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Chapter Six

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RNA: by transcription from DNA cloned in an expression vector. Probe is single stranded. ... Hybridization assays using cloned target DNA (libraries) and microarrays. ... – PowerPoint PPT presentation

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Title: Chapter Six


1
  • Chapter Six
  • Nucleic Acid Hybridization Principles
    Applications
  • Preparation of nucleic acid probes
  • - DNA from cell-based cloning or by PCR. Probe
    is double stranded. Labeling by DNA
    polymerase-based DNA strand synthesis.
  • - RNA by transcription from DNA cloned in an
    expression vector. Probe is single stranded.
    Labeling by run-off transcription.
  • - Oligonucleotide by chemical synthesis. Probe
    is single stranded. Labeling is by end labeling.

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  • DNA and RNA probes could be labeled in vitro by
    one of two methods
  • - Strand synthesis. By using DNA or RNA as a
    template to generate a labeled DNA strand. DNA or
    RNA polymerase are used and one of the four dNTPs
    in the reaction usually has a labeled group e.g.
    32P- dCTP. DNA could be labeled by
    nick-translation, random primed labeling, or
    PCR-mediated labeling. RNA probes are labeled by
    in vitro transcription.
  • - End-labeling Used in labeling single strand
    probes by adding one (kinase end-labeling) or
    very few (fill-in end-labeling) labeled groups at
    the 5 end.

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  • Isotopic labeling is detected by exposure to
    X-ray film (autoradiography) and also by counting
    the dpm of the labeled molecule using a
    scintillation counter.
  • Non-isotopic labeling includes
  • - Direct labeling using modified nucleotides
    containing a fluorophore (a chemical group that
    when exposed to light of certain wavelengths will
    fluoresce).
  • - Indirect labeling using a reporter molecule
    attached to a nucleotide precursor (a spacer of
    11-16 side C chain is used to distant the
    reporter from the nucleotide). An affinity
    molecule binds very strongly to the reporter
    molecule. Affinity molecules could be detected
    by a conjugated marker molecule. Two widely used
    methods are biotin-streptavidin (detected by
    fluorophores) and digoxigenin.

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  • 2. Principles of nucleic acid hybridization
    (NAH)
  • NAH is used to identify how close DNA molecules
    are. Factors to consider when performing a NAH
    assay between a probe and a target molecule are
  • - strand length
  • - base composition
  • - chemical environment monovalent cations
    stabilize the duplex while polar molecules such
    as formamide and urea are chemical denaturants.
  • - melting temperature
  • - hybridization stringency temperature and
    salt concentration (high NaCl conc. and low temp.
    is low stringency while low NaCl and high
    temperature is high stringency).

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  • 3. NAH assays
  • Dot-blot hybridization used with
    allele-specific oligonucleotides ASO probes where
    the probes are labeled and hybridized to
    immobilized target genomic DNA. In reverse blot
    hybridization, the ASO probes are not labellled
    and are immobilized on a membrane then hybridized
    to the labeled target DNA (genomic DNA).

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  • Southern and Northern blot hybridizatoions are
    used to hybridize a labeled probe to fractionated
    and immobilized DNA (Southern) or RNA (Northern).

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  • 3. Pulsed field gel electrophoresis (PFGE) used
    in conjugation with rare-cutter restriction
    endonucleases that recognize CpG islands in
    genomic DNA of vertebrates. CpG islands occur at
    low frequency in human (or other vertebrate) DNA
    which results in few recognition sites and a
    small number of large fragment (Mb range).

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  • In situ hybridization
  • a probe is hybridized to a chromosome
    preparation (on a microscopic slide) or to RNA of
    a tissue fixed on a slide

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  • Hybridization assays using cloned target DNA
    (libraries) and microarrays.
  • Colony hybridization and plaque lift
    hybridization.

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  • Gridded high density arrays of transformed cell
    clones or DNA clones now performed by robotic
    gridding devices. Membranes filters could be
    copied and distributed to a lrage number of
    laboratories.

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  • DNA microarray technology here the filters
    (membranes) have been replaced by a microscopic
    slide chemically-treated (e.g. nitrocellulose
    coated microscopic glass slides). Two type of
    microarrays depending on how the nucleic acid
    samples were generated and delivered to the
    microarray

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  • - microarrays of pre-synthesized nucleic acids
    individual DNA clones or oligonucleotides are
    spotted at individual locations (x, y coordinates
    of a miniaturized grid) using a robote.

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  • - micrarrays of oligonucleotides synthesized in
    situ here DNA chips are constructed by taking
    advantage of photolithography and the chemistry
    of oligonucleotide synthesis. The probe is a set
    of unlabeled nucleic acids fixed to the
    microarray. The target DNA (e.g. genomic DNA) is
    labeled with a fluorophore and allowed to mix
    with the micorarray to form heteroduplexes. After
    washing the microarray of excess hybridization
    solution, a laser scanner is used to acquire an
    image of the fluorophores (Cy3, green excitation
    Cy5 red excitation) to produce a ratio image.
    Digiatl imaging software is used to analyze a
    signal emitted by each spot on the microarray.

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  • DNA microarray technology has very important
    applications in biomedical research and
    diagnostic approaches. Two main principal
    applications are
  • 1. Expression screening here RNA expression
    levels are monitored by using using cDNA
    microarrays or gene-specific oligonucleotide
    microarrays.
  • 2. DNA variation screening Oligonucleotide
    microarrays are used. Will be used for assaying
    for mutations in known human disease genes
    (diagnostic). Will also be used to identify and
    catalog human single nucleotide polymorphism
    (SNP) markers.
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