Pulsed Field Gel Electrophoresis (PFGE) is a specialized laboratory technique used to separate exceptionally large DNA molecules, ranging from tens of thousands to several million base pairs. This method allows researchers to analyze DNA fragments far beyond the capabilities of traditional techniques, making it a widely accepted tool in various scientific disciplines.
The Challenge of Separating Large DNA
Standard gel electrophoresis separates DNA fragments based on their size and charge. In this process, DNA, which is negatively charged, moves through a gel matrix towards a positive electrode when a constant electric field is applied. Smaller DNA molecules navigate the pores of the gel more easily and thus travel faster and farther than larger ones, leading to their separation into distinct bands.
This conventional method works effectively for DNA fragments up to approximately 20 to 50 kilobases (kb). However, a significant problem arises with molecules exceeding roughly 50 kb. Beyond this size, the DNA molecules become so long that they essentially move through the gel as if they were all the same size, regardless of their actual length.
Larger DNA molecules tangle within the gel’s porous structure, causing them to migrate at a similar, low speed. Instead of discrete bands, these fragments accumulate at the top of the gel or appear as a continuous “smear,” making size distinction impossible. This limitation of standard gel electrophoresis highlighted the need for a more sophisticated technique capable of resolving megabase-sized DNA.
How Pulsed Field Gel Electrophoresis Works
Pulsed Field Gel Electrophoresis overcomes the limitations of standard methods by employing a unique approach to the electric field. Instead of a constant, unidirectional electric field, PFGE periodically changes the direction of the electric field applied to the gel. This alternating field allows very large DNA molecules to untangle and reorient themselves as they migrate through the gel matrix.
When the electric field direction changes, the elongated DNA molecules must first “relax” or untangle from their current orientation within the gel pores. Following this relaxation, they then reorient themselves to align with the new direction of the electric field before they can resume migration. The time required for this re-entanglement and re-alignment process is directly proportional to the size of the DNA molecule.
Smaller DNA molecules can reorient and move more quickly when the field changes direction. In contrast, larger DNA molecules take a longer time to untangle and realign, causing them to migrate more slowly through the gel over the entire duration of the run. This differential reorientation time allows for the effective separation of DNA fragments ranging from approximately 10 kilobases up to 10 megabases (Mb), a range significantly larger than that achieved by conventional electrophoresis.
The electric field switches between multiple directions. Pulse times can be adjusted; longer times are used for separating larger DNA fragments, while shorter times are better for smaller ones.
Key Applications of PFGE
PFGE’s ability to separate large DNA fragments makes it a valuable tool in several scientific and clinical applications. One use is in epidemiology and outbreak investigations, particularly for foodborne illnesses caused by bacteria like E. coli or Salmonella. By analyzing the unique DNA “fingerprints” of bacterial isolates from patients and suspected contaminated sources, PFGE helps to identify the origin and spread of infections. This allows public health officials to link cases and intervene rapidly during outbreaks.
The technique is also used in bacterial typing and strain differentiation. PFGE can distinguish between closely related bacterial strains, which is valuable for research, clinical diagnostics, and surveillance of antibiotic-resistant bacteria such as MRSA. It provides a highly reproducible restriction profile of the entire bacterial chromosome, allowing for the comparison of genetic patterns to determine relatedness between isolates. Strains with high similarity in their restriction fragment patterns are considered indistinguishable, indicating clonal relatedness.
PFGE has also been used for genome mapping and chromosomal analysis. It was instrumental in the early stages of constructing physical maps of large genomes and analyzing chromosomal rearrangements. It has provided insights into the geometry of bacterial chromosomes, revealing linear chromosomal DNA in some species. While newer sequencing technologies are used for detailed genomic analysis, PFGE remains a foundational technique for studying large-scale genomic structures and polymorphisms.