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Southern Blot

1975
  • Edwin Southern
Cena de laboratório demonstrando a técnica de Southern blot para análise de DNA em genética molecular.

(Imagem gerada apenas para fins ilustrativos)

The Southern blot is a método used for the detection of a specific DNA sequence in a DNA sample. It combines transfer of electrophoresis-separated DNA fragments to a filter membrane with subsequent fragment detection by a labeled DNA probe. The technique allows researchers to identify a specific gene within a complex DNA mixture based on size and sequence.

The Southern blot, named after its inventor Edwin Southern, is a powerful and specific method for detecting a target DNA sequence within a complex mixture of DNA. It was a foundational technique in molecular genetics before the widespread adoption of PCR and sequencing. The process is a multi-step procedure that combines size separation with specific molecular recognition. It begins with the digestion of the total genomic DNA sample using one or more restriction enzymes, which cleaves the DNA into fragments of varying sizes. These fragments are then separated according to their size by agarose gel electrophoresis. The gel now contains a smear of countless DNA fragments, sorted by length. The crucial next step is the transfer, or ‘blotting,’ of these separated DNA fragments from the fragile gel onto a more durable membrane, typically made of nitrocellulose or nylon. This is achieved through capillary action, which wicks a buffer solution through the gel and the membrane, carrying the DNA along with it and immobilizing it on the membrane’s surface. The DNA is first denatured into single strands so it can bind to the probe. The membrane now holds a faithful replica of the DNA fragment pattern from the gel. To find the specific gene of interest among the thousands of fragments, a labeled probe is used. This probe is a single-stranded piece of DNA (or RNA) that has a sequence complementary to the target gene and is tagged with a marker, such as a radioactive isotope ([latex]^{32}P[/latex]) or a fluorescent/chemiluminescent molecule. The membrane is incubated with this probe, which hybridizes (forms a double helix) only with its complementary DNA fragment on the membrane. After washing away any unbound probe, the location of the hybridized probe is detected, revealing a band that corresponds to the size of the DNA fragment containing the target gene.

UNESCO Nomenclature: 2406
Biologia Molecular

Tipo

Processo Químico

Interrupção

Incremental

Uso

Nicho/Especializado

Precursores

  • gel electrophoresis for DNA separation
  • discovery of restriction enzymes
  • understanding of DNA hybridization principles
  • development of radioactive labeling techniques for probes
  • techniques for creating nitrocellulose membranes

Aplicações

  • gene mapping
  • diagnosing genetic diseases (e.g., sickle cell anemia)
  • identifying gene mutations or rearrangements
  • forensic DNA analysis (RFLP)
  • paternity testing
  • detecting transgenes in GMOs

Patentes:

NA

Ideias de Inovação Potencial

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Related to: Southern blot, Edwin Southern, DNA detection, hybridization, DNA probe, RFLP, blotting, molecular biology, gene identification, electrophoresis.

Contexto histórico

Southern Blot

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(Caso a data seja desconhecida ou irrelevante, por exemplo, "mecânica dos fluidos", é fornecida uma estimativa aproximada de seu surgimento notável)

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