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DNA Technology and Genomics

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Title: DNA Technology and Genomics


1
DNA Technology and Genomics
2
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

3
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

4
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.

5
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.

6
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

7
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
8
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

9
Denaturation
Raise temperature to 94oC to separate the duplex
form of DNA into single strands
10
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

11
Annealing
  • Anneal primers at 50-65oC

12
Annealing
  • Anneal primers at 50-65oC

13
Extension
  • Extend primers raise temp to 72oC, allowing Taq
    pol to attach at each priming site and extend a
    new DNA strand

14
Extension
  • Extend primers raise temp to 72oC, allowing Taq
    pol to attach at each priming site and extend a
    new DNA strand

15
Repeat
  • Repeat the Denature, Anneal, Extension steps at
    their respective temperatures

16
Polymerase Chain Reaction
17
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.

18
RT-PCR
19
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

20
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.

21
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.

22
Blunt and Sticky Ends
23
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)

24
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.

25
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

26
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.

27
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!

28
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.

29
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?

30
(No Transcript)
31
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.

32
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.

33
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
34
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
35
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.

36
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.
37
DNA Microarray
  • Tagged probes become hybridized to the DNA
    chips microarray.

Millions of DNA strands build up on each
location.
38
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.

39
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
40
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.

41
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?

42
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.

43
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.

44
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.
45
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.

46
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.

47
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.
48
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.

49
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.

50
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.

51
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.

52
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.
53
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.

54
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)

55
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

56
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
58
Using bacteria to make proteins for human use
59
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
61
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

63
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)

65
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

69
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

71
Transformation using viral vectors
72
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

73
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

75
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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77
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.

78
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.
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