Topic 8, Southern and Northern blots.ppt.edu

Topic 8, Southern and Northern blots.ppt.edu - Topic...

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Topic 8—Hybridization expts (Southerns and Northerns)
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Learning Objectives Be able to: define the following terms: blotting, hybridization/annealing, denaturation/melting, probe, loading control, in situ hybridization explain how hybridization can be used to detect specific nucleic acid sequences explain the concept of denaturation/melting of DNA and what factors can control it describe how to label probes for hybridization diagram how electrophoresis and blotting are performed differentiate between targets of Northern, and Southern, and Western blots interpret results of a blotting experiment
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Blotting Blotting defined: transferring nucleic acids or proteins from a gel (after electrophoretic separation) onto a paper (a.k.a., a blot) Why blot? To detect and/or quantify specific molecules, since cells often have a huge diversity of molecules present Probing methods don’t work well with gels because probes/antibodies cannot readily reach target molecules due to thickness of the gel. If target is on the surface of paper, easy for probe/antibody to find and bind to target because it is on the surface.
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Southern blots—target DNA
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Small pieces of DNA digested with restriction enzymes
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Genomic DNA, cut with a restriction enzyme. No distinct bands are visible. Why? Because there are so many different size fragments produced from a large genome that you can’t see any individual bands You need to find a way to detect one specific fragment of DNA amongst many different fragments To find what you’re looking for, you use a technique called blotting and hybridization
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Hybridization defined: the annealing (coming together/sticking together) of 2 single-stranded nucleic acids to make 1 double-stranded molecule What would promote these 2 strands to stick together?
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Base-pairing is the key to nucleic acid hybridization CCTAGCGGA * If these 2 rules are met, then 2 single-stranded nucleic
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Topic 8, Southern and Northern blots.ppt.edu - Topic...

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