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20. 01. 14

Pulsed-field Gel Electrophoresis (PFGE)

 

Pulsed-field Gel Electrophoresis (PFGE) is a method which uses a controlled electric current to separate biomolecules, according to their size and electric charge, through an agarose matrix.

This technique was developed by Schartz and Cantor in 1984 as a tool to examine the chromosomal DNA of eukaryotic organisms. It has been one of the most useful advances of molecular epidemiology in the last decades, becoming a highly effective technique for the characterization of many bacteria, both Gram-positive and Gram-negative species.

PFGE achieves the separation of large DNA fragments by inducing its reorientation through periodic changes in the electric field. Duration of electric fields determines the range of sizes that can be separated- the larger the size of the fragments to separate, the longer the duration of the applied fields has to be.

The duration of the alternating electric fields is called the pulse duration and determines the range of DNA sizes which can be separated. These intervals are highly variable, from fractions of a second for very small fragments, to up to 1-2 hours for very large fragments (≥5Mb)

The preparation of agarose gels does not differ from the preparation of other DNA electrophoresis; D-5 agarose gels are used at 0,5-1% (20x20 or 15x15 cm).

Uses

  • Chromosome identification

  • Location of a particular gene

  • Polymorphism analysis of chromosome size

  • Isolation of a whole chromosome (for subsequent analysis by restriction enzymes, etc.)

D5 Agarose

D-5 Agarose is a linear polymer with a very high molecular weight, forming gel structures unlike those of traditional agaroses. This characteristic, together with the very low sulfate content, produces a strong intercatenary interaction, yielding a gel with a very high strength and higher exclusion limit.

Advantages

  • Extremely high gel strength: allowing lower gel concentrations (0.3%), enabling it to be applied not only with high molecular weight nucleic acids, including chromosomes, but also with large particles like viruses and ribosomes

  • High electrophoretic mobility: DNA mobility is greater when compared with D-1LE.Electrophoresis times are reduced depending on the buffer and agarose concentration used

  • Easy gel preparation: simple dilution in aqueous buffers by standard boiling or microwaving

  • Greater thermal stability due to high hysteresis

  • Exceptionally low absorption of staining agents

  • Absence of toxicity

 

 

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