Pulse Field Gel Electrophoresis

Introduction to PFGE
DNA
Genetic information of an organism

Chromosomal DNA
Sequence specificity for each species and even strain Circular DNA molecule within bacterial cell Carries all normal genes employed for growth and other practical functions

Introduction to PFGE
Chromosome size variety of base pairs and genes for bacteria:
Bacteria Chromosome size (base pairs) Estimated number of genes

Escherichia coli Campylobacter jejuni Bacillus subtillus Salmonella Typhimurium Listeria monocytogenes

4,639,211 1,641,481 4,214,814 4,857,432 2,866,709

4,279 1,654 4,112 4,450 2,873

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Introduction to PFGE
Chromosome size variety of base pairs and genes for L. monocytogenes:
L. monocytogenes strain 10403S EGD-e FSL J1-194 FSL-J2-071 FSL R2-503 Chromosome size (base pairs) 2,866,709 2,944,528 2,986,227 3,149,923 3,001,696 Estimated number of genes 2,873 2,867 3,692 3,373 4,767

How does one differentiate between strains in a rapid manner?

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What is PFGE?
Pulse Field Gel Electrophoresis
Molecular method to produce a genetic fingerprint or profile of a bacterial isolate Creates and visualizes segments of DNA from a bacterial sample to be compared with other samples Achieved through breaking of chromosomal DNA into segments by *restriction enzymes* Advanced method of gel electrophoresis

PFGE Background
First introduced in 1984 by Schwartz &Cantor in Cell 37:67-75
Described a way to differentiate yeast chromosomes
Segment chromosomal DNA, utilize non-uniform electric current, compare DNA band profile

Looked for a way to visualize segments of DNA Old size limit (50 kilobase) PFGE capability (10 megabase)

PFGE Applications
Food Safety
Epidemiological studies Tracking of outbreak strains

Food Quality
Monitoring subtle changes in fermentation cultures
Yogurt, beer, wine industries

PFGE Applications
Cancer Research
Observations on dsDNA structure alterations by suspect carcinogens Changes in DNA density for specific molecular weight regions indicate structure alterations

Genomics
Cloning fragments to be sequenced can be separated using PFGE DNA fingerprinting and physical chromosome mapping Location of promoter sites due to DNase sensitivities with chromatin

PFGE Applications
Epidemiological studies and strain differentiation
Agencies such as PulseNet utilize PFGE to identify similar or different bacterial strains to:
Differentiate food-related clinical cases based upon suspect pathogen strain Allows for accurate tracking of outbreak allowing for source identification Goal of improved food safety for the public

Segmentation of DNA
Nuclease enzymes that breakdown strands of nucleic acids Two ways to cut or restrict DNA to segments
Exonuclease
Works from the outside inwards, essentially dismantling DNA piece by piece Systematically removes one nucleotide at a time from the end of dsDNA Two versions of exonucleases (3 to 5 and 5 to 3) used to clean up polymerase products and other processes

Endonuclease
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 Cutting or restriction of dsDNA from within the molecule

RE works at a specific recognition site
Recognition site is a specific sequence of nucleotides that are identified and cut leaving fragments

Restriction Endonucleases (RE)

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Restriction Endonucleases
REs read through dsDNA from 5 to 3 ends on both strands (palindromes) until digestion site is recognized Thousands of different REs identified, some repetitive (same recognition site as another RE, called isoschizomers) Activity effected by
pH Ionic strength Temperature

Altered activity by changing the above conditions results in slight alterations in recognition sites (referred to as star activity)
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Restriction Endonucleases
Employed in PFGE to randomly break the chromosome into fragments to be visualized following electrophoresis The same RE is used for all samples to be compared (should be the same bacterial species)
Comparison between Listeria monocytogenes isolates from the same outbreak would use ApaI or AscI (example REs)

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DNA Orientation and Subsequent Digestion

RE Digestion of DNA Supercoiled Chromosomal DNA

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DNA Visualization through Gel Electrophoresis

Traditional electrophoresis
One dimensional application of electrical field DNA sample size < 50 kb can accurately be visualized Cause of method restrictions:
One direction voltage application and strength 1.0% and higher agarose is too complex/thick to allow large molecules (> 50kb) to move at different rates meaning one band represents multiple segments
DNA Fragments Gel Particle Pore

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DNA Visualization through Gel Electrophoresis

Pulse Field GE
Two dimensional application of pulses Variation in direction of electrical fields
These electric fields are altered throughout the same electrophoresis run Time between shifts or pulses allows for reorientation

Avoids the limitations of molecular sieving by forcing the molecules to reorient themselves upon shifts in directions
Application in one direction, pause, reorientation, application in another direction, repeat Causing zigzag transversals
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Comparison of Electrophoresis Fields

Traditional

PFGE

+
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Field Inversion Gel Electrophoresis (FIGE)

Periodical inversion of polarity of electrodes
Shift in pulse application at 180 Molecules spend part of the time moving backwards

-/+

+/-

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Transverse Alternating Field Electrophoresis (TAFE)

Employment of two different electrode groups creating simple geometrical pulse angles The pulse angle increases as the molecule moves downwards A (-) B (-)

B (+)

A (+)

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Rotating Gel Electrophoresis (RGE)

RGE is a new form of PFGE Instead of having multiple or altering charges of electrodes, the gel itself is moved

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Clamped Homogenous Electrical Field (CHEF)

Most commonly used form of PFGE Applies multiple pulses from varying sources to create additional vectors Varying angles result in increased accurate separation and more clear resolution
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Visualization
Similar to PCR product visualization, following electrophoresis, PFGE gels are:
Stained with ethidium bromide Visualized through exposure to UV light

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