Analytical Techniques - Pulsed Field Gel Electrophoresis (PFGE)

Pulsed-Field Gel Electrophoresis - PFGE

• Introduced in 1982 by Schwartz et al.
• Addresses the inability of conventional gel electrophoresis to separate fragments above 50 Kb due to loss of sieving action of the gel because of the large size of molecules
• Uses 2 alternating electric fields
• Provides means for routine separation of fragments exceeding 6000 Kb
• Ability to separate small, natural linear chromosomal DNAs from 50 Kb parasite minichromosomes to multimillion-bp long yeast chromosomes
• Basically separates DNAs from few Kb to more than 10 Mb

Types of PFGE

Field-Inversion Gel Electrophoresis (FIGE)

• Developed by Carle, Frank and Olsen in 1986
• 2 fields are 180º apart
• Electrode polarity is reversed at intervals with longer forward than reverse ones (3:1)
• Duration of pulse-times are increased progressively during a run to increase resolution "switch time ramping"
• Obtainment of straight lines using simple equipment
• Popular for small fragment separations
• Acceptable resolution = 800 Kb (600-750 Kb is optimal)

Transverse-Alternating field Gel Electrophoresis (TAFE)

• Simple, convenient format
• Gel is oriented vertically and simple 4-electrode array is placed in front and back of it
• Sample molecules are forced to zigzag through the thickness of the gel
• All lanes experience same effects, therefore get straight bands
• All lanes equally subjected to continual variations in field strength and reorientation angle
• Angle varies from top of the gel (115º) to the bottom (165º), therefore molecules do not move at constant velocity through the length of the gel
• Resolution = approximately 1600 Kb

Counter Clamped Homogenous Electric Fields (CHEF)

• Sophisticated, most widely used
• 24 point electrodes equally spaced around a hexagonal contour
• No "passive" electrodes → all connected to power supply via external loop of resistors
• Voltages set at these 24 points
• Electric fields sufficiently uniform, therefore get straight bands
• Orientation angle = 120º with gradations of electro-potential radiating from + → - poles
• Resolution = approximately 7000 Kb

Orthogonal Field Alternation Gel Electrophoresis (OFAGE)

• Carlen and Olsen (1984)
• Angle between electric fields varies to being more than 90º to less than 180º
• Resolution = 1000 Kb - 2000 Kb
• Electric field not uniform, DNA migrates at different rates

Rotating Gel Electrophoresis (RGE)

• Southern (1987)
• Gel rotates between 2 set angles while electrodes are off
• 1 set of electrodes is used, electric field is uniform, therefore get straight bands
• Uses a single homogenous field and changes orientation of electric field in relation to gel by discontinuously and periodically rotating gel
• Long switch times
• Angle of orientation can be easily altered by changing the angle of rotation
• Resolution = 50 Kb - 6000 Kb

Programmable Autonomously Controlled Electrodes (PACE)

• Precise control over all electric field parameters by independent regulation of voltages on 24 electrodes arranged in a closed contour
• Unlimited number of electric fields of controlled homogeneity, voltage gradient, orientation, and duration
• Can generate voltage clamped homogenous static fields
• Can alter reorientation angle between alternating fields → this increases the speed of separation for large molecules
• Resolution = 100 bp - 6 Mb

Pulsed-Homogenous Orthogonal Field Gel Electrophoresis (PHOGE)

• Field reorientation angle = 90º
• DNA molecules undergo 4 reorientations per cycle instead of 2
• Lanes do not run straight
• Resolution = 1 Mb

Equipment

Gel Box

• Immobilized gel with an array of electrodes
• Means of circulating electrophoresis buffer
• Voltage gradients = 10 volts/cm - 15 volts/cm
• Temperature controlled by heat exchange mechanism

High voltage power supply

• Electrodes 25-50 cm apart
• Maximum voltage rating = 750 volts
• Current drawn = 0.5 Amp at 14ºC using 0.5x TBE

Switch unit

• Ability to control reorientation angles between electric fields
• Not fast enough for separation of molecules less than 50 Kb

Computer program

• Controls switch time and run-time

Cooler

• DNA molecule migration is sensitive to temperature
• Buffer recirculated at 450mL/min and chilled with cold water (5ºC) thus maintaining temperature at 13-15ºC

Running Conditions

Pulse time

• At 10V/cm, t = 0.1 sec → 5 Kb resolved
• At 3V/cm, t = 1000 sec → 3-7 Mb resolved
• Selected so that DNA molecules of a targeted size spend most of the duration of the pulse reorienting rather than moving through the gel

Electric field shape

• Angles more than 110º = most effective
• Angles more than 90º = good resolution
• Angles between 120º - 150º = excellent resolution
• Angles less than 90º = not effective

Electric field strength

• Mobility = velocity/unit field
• Mobility independent of field strength but is affected by it in 2 ways:
• 100-500 Kb mobility → linear dependence on electric field strength
• Electric field strength affects DNA size of the transition between 2 zones of resolution

Reorientation angle

• Wide = sharper bands and better resolution

Voltage

• 6-10V/cm for 1 Mb
• 2V/cm for 3, 5, 6 Mb (S. pombe)
• 1.5V/cm for 12 Mb (N. crassa)
• When voltage gradient is low, switching intervals must be high

Temperature

• Between 4ºC - 15ºC = optimal
• Between 14ºC - 22ºC = best compromise between speed and resolution
• For example: at 34ºC, velocity is much higher than at 4ºC but the resolution is diminished

Switch interval

• If increased beyond time required to reorient, then the fragment will spend a large portion of gel run migrating
• For increased resolution, switch intervals should be decreased

Agarose concentration

• Low concentration = faster DNA migration
• For example: λDNA (48.5 Kb) migrates faster at 0.6% as compared to 1%
• Bands at 0.7% diffuse but are sharper at 1.4-1.8%

Restriction enzymes

• Base composition is an important factor in selection of restriction enzymes

Applications

• Quick resolution of bacterial genome into a small number of large fragments
• Obtain a discrete pattern of bands useful for fingerprinting and physical mapping of the chromosome
• Establish degree of relatedness among different strains of the same species
• Genome size estimation, construction of chromosome maps and characterization of bacterial species
• Genome characterization
• Construction of YAC libraries
• Construction of transgenic mice
• Study of radiation-induced DNA damage and repair, size organization and variation in mammalian centromeres
• Genome organization studies
• determining physical proximity of 2 genes
• determining size of large genes
• identifying location of chromosome breaks
• Mapping of human genome
• Determining order of markers more precisely than is possible with genetic linkage analysis
• Mapping a new mutation
• Precise selection of large DNA fragments for cloning
• use restriction enzymes specific for cutting infrequently occurring sequences
• Easy isolation of individual restriction fragments for further restriction mapping, gene insertion, and functional gene mapping