Blotting
    • All techniques use electrophoresis to 
      separate.
    • All are based on hybridization technique
    • Difference in techniques lies in the target
    • Four applications
       –Western
       –Southern
       –Northern
       –Southwestern
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                       Hybridization
       The binding between ss labeled probe to a 
       complementary nucleotide sequence on the target DNA.
       Degree of hybridization depends on method of probe 
       labeling (radioacitve or non-radioactive system e.g. 
       biotin-avidin.
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                                      Probes
           • Labeled material to detect a target.
           • For DNA: 20-30 nucleotides, complementary to a 
             region in the gene
                                 • Methods of labeling: 
             •                 32
              Radioactive e.g.  P                       • Non-radioactive e.g. Biotin
         •                                       •
          Sensitive                               Sensitive
         •                                       •
          Relatively cheap                        Relatively expensive
         •
          Hazardous
         You should follow the 
         radioactive waste disposal                                             *
                                                         Biotin          Avidin
         regulations.                          Probe
           Probe    *
                                              Target DNA
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           Target DNA
                   Southern blots
    • Named after British biochemist Edwin Southern
       – Southern EM. Detection of specific sequences among DNA fragments separated 
         by gel electrophoresis.
         J. Mol Biol. 1975 Nov 5;98(3):503-17. 
    •  A technique for identifying specific sequences of DNA in which DNA fragments are 
      separated by electrophoresis, transferred to a membrane, and identified with a 
      suitable probe.
    • Detects restriction fragments following a restriction enzyme digest of genomic DNA 
      (RFLPs)
    • Detects RT-PCR fragments
    • Can be used to screen cDNA libraries
    • Can be referred to as DNA blotting
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                   Southern Blot
    • Southern blot hybridization is one of the 
      most commonly used molecular 
      techniques to detect specific DNA 
      sequences using labeled probes.
    • Southern blotting is a process that transfer 
      the DNA fragments separated by 
      electrophoresis into a nitrocellulose 
      membrane/nylon
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