments can then be determined
  by comparing their migration in the gel to known
  size standards
• Ethidium bromide or other dyes that bind to DNA
  are added prior to electrophoresis in order to
  visualize migration of DNA
• Ethidium bromide flouresces bright orange when
  exposed to UV light

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Reading DNA DNA Sequencing

• DNA sequencing reactions are just like the PCR
  reactions for replicating DNA with the exception
  that the reactions are run in the presence of a
  dideoxynucleotide.
• Dideoxyribonucleotides are the same as
  nucleotides, with one exception. They do not
  have 3' hydroxyl group, so once a
  dideoxynucleotide is added to the end of a DNA
  strand, there's no way to continue elongating it.
  
• Sequencing reactions are set up in groups of
  four, e.g. one containing dideoxy-A, one
  containing dideoxy-C, one containing dideoxy-G
  and one containing dideoxy-T. Each reaction tube
  contains a mix of normal nucleotides (A,C,G, T)
  and a small amount of the particular
  dideoxynucleotide.
• Taking dideoxy-C as an example, replication of
  DNA will occur as per a PCR reaction. MOST of
  the time when a C' is required to make the new
  strand, the enzyme will get a good one and
  there's no problem. MOST of the time after adding
  a C, the enzyme will go ahead and add more
  nucleotides. However, 5 of the time, the enzyme
  will get a dideoxy-C, and that strand can never
  again be elongated. It eventually breaks away
  from the enzyme, a dead end product.

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Reading DNA DNA Sequencing

• Sooner or later ALL of the copies will get
  terminated by a T, but each time the enzyme makes
  a new strand, the place it gets stopped will be
  random. In millions of starts, there will be
  strands stopping at every possible T along the
  way.
• ALL of the strands we make started at one exact
  position. ALL of them end with a T. There are
  billions of them ... many millions at each
  possible T position. To find out where all the
  T's are in our newly synthesized strand, all we
  have to do is find out the sizes of all the
  terminated products!

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Reading DNA DNA Sequencing

• Gel electrophoresis can be used to separate the
  fragments by size and measure them.
• The dideoxynucleotides present in the fragments
  have been labelled with a radioisotope or a
  flourescent dye. In the case of the latter,
  these can be read by a laser and the information
  feed back to computer.
• Following electrophoresis and visualization of
  fragments we can determine the sequence.
  Smallest fragments are at the bottom, largest at
  the top. The positions and spacing shows the
  relative sizes. At the bottom are the smallest
  fragments that have been terminated by
  dideoxynucleotides.

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Assembling Genomes

• Based on sequencing data, we can take fragments
  and put them back together.
• Not as easy as it sounds!!!!!
• SCS Problem (Shortest Common Superstring)
• Some of the fragments will overlap
• We try to fit overlapping sequences together to
  get the shortest possible sequence that includes
  all fragment sequences
• Problems that may arise during this process
• DNA fragments contain sequencing errors
• There are two complements of DNA we need to
  take into account both directions of DNA
• The repeat problem - 50 of human DNA is just
  repeats. If you have repeating DNA, how do you
  know where it goes?

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Analyzing a Genome

• How to analyze a genome in four easy steps.
• Cut it
• Use enzymes to cut the DNA in to small fragments.
• Copy it
• Copy it many times to make it easier to see and
  detect.
• Read it
• Use special chemical techniques to read the small
  fragments.
• Assemble it
• Take all the fragments and put them back
  together. This is hard!!!
• Bioinformatics takes over
• What can we learn from the sequenced DNA.
• Compare interspecies and intraspecies.

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Nucleotide Hybridization

• Single-stranded DNA or RNA will naturally bind to
  complementary strands.
• Hybridization is used to locate genes, regulate
  gene expression, and determine the degree of
  similarity between DNA from different sources.
• Hybridization is also referred to as annealing or
  renaturation.
• Hybridization uses oligonucleotides to find
  complementary DNA or RNA seqments.
  Oligonucleotides are single-stranded DNA
  molecules of 20-30 nucleotides in length.
• Oligonucleotides are made with DNA synthesizers
  and tagged with a radioactive isotope or
  fluorescent dye
• Molecular techniques based on hybridization
  include Southern blotting, Northern blotting and
  microarrays.

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Create a Hybridization Reaction
T
C

• 1. Hybridization is binding two genetic
  sequences. The binding occurs because of the
  hydrogen bonds pink between base pairs.
• 2. When using hybridization, DNA must
  first be denatured, usually by using use heat or
  chemical.

T
A
G
C
G
T
C
A
T
T
G
T
TAGGC
ATCCGACAATGACGCC
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Create a Hybridization Reaction Cont.

• 3. Once DNA has been denatured, a
  single-stranded radioactive probe light blue
  can be used to see if the denatured DNA contains
  a sequence complementary to probe.
• 4. Sequences of varying homology stick to
  the DNA even if the fit is poor.

ACTGC
ACTGC
ATCCGACAATGACGCC
Great Homology
ACTGC
ATCCGACAATGACGCC
ATTCC
Less Homology

ATCCGACAATGACGCC
ACCCC
Low Homology
ATCCGACAATGACGCC
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Southern Blotting

• Cut total genomic DNA with restriction enzymes
  and separate by electrophoresis
• Blot the fragments onto nitrocellulose filter
  paper (the Southern blot)
• Probe the blot for a particular DNA region of
  interest using a specific labelled
  oligonucleotide.
• Wash blot to remove oligonucleotide that has not
  bound.
• Identify gene or region of interest by
  visualizing regions where probe has hybridised
  with DNA on Southern blot.
• Northern blotting follows a similar process to
  Southern blotting except that RNA is run on the
  initial gel and the oligonucleotide probes are
  used to detect expression of particular genes.

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DNA Microarray
Affymetrix
Microarray is a tool for analyzing gene
expression that consists of a glass slide.
Each blue spot indicates the location of a PCR
product. On a real microarray, each spot is about
100um in diameter.
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DNA Microarray

• Tagged probes become hybridized to the DNA
  chips microarray.

Millions of DNA strands build up on each
location.
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DNA Microarrays

• An array works by exploiting the ability of a
  given mRNA molecule to hybridize to the DNA
  template.
• Using an array containing many DNA samples in an
  experiment, the expression levels of hundreds or
  thousands genes within a cell by measuring the
  amount of mRNA bound to each site on the array.
• With the aid of a computer, the amount of mRNA
  bound to the spots on the microarray is precisely
  measured, generating a profile of gene expression
  in the cell.

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An experiment on a microarray
In this schematic GREEN represents Control
DNA RED represents Sample DNA  YELLOW
represents a combination of Control and Sample
DNA  BLACK represents areas where neither the
Control nor Sample DNA  Each color in an array
represents either healthy (control) or diseased
(sample) tissue. The location and intensity of a
color tell us whether the gene, or mutation, is
present in the control and/or sample DNA.
10
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ForensicsDNA technology in action

• Each of us is genetically unique, with the
  exception of identical (monozygotic) siblings.
  While phenotypic differences are apparent among
  us, the most fundamental expression of our
  uniqueness is in our genetic material, DNA.
• Today, individuals can be identified through a
  technique known as DNA profiling.
• The amount of DNA needed for DNA profiling is
  very small because DNA can be amplified through
  the polymerase chain reaction (PCR).
• One persons DNA profile is constant, regardless
  of the type of cell used to prepare the profile.
  A DNA profile prepared from a persons white
  blood cells is identical to that prepared from
  the same persons skin cells or other somatic
  cells.
• Because DNA molecules are only slowly degraded,
  DNA profiling can be carried out on biological
  samples from crime scenes from years ago, and
  this profiling has led to the solution of many
  cold cases worldwide.

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Forensic Identification

• Identification using DNA is a powerful tool that
  can be applied in many situations including
• forensic applications
• Can the DNA found at a crime scene be matched to
  a person on the national DNA database?
• Is this blood spot from the victim or from the
  possible assailant?
• In a rape case, is this semen from a previously
  convicted rapist?
• mass disasters, such as passenger aircraft
  crashes, the 9/11 terrorist attacks, the Bali
  bombings
• Can the various remains that have been recovered
  be matched to a particular person known to have
  been on-site?
• identification of human remains
• Are these remains those of a particular missing
  person?
• Who was the unknown child, tagged as body number
  4, recovered after the sinking of the Titanic in
  1912?

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What DNA is used for identification

• Depending on the purpose and circumstances of the
  identification, the DNA used comes from either
  the chromosomes (nuclear DNA) or from
  mitochondria (mtDNA).
• In both cases the identification depends on the
  existence of segments of DNA that vary greatly
  between individuals. Such regions of DNA are
  termed hypervariable.
• Hypervariable regions of DNA that are currently
  used for identification are
• short tandem repeats (STRs) in the nuclear DNA,
  also known as microsatellites.
• hypervariable regions (HVRs) in the non-coding
  region of mtDNA.

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STRs and HVRs

• Short Tandem Repeats (STRs) in the nuclear DNA,
  also known as microsatellites.
• A large number of STRs are present on different
  human chromosomes.
• DNA from STRs can identify one person uniquely
  (apart from identical siblings).
• DNA samples from relatives are not required.
• Used when there is a need to match a DNA sample
  from a crime scene to just one particular person.
• Hypervariable Regions (HVRs) in the non-coding
  region of mtDNA.
• mtDNA identification is less precise because
  persons from the same maternal line have
  identical mtDNA profiles.
• mtDNA is used only when chromosomal DNA cannot be
  recovered or when chromosomal DNA is degraded
  because of age.
• Identification using mtDNA is mainly applied
  either
• to identify victims of mass disasters where the
  names of the victims are known but where
  identification of the remains by conventional
  means, such as visual inspection or dental
  records, cannot be done, or
• to identify decomposed remains when the identity
  is suspected to be one of a few particular
  missing persons. In both cases, there must be
  living relatives on the maternal line to provide
  mtDNA for comparison with the mtDNA from the
  remains.

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DNA Fingerprinting - HVRs

• The original technique of identification using
  DNA was called DNA fingerprinting and was
  developed as an identification tool in 1985 by
  Professor Sir Alec Jeffreys
• This technique used DNA from hypervariable
  regions, known as minisatellites, that are
  located near the ends of chromosomes.
  Minisatellites are chromosomal regions where
  sequences of 9 to 80 base pairs are repeated tens
  or hundreds of times.
• DNA fingerprinting involved cutting
  minisatellites from the chromosomal DNA with a
  restriction enzyme (Hin fI), separating the DNA
  fragments by electrophoresis, transferring them
  to a membrane using Southern blotting and
  exposing the fragments to one probe that
  hybridised to a base sequence present in all the
  minisatellites.
• This probe, known as a multi-locus probe, carried
  a radioactive label. The final result seen on an
  autoradiograph was a pattern of up to 36 bands,
  something like a barcode, with each band being
  one allele of one of the minisatellites. Because
  of the variation between individuals, each DNA
  fingerprint is unique.

The figure above shows the simplified DNA
fingerprints of two people based on just four
hypothetical minisatellites. In actual DNA
fingerprinting, the pattern for each individual
has many more bands.
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DNA profiling - STRs

• DNA fingerprinting has been replaced by a
  technique known as DNA profiling that uses short
  tandem repeats (STRs). These are
• STRs are hypervariable regions of chromosomes
  where sequences of just two to five base pairs
  are repeated over and over. These regions are
  very common and hundreds are scattered throughout
  the human chromosomes.
• STRs are termed short because the repeat
  sequences are only 2 to 5 base pairs long, and
  tandem because the repeats occur one after the
  other. However, the number of repeats at an STR
  locus can vary between people and each variation
  is a distinct allele.
• The number of repeats of a 4-base pair sequence
  at one STR locus on the number-5 chromosome
  ranges from 7 to 15.
• In most cases, the alleles at an STR locus on a
  human chromosome are named according to the
  number of repeats and so are identified as allele
  7, allele 8 and so on. The figure below shows an
  STR with 7 repeats of the sequence CATT.

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DNA profiling - STRs

• At each STR locus, one individual is either
  homozygous or heterozygous and so can have a
  maximum of just two different alleles. These
  alleles are inherited in a Mendelian fashion.
• The figure below shows that a person who is
  heterozygous 5/7 at one particular STR locus has
  one allele with 5 repeats and another allele with
  7 repeats.
• Within the gene pool of a population, however,
  many different alleles can exist at each STR
  locus.

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Frequencies of the alleles at the D5 STR locus on
the number-5 chromosome for three sample
populationsin Australia.
Note that the allele frequencies vary within a
population, with allele 11 being far more common
than allele 15. Note also that the frequencies
vary between populations, with allele 7 being
about 20 times more common in Asian populations
than in the other two populations.
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Why use STRs rather than minisatellites?

• Compared with DNA fingerprinting, DNA profiling
• is far more sensitive and requires smaller
  quantities of DNA (even a pinhead sized spot of
  blood can provide sufficient DNA) and the STRs
  can be amplified by the polymerase chain reaction
  (PCR)
• is based on alleles whose sizes allow fragments
  differing by just one base pair to be
  distinguished
• is carried out in a much shorter time hours
  rather than days
• uses several single-locus probes rather than one
  multi-locus probe
• uses coloured fluorescent labels to visualise the
  STRs rather than radioactive labels so that each
  different STR allele can be identified by colour
  as well as by size
• produces less complex patterns that are more
  easily interpreted
• In addition, unlike minisatellites, population
  data on allele frequencies of STR alleles can be
  obtained.

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DNA Profiling in Australia

• All Australian states use a common method of DNA
  profiling for forensic purposes that involves
  nine STRs from different human chromosomes.
• These STR markers were chosen for this purpose
  because they are reproducible and robust, easy to
  score, are highly informative and have low
  mutation rates.
• In addition, a tenth marker (that is not an STR)
  is used to identify the gender of the individual.
  This gender marker is the Amel locus that is
  present on both the X chromosome and the Y
  chromosome.
• The Amel gene on the X chromosome is just 107
  base pairs long while that on the Y chromosome
  contains 113 base pairs. As a result, the gender
  of a person can be identified from this marker.

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Loci currently used for DNA profiling in Australia

• For simplicity, STR loci that start with the
  letter D are identified by their chromosomal
  location only, for example, D13 or D7. In
  reality, the naming of STRs is more complex
  because there are multiple STR regions on the one
  chromosome and these two STRs (D13 and D7) are
  formally identified as D13S317 and D7S820.

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STR Profiling

• To produce a DNA profile, multiple copies of the
  alleles at these nine STRs are simultaneously
  produced using the polymerase chain reaction and
  the various alleles are then separated and made
  visible with fluorescent dyes.
• The resulting DNA profile is a series of coloured
  peaks at different locations, with each peak
  being one allele of one specific STR. The
  location of each peak indicates the size of the
  allele and hence the number of repeats.
• Where sizes overlap, alleles of different STRs
  are distinguished by fluorescent labels of
  different colours.

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STR Profiling

• A person shows either one or two peaks at each
  STR loci, where a peak corresponds to an allele,
  depending on whether the individual is homozygous
  or heterozygous at that locus.
• For the Amel gender marker, if just a single peak
  with a size of 107 base pairs appears on the
  profile, the person is female if two peaks are
  detected, one at 107 and the second at 113 base
  pairs, then the person is male.

This person is female. They are heterozygous
for loci D3, vWA, FGA, D18 and D7. They are
homozygous for loci D8, D21, D5 and D13.
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Is STR Profiling reliable?

• STR loci generate many different genotypes
  (profiles)
• For one gene locus with n different alleles, the
  number of different genotypes possible is
• n x (n 1)/2.
• An STR locus with 14 alleles can produce 105
  different genotypes in a population and a
  different STR locus with nine alleles can have 45
  different genotypes. Together, these two STR loci
  produce 105 45 4725 different genotypes.
• As the number of STR loci increases, the number
  of different genotypic combinations in a
  population increases enormously.
• As a result, a DNA profile based on nine STRs
  will be a unique combination that allows a person
  to be identified with a very high level of
  probability.
• The chance that the DNA profile of one person
  will be identical with that of another person
  (except for an identical sibling) is one in many
  billions.

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Genetic Engineering

• Genetic engineering refers to scientific methods
  for the artificial manipulation of genes
• Since these methods involve the recombining of
  DNA from different individuals and even different
  species, it is often referred to as recombinant
  DNA technology
• Genetic engineering was made possible by the
  discovery of a number of techniques and tools
  during the 1970s and 1980s
• Restriction enzymes can be used to cut DNA (from
  different sources) into pieces that are easy to
  recombine in a test tube
• Methods were developed to insert the recombinant
  DNA into cells, by using so-called vectors
  self-replicating DNA molecules that are used as
  carriers to transmit genes from one organism to
  another
• Organisms such as bacteria, viruses and yeasts
  have been used to propagate recombinant genes
  and/or transfer genes to target cells (cells that
  receive the new DNA)

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Gene Cloning

• Gene cloning is a process of making large
  quantities of a desired piece of DNA once it has
  been isolated
• Cloning allows 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
• 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
• Large quantities of the desired gene can be
  obtained if the recombinant DNA is allowed to
  replicate in an appropriate host

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Gene cloning using plasmids

• Plasmid vectors, found in bacteria, are prepared
  for cloning in the following manner
• A gene of interest (DNA fragment) is isolated
  from human tissue cells
• An appropriate plasmid vector isolated from a
  bacterial cell
• Human DNA and plasmid are treated with the same
  restriction enzyme to produce identical sticky
  ends
• DNAs are mixed together and the enzyme DNA ligase
  used to bond the sticky ends
• 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 the solution
• The actual gene cloning process (making multiple
  copies of the human gene) occurs when the
  bacterium with the recombinant plasmid is allowed
  to reproduce
• Colonies of bacteria that carry the recombinant
  plasmid can be identified by a genetic marker
  such as ampicillin resistance

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Gene cloning using plasmids
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Using bacteria to make proteins for human use
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Gene cloning using viruses

• Some bacteriophages are convenient for cloning
  large fragments of DNA (15 to 20kbp)
• Main steps in preparing a clone using viral
  vectors
• A gene is isolated from human tissue cells
• An appropriate bacteriophage vector is selected
  that is capable of infecting the target cell
• Human and the viral DNA are cut with same
  restriction enzyme
• DNAs are mixed together and the enzyme DNA ligase
  used to bond the sticky ends
• The recombinant DNA is packaged into phage
  particles by being mixed with page proteins
• 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 neighbouring bacteria.

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Gene cloning using viruses
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Transgenesis

• Trangenesis, 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
• Because of their immense economic importance,
  plants have been the subject of traditional
  breeding programmes aimed at developing improved
  varieties
• Recombinant DNA technology now allows direct
  modification of a plants genome allowing traits
  to be introduced that are not even present in the
  species naturally
• DNA can now be introduced from other plant
  species, animals or even bacteria
• Micropropagation techniques allow introduced
  genes to become par of the germ line for plants
  (the trait is inherited)
• Animal cells may become transformed (receive
  foreign DNA) to provide new enhanced
  characteristics in livestock as well as providing
  a means of curing genetic defects in humans
  through gene therapy

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Transformation using a plasmid

• Ti plasmid isolated from bacteria Agrobacterium
  tumefaciens. Agrobacterium tumefaciens causes
  tumours (galls) in plants.
• The Ti plasmid can be succesfully transferred to
  plant cells where a segment of its DNA can be
  integrated into the plants chromosome.
• Restriction enzyme and DNA ligase splice the gene
  of interest into the plasmid as discussed
  previously for cloning into plasmids
• Introduce plasmid into plant cells
• Part of the plasmid containing the gene of
  interest integrates into the plants chromosomal
  DNA
• Transformed plant cells are grown by tissue
  culture

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Transformation using a plasmid
64
Transformation by protoplast fusion

• This process requires the cell walls of plant to
  be removed by digesting enzymes
• The resulting protoplasts (cells that have lost
  their cell walls) are then treated with
  polyethylene glycol (PEG) which causes them to
  fuse
• In the new hybrid cell, the DNA derived from the
  2 parent cells may undergo natural
  recombination (they may merge)

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Transformation by protoplast fusion
66
Transformation using a gene gun

• This method of introducing foreign DNA into plant
  cells, literally shoots it directly through cell
  walls using a gene gun
• Microscopic particles of gold or tungsten are
  coated with DNA and propelled by a burst of
  helium through the cell wall and membrane
• Some of the cells express the introduced DNA as
  if it were their own

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Transformation using a gene gun
68
Transformation using 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

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Transformation using liposomes
70
Transformation using viral vectors

• Some viruses are well suited for gene therapy
  they can accommodate up to 7.5kbp of inserted DNA
  in their protein capsule
• When viruses infect and reproduce inside the
  target cells, they are also spreading the
  recombinant DNA gene
• A problem with this method involves the hosts
  immune system reacting to and killing the virus
• Common viruses used for viral transformation of
  target cells are retroviruses, lentiviruses and
  adenoviruses

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Transformation using viral vectors
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Transformation using microinjection

• DNA can be introduced directly into an animal
  cell (usually an egg cell) by microinjection
• This technique requires the use a glass
  micropipette with a diameter that is much smaller
  than the cell itself the sharp tip can then be
  used to puncture the cell membrane
• The DNA is then injected through it and into the
  nucleus

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Transformation using microinjection
74
Making an artificial gene

• Biologists get genes for cloning from two main
  sources
• DNA isolated directly from an organism
• complementary DNA (cDNA) made in the laboratory
  from mRNA templates
• One problem with cloning DNA directly from an
  organisms cell is that it often contains long
  non-coding regions called introns
• These introns can be enormous in 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 extra long DNA into their
  protein coats
• To avoid this problem, it is possible to make an
  artificial gene that lacks introns
• This is possible by using the enzyme reverse
  transcriptase which is able to reverse the
  process of transcription
• The important feature of this process is that
  mRNA has already had the introns removed, so by
  using them as the template to recreate the gene,
  the cDNA will also lack the intron region

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Gene Therapy

• By using the techniques of recombinant DNA
  technology, medical researchers attempt to insert
  a functional gene into a patients somatic cells
• This should make the patient capable of producing
  the protein encoded by that allele
• Genetic material delivered to a patients cells
  could be used to treat a number of conditions
• Restore the function of a gene that has been lost
  as a result of a mutation (i.e. possesses a
  harmful allele)
• Kill abnormal cells such as those in cancerous
  tumours
• Introduce genes that inhibit the reproduction of
  infectious agents such as viruses, bacteria and
  endoparasites
• Render cells resistant to toxic drugs used in the
  medical treatment of diseases
• By replacing missing genes or modifying faulty
  genes, it may be possible to treat genetic
  diseases
• There have been suggestions that the techniques
  of gene therapy may also be put to use to create
  designer babies that have traits that are
  selected by the parents

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Gene Therapy

• Genetic disorders that are currently undergoing
  clinical trials include
• SCIDS
• Cancers (including melanoma, breast and colon)
• Cystic fibrosis
• Haemophilia
• Rheumatoid arthritis
• Peripheral vascular disease
• Inherited high blood cholesterol
• First attempt at gene therapy was when Ashanti
  DeSilva was treated for adenosine deaminase (ADA)
  deficiency on 14 September 1990
• She received new infusions of ADA restored cells
  every 1-2 months for the first year, then every
  3-6 months thereafter.
• Ashanti is not completely cured - she still takes
  a low dose of PEG-ADA. Normally the dose size
  would increase with the patient's age, but her
  doses have remained fixed at her four-year-old
  level. It's possible that she could be taken off
  the PEG-ADA therapy entirely, but her doctors
  don't think it's yet worth the risk.
• The fact that she's alive today-let alone healthy
  and active-is due to her gene therapy, and also
  helps prove a crucial point genes can be
  inserted into humans to cure genetic diseases.

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Gene Therapy

• In contrast, eighteen-year-old Jesse Gelsinger
  died on September 17th, 1999 while enrolled in
  gene therapy trial.
• Jesse Gelsinger was not sick before died. He
  suffered from ornithine transcarbamylase (OTC)
  deficiency, a rare metabolic disorder, but it was
  controlled with a low-protein diet and drugs, 32
  pills a day.
• He was not expecting that he would benefit from
  the study, its purpose was to test the safety of
  a treatment for babies with a fatal form of his
  disorder.
• Still, it offered hope, the promise that someday
  Jesse might be rid of the cumbersome medications
  and diet so restrictive that half a hot dog was a
  treat. "What's the worst that can happen to me?"
  he told a friend shortly before he left for the
  Penn hospital, in Philadelphia. "I die, and it's
  for the babies."
• The researchers had tested their vector, at the
  same dose Jesse got, in mice, monkeys, baboons
  and one human patient, and had seen expected,
  flulike side effects, along with some mild liver
  inflammation, which disappeared on its own.
• When Jesse got the vector, he suffered a chain
  reaction that the testing had not predicted
  jaundice, a blood-clotting disorder, kidney
  failure, lung failure and brain death. It is
  thought that the adenovirus triggered an
  overwhelming inflammatory reaction -- in essence,
  an immune-system revolt.