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Dot Blot Technique - This technique is used to detect the presence of a given sequence of DNA/ RNA in the nonfractionated (not subjected to gel electrophoresis) DNA. Sample DNA's from several tissues/individuals can be tested in a single test run.

Dot blots are useful in detecting the presence of the sequence being transferred in a number of suspected transgenic individuals, and the presence of a specific mRNA in several such individuals or in different tissues of a single individual. The generalized procedure of dot blot is briefly described below.

1. The sample DNAs (or RNAs) from different individuals or tissues are transferred onto a nitrocellulose filter in form of dots; several samples are dot blotted onto a single filter. A sample

DNA or RNA is the total DNA or RNA extracted from an individual or a tissue.

2. The DNA is first denatured and then the filter is backed at 80ºC to fix the DNA firmly onto the filter. The filter is now pretreated appropriately to prevent nonspecific binding of the probe to the filter (see, Southern hybridization).

3. The filter is then treated with the appropriate radioactive single stranded DNA probe under conditions favouring hybridization. The filter is now washed repeatedly to remove the free probe.

4. Dots having the appropriate DNA or RNA sequence will hybridize with the radioactive probes. These dots are detected by autoradiography; the intensity of dot in the autoradiograph corresponds fairly well with the extent to which DNA or RNA is represented in the sample.

The dots that show up in the autoradiograph denote the individuals or tissues in which the DNA or RNA sequence corresponding to the probe is represented. In case of transgenic individuals, Southern hybridization may then be used for the dot blot positive individuals to derive more precise information on whether and where the sequence in question is integrated into the genome.

 
 

 
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