Southern blotting procedure

Southern Blotting.

Southern Blotting is a technology that can be found in DNA compromise. It brings together the use of limitation enzymes, electrophoresis, and DNA probes.

southern blotting procedure

A. Experimental Procedure:

This method, named after its founder, Edward The southern part of, involves the following steps. First, DNA is extracted from cells, for example, a patient’s leukocytes. Second, the DNA is cleaved into many fragments using a restriction enzyme, Third, the resulting fragments are separated on the basis of size by electrophoresis. [Note: As the large fragments move less quickly than small fragments, the measures of the pieces, usually indicated as the number of platform sets, can be measured from evaluation of the place of the group comparative to plain fragments of known size.] The DNA nitrocellulose in the gel are denatured and moved (blotted) to a nitrocellulose tissue layer for analysis. If the original DNA presents the individual’s entire genome, the enzyme digest contains a million or more fragments. The gene of interest is on only one (or a few of the gene itself was fragments) of these pieces of DNA. If all the DNA segments were visualized by a nonspecific technique, they would appear as an unresolved blur of overlapping bands. To avoid this, the last step in Southeast blotting uses a sensor/probe to recognize the DNA pieces of interest. The styles noticed on Southeast mark research depend both on the specific limitation endonuclease and on the sensor/probe used to imagine the limitation fragments. [ Note: Variants of the southern blot have been facetiously named “Northern” (electrophoresis of mRNA followed by hybridization with a specific probe), and “western” (electrophoresis of protein followed by detection with an antibody directed against the protein of interest), neither of which relate to anyone’s name or to points of the compass.]

B. Detection of mutations:

The existence of a mutation impacting a set limit site causes the design of groups to stand out from those seen with a normal gene. For example, a change in one nucleotide may alter the nucleotide sequence so that the restriction endonuclease fails to recognize and cleave at that site (for example Person 2 lacks a restriction site present in Person 1), Alternatively, the change in a single nucleotide may create a new cleavage site that results in new restriction fragments. A mutation may not affect a restriction site of one specific restriction enzyme but may be revealed by using a different restriction enzyme whose recognition sequence is affected by the mutation. [Note: Most sequence differences at restriction sites represent normal variation present in the DNA, and those found in the noncoding regions are often silent and, therefore, are generally not clinically significant.]

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