Pulsed Field Gel Electrophoresis

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Overview

The ability to separate DNA fragments in the size range from 50 kb to 10 Mb has enabled otherwise difficult analyses and resulted in various applications of pulsed field gel electrophoresis (PFGE) in cancer research, food safety, public health, quality control, and genome mapping. It is widely used in molecular epidemiology for strain typing and it has been adopted by PulseNet, a global network of health and food regulatory agency laboratories coordinated by the Centers for Disease Control and Prevention (CDC).

Research Areas Where PFGE Is Used

Molecular Epidemiology

Strain Typing in Public Health and Food Safety
PFGE is used for epidemiological studies of pathogenic organisms such as Escherichia coli O157:H7, Salmonella, Shigella, Listeria, Campylobacter, or Vibrio cholerae. When epidemiologists need to precisely identify the strain variants from a sample, genetic fingerprinting is the method of choice. Rare cutting restriction enzymes yield large DNA fragments which are analyzed using the CHEF system; the variant-specific electrophoretic gel pattern, which is usually highly reproducible and distinctive among the pathogenic strains, is then compared to the PulseNet database.

The PulseNet database, which contains thousands of gel patterns, is used by member laboratories to identify and track foodborne infections worldwide.

Food Quality Control
PFGE is in widespread use as a quality control method in the food industry. For example, the beer and wine industries use it to monitor the genetic stability of organisms in fermentation processes.

Cancer Research

DNA Damage and Repair Studies
Research efforts using PFGE are focused on better understanding the factors mediating the damage to DNA caused by ionizing radiation and chemical treatment. It is important to quantitatively measure the dsDNA breaks due to these treatments. DNA from treated cells is subjected to PFGE and the density of the DNA in different molecular weight regions indicates the integrity of DNA and the extent of its repair.

Apoptosis Assays
Apoptotic DNA fragmentation is a key characteristic of programmed cell death. Analysis of the fragmentation that occurs in the apoptosis process demonstrates either a "ladder" pattern at ~200 bp intervals (200-600 bp) or the formation of larger fragments (50-300 kb). Both of these size ranges can be visualized on one gel using field inversion gel electrophoresis (FIGE), which is available on the CHEF Mapper® XA system and enables easier assessment of the fragmentation process.

Genomics Applications

Generation of Artificial Chromosome Libraries
Cloning large DNA (100 kb-1 Mb) is the first step in sequencing complex genomes. FIGE is often used to separate and isolate the large digested DNA fragments. These are then cloned into artificial chromosomes to generate yeast, bacterial, human, and mammalian libraries.

Genome and Genetic Mapping
PFGE is still the benchmark for mapping applications. The libraries constructed using FIGE can also be used for mapping applications and specific assays used in research areas, including mapping specific disease loci, identifying chromosome rearrangements, and RFLP and DNA fingerprinting. Long-range genetic mapping is possible through the use of infrequently cleaving restriction endonucleases in conjunction with PFGE.

Chromosomal Assignment of Genes
Lower eukaryotes such as Saccharomyces have chromosomes in this size range and chromosomal assignment of genes may be determined simply through the combination of PFGE and southern blotting (Carle et al. 1984).

Yeast chromosomes separated by PFGE.

Yeast chromosomes separated by PFGE.

DNase I Hypersensitivity Assay
PFGE is used in mapping genomes for DNase I hypersensitive sites. This involves identifying different types of regulatory domains, such as active promoters and enhancers, where DNA-binding proteins are bound within nuclear chromatin. The conformation of chromatin at these sites causes them to be sensitive to DNase I cleavage. These sites are often located near active genes and play a role in eukaryotic gene regulation.

References

Carle GR and Olson MV (1984). Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res 12, 5647–5664.

Related Content

Literature
Number Description Download
1589 Preparation and Separation of Mammalian DNA by Pulsed Field Gel Electrophoresis Bulletin, Rev A Click to download
1648 Application of Two-Dimensional Pulsed Field Electrophoresis for Determining Molecular Karyotypes Bulletin, Rev A Click to download
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