Lec12_W11

Lec12_W11 - 1. Hybridization techniques Nucleic acid...

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1. Hybridization techniques Nucleic acid hybridization based techniques use complementary DNA probes = pieces of ssDNA designed to be complementary to a target DNA sequence. So probes are similar to PCR primers, except that probes have some kind of tag – usually radioactivity so that probe can be localized. In hybridization methods, a labeled probe is used to detect a clone or DNA fragment that contains a sequence complementary to the probe
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Southern blots Say you have a vector with a large insert that you have identified as containing your sequence of interest and now you want to narrow down the area in this piece of DNA as to where a particular gene or smaller sequence is located: do a Southern blot. 1. In the first step, the high molecular weight DNA is cut by restriction enzymes to create smaller fragments. These fragments are separated on a agarose gel. 2. The restriction fragments within the gel are denatured by soaking in alkaline solution The alkali causes the DNA strands to denature – you need to do this so probe has access to its complementary strand. The gel is then covered by a membrane and put in contact with a solution containing salt – the DNA is then wicked up from the gel to the nylon membrane where it binds.
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The transfer creates an exact replica of the DNA within the gel on a solid support, the nylon membrane, that is easier to work than a gel.
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Southern blot cont. 3. The next step is called prehybridization. During this step the membrane containing the DNA is pretreated with a buffer containing blocking reagents such as albumin or salmon sperm DNA. These treatments block any areas on the membrane that might bind the probe nonspecifically. If prehybridization is inadequate, the blot may
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Lec12_W11 - 1. Hybridization techniques Nucleic acid...

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