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Application Overview


Nucleic acid blotting is a well established technique that is used to identify the presence of a specific nucleotide sequence that was previously separated in a gel. (For more information on separation of mixtures of nucleic acid by gel electrophoresis, please refer to the section called “Nucleic acid electrophoresis”.)

Southern blotting was named after its inventor, the British biologist Edwin Southern. In Southern blotting, DNA is analyzed, and in Northern blotting if it is RNA. In both cases, the DNA or RNA from the intact gel is transferred onto a piece of nitrocellulose or nylon membrane. The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA (or RNA) is to be determined. The probe DNA is labeled so that it can be detected by radiography, fluorescence, or chemiluminescence.

The first step in blotting is the transfer of the nucleic acids from the gel to a membrane, which is conventionally done using the capillary transfer method. In this procedure, the gel is placed on top of a paper wick that dips into a reservoir containing the transfer buffer. The membrane is sandwiched between the gel and a stack of absorbent material (usually dry paper towels) that serves to draw the transfer buffer through the gel by capillary action. The nucleic acid molecules in the gel are then carried by the buffer and become immobilized on the membrane. Vacuum blotting is another blotting procedure that has grown in popularity because it is faster and gives excellent recoveries. Vacuum blotting also uses a flow of buffer to transfer nucleic acid bands onto the membrane, but it uses a vacuum instead of capillary action to create the flow. The membrane is placed on a porous support, the gel is placed over it, vacuum is applied and the buffer is added to submerge the gel.

Once the transfer is complete, all of the nucleic acid bands originally on the gel is immobilized (blotted) on the membrane and the process of hybridization may begin. The membrane is incubated with small piece of DNA or RNA (the probe), which has a sequence that is complementary to a particular DNA or RNA sequence in the membrane. This allows the probe to hybridize, or bind, to a specific DNA fragment on the membrane. The probe has previously been labeled, typically with radioactive atom, fluorescent dye or tagged with an enzyme that generates a chemiluminescent signal when incubated with an appropriate substrate. After hybridization, the labeled probe allows the DNA fragment of interest to be detected from among the many different DNA fragments on the membrane.


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