Molecular Genetics

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• In Southern blotting, denatured DNA is transferred from an electrophoresis gel to a sheet of nitrocellulose or derivatized nylon.
• The exploded view at left shows that transfer is accomplished by the flow of buffer through wick, gel, sheet and into adsorbent paper layers.
• DNA is retained by the derivatized nylon or nitrocellulose. After transfer, the DNA is permanently attached by heating or by cross-linking using UV radiation.
• Spatial arrangement of DNA in the gel is preserved in the sheet.

• In hybridization, the sheet with attached DNA can be incubated with a tagged denatured DNA population (probe).
• If probe molecules are complementary to molecules bound on the sheet, double-stranded DNAs will form, thus binding the probe to the sheet.
• After excess probe and poorly bound probe molecules are removed by washing, the only tagged molecules left are those that have formed hybrids with the target DNA.

• Uses:
  • RFLP
  • VNTR
  • Gene cloning
  • chromosome walking
  • genome mapping
  • gene identification

• Variations:
  • Dot and slot blots, in which the sample is spotted directly on the nylon or nitrocellulose sheet without prior separation.
  • Northern blots in which the molecules separated by electrophoresis are RNAs instead of DNAs.
  • Colony lifts, in which the pattern of colonies on an agar plate is reproduced as a pattern of DNA spots on a nitrocellulose or nylon sheet.
  • Plaque lifts, similar to colony lifts except the material lifted is from a phage plating.
• Further information:
  • A "Shockwave" animation is available.
  • Technical hints provided by commercial concerns are available.