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Updated: Mar 8 2020


  • Blotting Procedures
    • Function
      • probe for specific substance in solution
        • Southern blot - used to analyze DNA
          • normally used to examine the presence of a particular DNA sequence
          • remember: Southern Dixieland
        • Northern blot - used to analyze RNA
          • normally used to examine gene expression
        • Western blot (immunoblot) - used to analyze protein
    • Process
      • run a gel electrophoresis to separate the components of the solution
      • bands are transferred ("blotted") to a filter/membrane
      • radiolabeled or fluorescently labeled probe is incubated with the membrane
        • Southern blot - 32P-DNA
        • Northern blot - 32P-DNA
        • Western blot - enzyme-linked or 131I antibody
      • specific probe binds with high specificity to DNA/RNA segment or protein of interest
      • membrane is visualized under conditions to illuminate the probe
    • Clinical use
      • restriction length polymorphism (RFLP)
        • can be used for genetic testing
        • based on the principle that individuals have unique/heritable variations in RFLPs
          • loss or gain of a restriction site changes the fragment lengths after digestion by an endonuclease
          • variation provided in part by variable number tandem repeats (VNTRs)
        • Southern blot used to visualize results
        • ex.) testing for sickle-cell anemia
          • wild type (wt) gene contains a restriction site in the gene fragment
          • sickle cell (sc) gene lacks this restriction site
          • the diagnostic results following a Southern blot
            • wt homozygote would have only medium and small fragments
            • heterozygote would have large, medium and small fragments
            • sc homozygote would have only large fragments
      • gene expression profiling
        • measure whether a particular region of DNA is being expressed
        • Northern blot used to visualize results
        • ex.) testing for fragile X syndrome
          • ↓ expression of FMR1 gene
          • trinucleotide repeat disorder
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