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Pulsed Field Gel Electrophoresis
What is PFGE?
Conventional methods of gel electrophoresis are carried out by placing DNA samples in a solid matrix (agarose or polyacrilamide) and inducing the molecules to migrate through the gel under a static electric field. When DNA molecules are under the influence of this electric field, they elongate and align themselves with the field, migrating toward the anode in a process called reptation. There are several parameters that affect the migration of DNA through the gel: concentration and composition of the gel, the buffer, the temperature, and the voltage gradient of the electric field. In DNA electrophoresis by the standard method however, DNA molecules larger than 20kb show essentially the same mobility in a static electric field, making differentiation between these DNA molecules impossible. The first attempts to resolve these larger fragments included using low percentage agarose gels and low voltage gradients. Even under these extreme conditions, separation of large DNA molecules was difficult. In 1984, David Schwartz was able to offer a new technique. He suggested that periodically changing the orientation of the electric field would force DNA molecules in the gel to relax upon the removal of the first field and elongate to allign with the new field. It was his assumption that this process should be size dependent. Schwartz was finally able to demonstrate the effectiveness of this technique when he successfully separted yeast chromosomes that were several hundred kilobases in length. (Birren et al., 1993)
Development of the Technique
The method of pulsed field gel electrophoresis was first utilized in 1982, and since then several apparatuses have been developed for separating large molecules of DNA, all using multiple electric fields. All systems seaparte DNA molecules within the same size range but differ in the speed of separation and the resolution. Below are schematic diagrams of the various apparatuses:
Figure 1: Schematic diagrams of published pulsed field gel systems. Nomenclature: PFGE-pulsed field gradient gel electrophoresis, OFAGE-orthogonal field alternation gel electrophoresis, TAFE- transverse alternating field electrophoresis, FIGE- field inversion gel electrophoresis, CHEF- contour clamped homogeneous electric field, crossed field gel electrophoresis (Waltzer), and ST/RIDE- simultaneous tangential/rectangular inversion decussate electrophoresis. (Figure 2.1, pg. 8, Pulsed Field Gel Electrophoresis: A Practical Guide).
In order to better understand the differences between these techniques, some vocabulary terms must first be defined:
Pulsed Field - any electrophoresis process that uses more than one electric field alternatingly
Switch Interval - amount of time each of the alternating fields is active
Reorientation Angle - acute angle between the two alternating electric fields
Field Inversion - PFGE system in shich the two alternating fields are oriented opposite each other
Voltage Gradient - electrical potential applied to the gel
Homogeneous Field - electric field that has uniform potential differences across the whole field
The first PFGE apparatus used two alternating electric fields, one homogeneous and the other nonhomogeneous. The OFAGE apparatus uses two nonhomogeneous electric fields and was developed shortly after PFGE was developed. A problem with these first two methods arose due to the fact that the DNA molecules ran in a curved trajectory, making lane-to-lane comparisons difficult. Thus, the TAFE system used homogeneous electric fields produced across the width of the gel to eliminate the "bent" lanes. This system also has its drawbacks because the reorientation angles are not constant throughout the gel, thus molecules do not move at constant velocity throughout the gel and liquid samples cannot be used. A modification of the TAFE system is found in the ST/RIDE system which allows for the changing of reorientation angles while the gel is running. This technique minimizes band stacking and liquid samples can be used. The FIGE apparatus is the simplest to construct and operates by periodically inverting a uniform electric field in one dimension. Another more complex approach using the same method is ZIFE or zero integrated field electrophoresis. ZIFE (not shown) is slower than FIGE but has the ability to resolve larger molecules of DNA. The advantage of the CHEF apparatus is that it is capable of separating a large number of DNA samples in straight lines by generating homogeneous electric fields using multiple electrodes arranged around a closed contour. Finally, in the crossed field apparatus, the gel is simply placed on a moblile platform that can be rotated in order to change the orientation of the electric field relative to the DNA. (Birren et al., 1993)
How do you prepare the DNA?
Large DNA is very easily sheared and often difficult to pipet due to its high viscosity. Thus, DNA preparation for PFGE is a bit different from standard DNA preparation methods. Chromosomal DNA must first be embedded in agarose plugs and these plugs are treated with enzymes to digest the proteins, leaving behind the naked DNA. The plugs are then cut to size, treated with restriction enzymes, loaded into the wells of the gel and sealed into place with agarose. The link below provides a more detailed description of this procedure as well as a detailed protocol for running the gel.
Purifying Large E.Coli Restriction Fragments from Pulsed Field Gels
Setting the Parameters
When running a gel using a PFGE system there are several parameters that must be considered in order for the proper setup to be established. For example, the voltage gradient must be altered according to the size of the sample to be electrophoresed. Larger DNA samples require lower voltage gradients in order to migrate properly through the gel. When choosing an agarose it is also important to keep the size of the sample in mind. For separation of molecules larger than 2.5Mbp a low EEO agarose certified for molecular biology is suffieient, however, for larger molecules "pulsed field" agaroses are better because of the reduced run times. Temperature also affects the DNA mobility within the gel. Raising the temperature increases the mobility and 12-15 degrees Celcius is the most frequently used temperature range. Furthermore, it must be taken into consideration that DNA will migrate more quicly in buffers of low ionic strength. Finally, one of the PFGE apparatuses shown above must be selected (Birren et al., 1993). Seen below are examples of gels run using the FIGE system (Figure 2) and the RGE system (Figure 3).
Figure 2. Increased separation of the 20-50 kb range with field inversion gel electrophoresis (FIGE). Run conditions: 230 V, 7.9 V/cm, 16 hrs., 50 msec. pulse, forward:reverse pulse ratio = 2.5:1, 1% GTG agarose, 0.5X TBE, 10 C. a) 1 kb ladder, 0.5-12 kb; b) Lambda/Hind III, 0.5-23 kb; and c) High molecular weight markers, 8.3-48.5 kb (Permission pending for the use of this image).
Increasing both the separation range and the resolution of large DNA requires smaller reorientation angles, generally 96-140ø, with 120ø most common. Smaller angles (e.g., 100ø) increase the mobility of the DNA generally without seriously affecting resolution. The lower limit is approximately 96ø. Below this, separation is seriously compromised. (HSI Laboratories, Hoefer Scientific Instruments)
Figure 3. Rotating gel electrophoresis (RGE) separation Saccharomyces cercevisiae chromosomes (245-2190 kb). Run conditions: 180 V, 5.1 V/cm, 34 hrs., 120 angle, 60-120 sec. pulse ramp, 0.5X TBE, 1.2% GTG agarose, 10 C. Two combs were used on the same gel to load 32 samples, a maximum of 72 are possible (Permission pending for the use of this image).
Most PFGE systems separate DNA over a relatively small area, limiting the resolution of complex samples. RGE is an exception to this, with a useful separation distance up to 20 cm and a maximum gel size of 18 x 20 cm. (HSI Laboratories, Hoefer Scientific Instruments)
Applications of PFGE
- Pulsed field gel electrophoresis has been used as a means of identifying the genetic defects that cause many hereditary diseases. In principle, detection of chromosomal rearrangements should be easy since, when run on a gel, they produce size differences when the normal gene and the defective gene are compared. However, when using the standard method of the gel electrophoresis, only size differences up to 30kbp can be detected. Below is a figure that demonstrates the difference between the hybridization pattern of a conventional gel analysis and the hybridization pattern of a PFGE gel analysis (Mathew, 1991).
Figure 4: (Fig. 1, pg. 316, Protocols in Human Molecular Genetics)
- Pulsed Field Gel Electrophoresis was applied to the study of Duchenne Muscular Distrophy. Since the DMD gene is 2.3Mbp, it was necessary to use PFGE in order to uncover the genetic defect. The use of PFGE analysis on patients with the disease soon revealed that 50% of the cases large deletions or duplications were a responsible for the disease (Mathew, 1991).
- From December 1994 to January 1995 Salmonella agona infections increased dramatically in England and Wales. It was necessary to characterize those who fell victim to the outbreak through genotipic methods and PFGE was the best method to provide a genotypic fingerprint of each patient. The case is described further in an article from the Journal of Emerging Infectious Diseases.
Other Useful Resourses
There are several sites which provide additional information on the techniques and usage of Pulsed Field Gel Electrophoresis:
Several Recent Journal Articles have been published using the technique of Pulsed Field Gel Electrophoresis
References:
Application of Pulsed Field Gel Electrophoresis to an Outbreak of Salmonella Agona. <http://ftp.cdc.gov/pub/EID/vol2no2/adobe/threlfal.pdf>. Accessed 2003 feb 17.
ARUP Laboratories. 2003 Feb. <http://www.aruplab.com/guides/clt/tests/clt_b3.htm>. Accessed 2003 Feb 17.
Austin, Robert. 2002. A DNA Prism: Rapid Continuous Gractionation and Separation of Genomic DNA molecules [abstract]. In NNUN Abstracts, Biology and Chemistry pg. 6.<http://www.nnf.cornell.edu/nnun/2002NNUNpg6.pdf> Accessed 2003 Feb. 17.
Birren, Bruce and Lai, Eric.Pulsed Field Gel Electrophoresis: A Practical Guide. San Diego, California: Academic Press Inc. 1993.
Joppa, Barbara; Li, Samantha; Cole, Scott; Gallagher, Sean. Pulsed Field Electrophoresis
for Separation of Large DNA.
<http://www.nal.usda.gov/pgdic/Probe/v2n3/puls.html>
Accessed 2003 Feb 17.
Mathew, Christopher G., Protocols in Human Molecular Genetics. Clifton, New Jersey: The Humana Press Inc. 1991. pp.316-325.
Romalde JL, Castro D, Magarinos B, Lopez-Cortes L, Borrego JJ. 2002 Dec. Comparison of ribotyping, randomly amplified polymorphic DNA, and pulsed-field gel electrophoresis for molecular typing of Vibrio tapetis [abstract]. In Syst Appl Microbiol 2002 Dec: 25(4):544-50. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12583715&dopt=Abstract>. Accessed 2003 Feb 17.
Schalch, B., Bader, L., Schau, H.P., Bergmann, R., Rometsch, A., Maydl, G., Kessler, S. 2003 Feb. Molecular Typing of Clostridium perfringens from a Food-Borne Disease Outbreak in a Nursing Home: Ribotyping versus Pulsed-Field Gel Electrophoresis [abstract]. J Clin Microbiol 2003 Feb: 41(2):892-895. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12574310&dopt=Abstract>. Accessed 2003 Feb 17.
Stanford University. 1996 Dec 12. Stanford Genome Technology Center Homepage. <http://www-sequence.stanford.edu/protocols/pulsed-fieldgels.html>. Accessed 2003 Feb 17.
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