pulsed-field gel electrophoresis

pulsed-field gel electrophoresis
Acronym	PFGE
Classification	Electrophoresis
Related	Agarose gel electrophoresis, Southern blot
Uses	Molecular biology, Microbiology, Genomics

pulsed-field gel electrophoresis is a specialized electrophoresis technique used to separate very large DNA molecules, such as entire chromosomes or large genomic fragments, which cannot be resolved by standard agarose gel electrophoresis. It was pioneered in the 1980s by David C. Schwartz and Charles Cantor at Columbia University. The method is a cornerstone technique in molecular genetics and epidemiology, particularly for bacterial strain typing and constructing physical maps of genomes.

Principle and mechanism

The fundamental principle relies on periodically changing the direction of the electric field applied across an agarose gel. Very large DNA molecules become trapped in the gel matrix when a constant field is applied, a phenomenon known as reptation. By alternating the field direction at timed intervals, these large molecules are forced to reorient themselves to move through the pores of the gel. The time required for reorientation is size-dependent, allowing for the separation of megabase-sized fragments. This process is controlled by sophisticated electrophoresis apparatus like the CHEF system.

Types and techniques

Several instrument configurations have been developed to generate the alternating fields. The most common is contour-clamped homogeneous electric field electrophoresis, invented by G. Chu and colleagues at Stanford University, which uses a complex array of electrodes to create a uniform field that changes angle. Other historical methods include field inversion gel electrophoresis, developed by G. Carle and M. Olson, and rotating gel electrophoresis. The choice of technique depends on the desired resolution range and the specific DNA size standards being used.

Applications

Its primary application is in molecular epidemiology for tracking the source and spread of pathogens. It is considered the "gold standard" for outbreak investigation of bacteria like Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes by agencies such as the Centers for Disease Control and Prevention. In genomics, it is used for constructing physical maps of yeast artificial chromosomes and for analyzing chromosomal aberrations in diseases like chronic lymphocytic leukemia. It also plays a role in studying protozoan parasites like Plasmodium falciparum.

Advantages and limitations

The major advantage is its unparalleled ability to separate entire chromosomes from organisms like Saccharomyces cerevisiae and Candida albicans, providing a macroscopic view of the genome. It offers high discriminatory power for strain differentiation, crucial for public health surveillance. However, the technique is labor-intensive, requiring several days to complete, and demands highly skilled technicians. The specialized equipment, such as the Bio-Rad Laboratories CHEF-DR II system, is costly, and the process can be less reproducible than newer methods like whole-genome sequencing.

Historical development

The technique was conceived in 1983 by David C. Schwartz while working in the laboratory of Charles Cantor at Columbia University College of Physicians and Surgeons. Their initial work, published in the journal Cell (journal), demonstrated the separation of chromosomes from the yeast Saccharomyces cerevisiae. This breakthrough solved a major problem in molecular biology and paved the way for the Human Genome Project. Subsequent refinements, notably the CHEF system, were developed by groups at Stanford University and the University of California, Berkeley.

Data analysis and interpretation

The resulting gel patterns, often called DNA fingerprints, are analyzed using specialized software like BioNumerics from Applied Maths. Bands are compared to standardized molecular weight markers, such as those from the bacterium Salmonella Braenderup strain H9812. Interpretation involves determining clonal relatedness between isolates; indistinguishable patterns suggest a common source. Standardized protocols from PulseNet, an international network coordinated by the Centers for Disease Control and Prevention, ensure data can be shared and compared globally among public health laboratories like the Robert Koch Institute.

Category:Molecular biology techniques Category:Microbiology techniques Category:Laboratory techniques