Pulsed-field gel electrophoresis explained

Pulsed-field gel electrophoresis (PFGE) is a technique used for the separation of large DNA molecules by applying an electric field that periodically changes direction to a gel matrix.[1] [2] Unlike standard agarose gel electrophoresis, which can separate DNA fragments of up to 50 kb, PGFE resolves fragments up to 10 Mb. This allows for the direct analysis of genomic DNA.

History

In 1984, David C. Schwartz and Charles Cantor published the first successful application of alternating electric fields for the separation of large DNA molecules.[3] [4] This technique, which they named PFGE, resulted in the development of several variations, including Orthogonal Field Alternation Gel Electrophoresis (OFAGE), Transverse Alternating Field Electrophoresis (TAFE), Field-Inversion Gel Electrophoresis (FIGE), and Clamped Homogeneous Electric Fields (CHEF), among others.

Procedure

The procedure for PFGE is similar to that of standard agarose gel electrophoresis, with the main exception being the application of the electric current. Generally, in PFGE electrophoresis chambers, the voltage periodically switches between three directions: one along the central axis, and two at a 60 degree angle along each side.[5] The application of the voltage can change depending on the variation of PFGE used.[6] [7]

Applications

PFGE may be used for genotyping or genetic fingerprinting. It has commonly been considered a gold standard in epidemiological studies of pathogenic organisms for several decades. For instance, subtyping bacterial isolates with this method has made it easier to discriminate among strains of Listeria monocytogenes, Lactococcus garvieae[8] and some clinical isolates of Bacillus cereus [9] group isolated from diseases aquatic organisms and thus to link environmental or food isolates with clinical infections. It is now in the process of being superseded by next generation sequencing methods.[10]

See also

External links

Notes and References

  1. Book: 10.1385/0-89603-498-4:33 . Pulsed-Field Gel Electrophoresis . Molecular Bacteriology . 1998 . Kaufmann . Mary Elizabeth . Methods in Molecular Medicine . 15 . 33–50 . 21390741 . 0-89603-498-4 .
  2. Herschleb . Jill . Ananiev . Gene . Schwartz . David C . Pulsed-field gel electrophoresis . Nature Protocols . March 2007 . 2 . 3 . 677–684 . 10.1038/nprot.2007.94 . 17406630 . 13265518 .
  3. Lopez-Canovas . Lilia . Martinez Benitez . Maximo B. . Herrera Isidron . Jose A. . Flores Soto . Eduardo . Pulsed Field Gel Electrophoresis: Past, present, and future . Analytical Biochemistry . May 2019 . 573 . 17–29 . 10.1016/j.ab.2019.02.020.
  4. Schwartz . David C. . Cantor . Charles R. . Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis . Cell . 1984 . en . 37 . 1 . 67–75 . 10.1016/0092-8674(84)90301-5. 6373014 . 30743288 .
  5. Web site: What is Pulsed Field Gel Electrophoresis? . New England Biolabs . 1 July 2024.
  6. Nassonova . E. S. . Pulsed field gel electrophoresis: Theory, instruments and application . Cell and Tissue Biology . 1 December 2008 . 2 . 6 . 557–565 . 10.1134/S1990519X08060011 . en . 1990-5203.
  7. Goering . Richard V. . Pulsed field gel electrophoresis: A review of application and interpretation in the molecular epidemiology of infectious disease . Infection, Genetics and Evolution . October 2010 . 10 . 7 . 866–875 . 10.1016/j.meegid.2010.07.023.
  8. Rao . Shreesha . Chen . Mei‐Yun . Sudpraseart . Chiranan . Lin . Peiry . Yoshida . Terutoyo . Wang . Pei‐Chi . Chen . Shih‐Chu . Genotyping and phenotyping of Lactococcus garvieae isolates from fish by pulse‐field gel electrophoresis (PFGE) and electron microscopy indicate geographical and capsular variations . Journal of Fish Diseases . June 2022 . 45 . 6 . 771–781 . 10.1111/jfd.13601.
  9. Cheng . Li‐Wu . Rao . Shreesha . Poudyal . Sayuj . Wang . Pei‐Chi . Chen . Shih‐Chu . Genotype and virulence gene analyses of Bacillus cereus group clinical isolates from the Chinese softshell turtle (Pelodiscus sinensis) in Taiwan . Journal of Fish Diseases . October 2021 . 44 . 10 . 1515–1529 . 10.1111/jfd.13473.
  10. Ribot . Efrain M. . Freeman . Molly . Hise . Kelley B. . Gerner-Smidt . Peter . PulseNet: Entering the Age of Next-Generation Sequencing . Foodborne Pathogens and Disease . July 2019 . 16 . 7 . 451–456 . 10.1089/fpd.2019.2634 . 31241352 . 6653803 .

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