PFGE

1. 본문

Pulsed-field gel electrophoresis (PFGE) is a laboratory technique used to separate very large DNA molecules. Here's a breakdown of what it is and how it works:
What is PFGE?

• Separation of Large DNA: PFGE is a type of electrophoresis that separates large DNA molecules, including entire genomic DNA, by applying an electric field to a gel matrix. The electric field periodically changes direction.
• Larger than Standard Electrophoresis: Standard gel electrophoresis can only separate DNA fragments up to about 50 kb (kilobases). PFGE, however, can resolve fragments up to 10 Mb (megabases), enabling the analysis of genomic DNA.
• Gold Standard: PFGE is often considered the "gold standard" in epidemiological studies of pathogenic organisms due to its ability to discriminate between different strains of bacteria.

• Alternating Electric Field: Unlike standard gel electrophoresis, which uses a constant, unidirectional electric field, PFGE applies an electric field that periodically changes direction. The apparatus uses multiple electrodes that surround the gel, and activating specific electrodes allows modulation of the electric current.
• Reorientation and Movement: The alternating field forces large DNA molecules to reorient themselves before continuing to move through the gel. Larger DNA molecules take longer to realign to the changes in field direction compared to smaller molecules. This difference in reorientation time leads to a greater degree of separation.
• Procedure:
• Intact chromosomal DNA is isolated from cells embedded in agarose plugs. This avoids mechanical shearing of the large DNA molecules.
• The DNA within the agarose plug is digested with rare-cutting restriction enzymes, producing large DNA fragments.
• The digested DNA is subjected to PFGE, where the alternating electric field separates the fragments based on size.
• Pulse times are usually equal for each direction, creating a net forward migration of DNA.

• Genotyping/Genetic Fingerprinting: PFGE is used for genotyping and genetic fingerprinting in epidemiological studies.
• Physical Mapping: It enables the construction of physical maps of chromosomes from various bacterial species by characterizing large DNA fragments.
• Epidemiological Studies: It's considered a gold standard for subtyping and discriminating among strains of pathogenic organisms, helping to link environmental or food isolates with clinical infections.