Definition: It is a technique used for the separation of
large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an
electric field that periodically changes direction.
Developed David C. Schwartz and Charles Cantor at Columbia University in 1984.
How is PFGE different from conventional agarose gel electrophoresis?
In conventional gels, the current is applied in a single direction (from top to bottom). But in PFGE, the direction of the current is altered at a regular interval.
Conventional electrophoresis can separate DNA fragments up to 20 kb. DNA molecules larger than 20kb can be separated by PFGE. DNA fragments of up to ~10 Mb can be effectively separated using PFGE.
Procedure:
General procedure is same as conventional agarose gel electrophoresis. The differences are in sample preparation and in direction of current flow.
Sample preparation: High molecular weight DNA t be separated using PFGE is easily cleaved through shearing and has very high solution viscosity. For these reasons, DNA samples for PFGE are generally prepared by embedding in gel medium. Cellular source material is suspended in low gelling agarose and the gelled suspension is poured into molds. All subsequent manipulations (for example, cell lysis, protein removal, and restriction digestion) are performed by diffusing reagents into the resultant gel plugs. The processed gel plugs are then carefully loaded into wells of an agarose gel used for PFGE.*
Direction of current: Instead of constantly running the voltage in one direction as in conventional agarose gel electrophoresis, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 60 degrees either side.
PFGE Application:
• First used to separate yeast chromosomal DNA. Therefore ideal for separating chromosomal DNAs of different organisms for genome projects
• Pulsed field gel electrophoresis has been used as a means of identifying the genetic defects that cause many hereditary diseases**.
• used for genotyping or genetic fingerprinting.
• It is commonly considered a gold standard in epidemiological studies of pathogenic organisms. PFGE applied as a universal generic method for subtyping of bacteria. Only the choice of the restriction enzyme and conditions for electrophoresis need to be optimized for each species.
References:
Schwartz DC and Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67–75 (original paper on PFGE).
*http://www.bio-rad.com/en-is/applications-technologies/pulsed-field-gel-electrophoresis
**http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Cobain/method.html
Developed David C. Schwartz and Charles Cantor at Columbia University in 1984.
How is PFGE different from conventional agarose gel electrophoresis?
In conventional gels, the current is applied in a single direction (from top to bottom). But in PFGE, the direction of the current is altered at a regular interval.
Conventional electrophoresis can separate DNA fragments up to 20 kb. DNA molecules larger than 20kb can be separated by PFGE. DNA fragments of up to ~10 Mb can be effectively separated using PFGE.
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| Image credit |
General procedure is same as conventional agarose gel electrophoresis. The differences are in sample preparation and in direction of current flow.
Sample preparation: High molecular weight DNA t be separated using PFGE is easily cleaved through shearing and has very high solution viscosity. For these reasons, DNA samples for PFGE are generally prepared by embedding in gel medium. Cellular source material is suspended in low gelling agarose and the gelled suspension is poured into molds. All subsequent manipulations (for example, cell lysis, protein removal, and restriction digestion) are performed by diffusing reagents into the resultant gel plugs. The processed gel plugs are then carefully loaded into wells of an agarose gel used for PFGE.*
Direction of current: Instead of constantly running the voltage in one direction as in conventional agarose gel electrophoresis, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 60 degrees either side.
PFGE Application:
• First used to separate yeast chromosomal DNA. Therefore ideal for separating chromosomal DNAs of different organisms for genome projects
• Pulsed field gel electrophoresis has been used as a means of identifying the genetic defects that cause many hereditary diseases**.
• used for genotyping or genetic fingerprinting.
• It is commonly considered a gold standard in epidemiological studies of pathogenic organisms. PFGE applied as a universal generic method for subtyping of bacteria. Only the choice of the restriction enzyme and conditions for electrophoresis need to be optimized for each species.
References:
Schwartz DC and Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67–75 (original paper on PFGE).
*http://www.bio-rad.com/en-is/applications-technologies/pulsed-field-gel-electrophoresis
**http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Cobain/method.html
Tags:
Molecular Biology Techniques
PFGE Definition
PFGE Application
PFGE Procedure
Pulse Field Gel Electrophoresis