Pulsed-field gel electrophoresis: Difference between revisions
m →Applications: Added qualification to "gold standard" statement. PFGE is still in use, but it is rapidly being pushed out by quicker and better whole genome sequencing methods (reference supplied) |
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{{short description|Lab technique for separation of DNA}} |
{{short description|Lab technique for separation of DNA}} |
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[[Image:Microbiologist 01.jpg|right|thumb|250px|A microbiologist runs a pulsed-field gel electrophoresis test used in bacterial typing]] |
[[Image:Microbiologist 01.jpg|right|thumb|250px|A microbiologist runs a pulsed-field gel electrophoresis test used in bacterial typing]] |
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'''Pulsed field gel electrophoresis''' is a technique used for the separation of large [[DNA]] [[molecule]]s by applying to a [[gel]] matrix an [[electric field]] that periodically changes direction.<ref name="Kaufmann1998">{{cite book|last1=Kaufmann|first1=Mary Elizabeth|title=Molecular Bacteriology|chapter=Pulsed-Field Gel Electrophoresis|journal=Methods in Molecular Medicine|volume=15|year=1998|pages=33–50|doi=10.1385/0-89603-498-4:33|pmid=21390741|isbn=0-89603-498-4}}</ref><ref name="HerschlebAnaniev2007">{{cite journal|last1=Herschleb|first1=Jill|last2=Ananiev|first2=Gene|last3=Schwartz|first3=David C|title=Pulsed-field gel electrophoresis|journal=Nature Protocols|volume=2|issue=3 |
'''Pulsed field gel electrophoresis''' is a technique used for the separation of large [[DNA]] [[molecule]]s by applying to a [[gel]] matrix an [[electric field]] that periodically changes direction.<ref name="Kaufmann1998">{{cite book|last1=Kaufmann|first1=Mary Elizabeth|title=Molecular Bacteriology|chapter=Pulsed-Field Gel Electrophoresis|journal=Methods in Molecular Medicine|volume=15|year=1998|pages=33–50|doi=10.1385/0-89603-498-4:33|pmid=21390741|isbn=0-89603-498-4}}</ref><ref name="HerschlebAnaniev2007">{{cite journal |last1=Herschleb |first1=Jill |last2=Ananiev |first2=Gene |last3=Schwartz |first3=David C |title=Pulsed-field gel electrophoresis |journal=Nature Protocols |date=March 2007 |volume=2 |issue=3 |pages=677–684 |doi=10.1038/nprot.2007.94 |pmid=17406630 }}</ref> |
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==Historical background== |
==Historical background== |
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Standard [[DNA electrophoresis|gel electrophoresis]] techniques for separation of DNA molecules provided huge advantages for molecular biology research. However, it was unable to separate very large molecules of DNA effectively. DNA molecules larger than 15–20 kb migrating through a gel will essentially move together in a size-independent manner. At [[Columbia University]] in 1984, David C. Schwartz and [[Charles Cantor]] developed a variation on the standard protocol by introducing an [[Alternating current|alternating]] voltage gradient to improve the resolution of larger molecules.<ref>{{cite journal |vauthors=Schwartz DC, Cantor CR |title=Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis |journal=Cell |volume=37 |issue=1 |pages=67–75 |date=May 1984 |pmid=6373014 |doi= |
Standard [[DNA electrophoresis|gel electrophoresis]] techniques for separation of DNA molecules provided huge advantages for molecular biology research. However, it was unable to separate very large molecules of DNA effectively. DNA molecules larger than 15–20 kb migrating through a gel will essentially move together in a size-independent manner. At [[Columbia University]] in 1984, David C. Schwartz and [[Charles Cantor]] developed a variation on the standard protocol by introducing an [[Alternating current|alternating]] voltage gradient to improve the resolution of larger molecules.<ref>{{cite journal |vauthors=Schwartz DC, Cantor CR |title=Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis |journal=Cell |volume=37 |issue=1 |pages=67–75 |date=May 1984 |pmid=6373014 |doi=10.1016/0092-8674(84)90301-5 |doi-access=free }}</ref> |
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This technique became known as pulsed-field gel electrophoresis (PFGE). The development of PFGE expanded the range of resolution for DNA fragments by as much as two orders of magnitude. |
This technique became known as pulsed-field gel electrophoresis (PFGE). The development of PFGE expanded the range of resolution for DNA fragments by as much as two orders of magnitude. |
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==Applications== |
==Applications== |
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PFGE may be used for [[genotyping]] or [[genetic fingerprinting]]. It has commonly been considered a [[gold standard (test)|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]]'' 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<ref> |
PFGE may be used for [[genotyping]] or [[genetic fingerprinting]]. It has commonly been considered a [[gold standard (test)|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]]'' 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.<ref>{{cite journal |last1=Ribot |first1=Efrain M. |last2=Freeman |first2=Molly |last3=Hise |first3=Kelley B. |last4=Gerner-Smidt |first4=Peter |title=PulseNet: Entering the Age of Next-Generation Sequencing |journal=Foodborne Pathogens and Disease |date=July 2019 |volume=16 |issue=7 |pages=451–456 |doi=10.1089/fpd.2019.2634 |pmid=31241352 |pmc=6653803 }}</ref> |
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==See also== |
==See also== |
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Revision as of 18:13, 17 February 2021
Pulsed field gel electrophoresis is a technique used for the separation of large DNA molecules by applying to a gel matrix an electric field that periodically changes direction.[1][2]
Historical background
Standard gel electrophoresis techniques for separation of DNA molecules provided huge advantages for molecular biology research. However, it was unable to separate very large molecules of DNA effectively. DNA molecules larger than 15–20 kb migrating through a gel will essentially move together in a size-independent manner. At Columbia University in 1984, David C. Schwartz and Charles Cantor developed a variation on the standard protocol by introducing an alternating voltage gradient to improve the resolution of larger molecules.[3] This technique became known as pulsed-field gel electrophoresis (PFGE). The development of PFGE expanded the range of resolution for DNA fragments by as much as two orders of magnitude.
Procedure
The procedure for this technique is relatively similar to performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, 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. The pulse times are equal for each direction resulting in a net forward migration of the DNA. For extremely large molecules (up to around 2 Mb), switching-interval ramps can be used that increases the pulse time for each direction over the course of a number of hours—take, for instance, increasing the pulse linearly from 10 seconds at 0 hours to 60 seconds at 18 hours.
This procedure takes longer than normal gel electrophoresis due to the size of the fragments being resolved and the fact that the DNA does not move in a straight line through the gel.
Theory
While in general small fragments can find their way through the gel matrix more easily than large DNA fragments, a threshold length exists above 30–50 kb where all large fragments will run at the same rate, and appear in a gel as a single large diffuse band.
However, with periodic changing of field direction, the various lengths of DNA react to the change at differing rates. That is, larger pieces of DNA will be slower to realign their charge when field direction is changed, while smaller pieces will be quicker. Over the course of time with the consistent changing of directions, each band will begin to separate more and more even at very large lengths. Thus separation of very large DNA pieces using PFGE is made possible.
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 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.[4]
See also
References
- ^ Kaufmann, Mary Elizabeth (1998). "Pulsed-Field Gel Electrophoresis". Molecular Bacteriology. Vol. 15. pp. 33–50. doi:10.1385/0-89603-498-4:33. ISBN 0-89603-498-4. PMID 21390741.
{{cite book}}:|journal=ignored (help) - ^ Herschleb, Jill; Ananiev, Gene; Schwartz, David C (March 2007). "Pulsed-field gel electrophoresis". Nature Protocols. 2 (3): 677–684. doi:10.1038/nprot.2007.94. PMID 17406630.
- ^ Schwartz DC, Cantor CR (May 1984). "Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis". Cell. 37 (1): 67–75. doi:10.1016/0092-8674(84)90301-5. PMID 6373014.
- ^ Ribot, Efrain M.; Freeman, Molly; Hise, Kelley B.; Gerner-Smidt, Peter (July 2019). "PulseNet: Entering the Age of Next-Generation Sequencing". Foodborne Pathogens and Disease. 16 (7): 451–456. doi:10.1089/fpd.2019.2634. PMC 6653803. PMID 31241352.
External links
- Pulse field method
- Applied Maths BioNumerics PFGE typing
