Chapter Six

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• 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.