Southern blotting

Southern blotting

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  ©2000 Timothy G.Standish Southern Blot Pankaj Kapruwan M.Sc + M.tech (NT)
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  ©2000 Timothy G.Standish What is southern blotting?  Reveals information about DNA identity, size and abundance.  Developed by Edwin Southern in 1975.  Classic technique that involves separating DNA fragments based on size via electrophoresis, transferring them into a membrane, hybridization with a labelled sequence specific probe, washing and finally detection of labelled DNA band (s).
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  ©2000 Timothy G.Standish Method- Involve four steps: Digestion of DNA using restriction enzymes. Separation of the DNA fragments by size using gel electrophoresis. Transfer of fragments to a nitrocellulose or nylon membrane. Hybridization of a probe to the fragment or fragments of interest Probe detection.
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  ©2000 Timothy G.Standish  Restriction enzyme: An enzyme found in bacteria that cuts DNA at a certain sequence Restriction fragment: a small segment of a DNA strand that was “cut” with a restriction enzyme. Sticky end Blunt end CCGG GGCC
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  ©2000 Timothy G.Standish Step1: Preparing restriction fragments Each sample of DNA is mixed with the same restriction enzyme, resulting in thousands of restriction fragments.
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  ©2000 Timothy G.Standish 1 2 3 Digestion and Electrophoresis Experimental 3 Marker 1 Control 2
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  ©2000 Timothy G.Standish Membrane DNA Transfer To Membrane DNA Gel Buffer Gel Membrane Paper Towels
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  ©2000 Timothy G.Standish Membrane with bound DNA Addition of blocking reagent Parts of the membrane not already covered with DNA now bind blocking reagent Probe covers the membrane, but only binds to complimentary DNA Probe addition After washing Probe only remains annealed to complimentary DNA Probe Hybridization
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  ©2000 Timothy G.Standish Fragments complimentary to the probe appear as bands on the autorad Autorad Development Membrane with probe bound to complimentary DNA X-ray film is placed over the membrane and left until radiation from the probe has exposed the film
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  ©2000 Timothy G.Standish Applications  To identify a single gene among thousands of fragments of DNA and to detect sequence of DNA in an organic genome.  In gene discovery and gene mapping.  Can be used to identify methylated sites in particularly genes.  In DNA finger printing, genetic engineering.
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  ©2000 Timothy G.Standish