Nucleic Acidbased Methods

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					Nucleic acid-based methods (I)
Structure of DNA
   Complementality of DNA
(high specificity in base paring)
Replication of DNA
Easy manipulation of DNA
Hybridization
      Nucleic acid-based methods
• Gene probing
  – Colony hybridization
  – Southern/Northern hybridization
  – Microarray
• Polymerase Chain Reaction (PCR)
  –   PCR
  –   RT-PCR
  –   Multiplex PCR
  –   Nested PCR
               Gene probes
• Small pieces of DNA complementary to the
  target sequence of interest
• Labeling options
  – Radioactive chemicals
  – Nonradioactive alternatives (DIG, biotin, or
    fluorescein)
Gene probe detection
Colony hybridization
Southern hybridization
Gel electrophoresis
Gel electrophoresis (I)
Gel electrophoresis (II)
DNA on a gel
Southern hybridization
  An application of southern
hybridization (forensic science)
   Microbe Nucleic Acid Detection by DNA Microarrays
              or “Gene Chip” Technology
Generate/obtain DNA complimentary to genes
   (sequences) of interest;
    – 1000s of different ones
Apply tiny quantities of each different one onto
   solid surfaces at defined positions
    – “gene chip” or “DNA microarray”
Isolate or amplify target NA of interest and label
    with a fluorescent probe
Apply sample NA to the “gene chip” surface
    – Sample NA binds to specific DNA probes on
      chip surface; wash away unbound NA
Detect bound DNA or RNA by fluorescence after        Fluorescing Gene Chip or
   laser excitation
                                                     DNA Microarray
Analyze hybridization data using imaging
   systems and computer software
Polymerase Chain Reaction
         (PCR)
         Primers and enzymes
• Primers
  – Short nucleotides complementary to a target DNA
  – Upstream and downstream primers
• Taq polymerase
  – Heat stable DNA-dependant DNA polymerase
  – Isolated from thermophilic bacteria (Thermus
    aquaticus)
  – Withstand temperature up to 98oC
      Different PCR techniques
•   PCR
•   RT-PCR
•   Multiplex PCR
•   Nested PCR
           Agarose Gel Electrophoresis
• Separate nucleic acid fragments in
  an agarose gel
• Resolves small DNA molecules:
  0.1 to 50 kb
• % agarose determines resolution
  of DNA size:
   – 0.3% w/v: resolves 5 to 50 kb
   – 2% w/v resolves 0.1 to 2 kb
• Resolving large molecules (up to
  500 kb) requires specialized
  methods                              DNA      Specific
   – Pulse-field gel electrophoresis   marker   DNA
     (PFGE)                            ladder   fragment
Example: RT-PCR and Oligoprobe Detection of
          Enteroviruses in Water
  Real-Time PCR and Quantitative Fluorogenic Detection
• Molecular beacon. Several 5'
  bases form base pairs with
  several 3' bases; reporter and
  quencher in close proximity.
   – If reporter is excited by light,
     its emission is absorbed by
     quencher & no fluorescence is
     detected.
• Detection of PCR product by
  molecular beacon.
   – Beacon binds to PCR product
     and fluoresces when excited
     by the appropriate  of light.
   – [Fluorescence] proportional to
     [PCR product amplified]

				
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posted:9/13/2012
language:English
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