pfge theory
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Pulse Field Gel Electrophoresis
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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
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Introduction to PFGE
Chromosome size variety of base pairs and genes forbacteria:
Bacteria Chromosome size (base pairs) Estimated number of genes
Escherichia coli 4,639,211 4,279
Campylobacter
jejuni
1,641,481 1,654
Bacillus subtillus 4,214,814 4,112
SalmonellaTyphimurium
4,857,432 4,450
Listeria
monocytogenes
2,866,709 2,873
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Introduction to PFGE
Chromosome size variety of base pairs and genes for
L. monocytogenes:
How does one differentiate between strains in a
rapid manner?
L. monocytogenes
strain
Chromosome size (base
pairs)
Estimated number of genes
10403S 2,866,709 2,873EGD-e 2,944,528 2,867
FSL J1-194 2,986,227 3,692
FSL-J2-071 3,149,923 3,373
FSL R2-503 3,001,696 4,767
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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 bacterialsample to be compared with other samples
Achieved through breaking of chromosomal DNA into
segments by *restriction enzymes*
Advanced method of gel electrophoresis
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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)
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PFGE Applications
Food Safety
Epidemiological studies
Tracking of outbreak strains
Food Quality
Monitoring subtle changes in fermentation cultures
Yogurt, beer, wine industries
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PFGE Applications
Cancer Research Observations on dsDNA structure alterations by suspect
carcinogens
Changes in DNA density for specific molecular weight regionsindicate structure alterations
Genomics
Cloning fragments to be sequenced can be separated usingPFGE
DNA fingerprinting and physical chromosome mapping
Location of promoter sites due to DNase sensitivities withchromatin
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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
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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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Restriction Endonucleases (RE) 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
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Restriction Endonucleases
REs read through dsDNA from 5 to 3 ends on bothstrands (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 conditionsresults 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 useApaI orAscI (example REs)
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DNA Orientation and Subsequent Digestion
Supercoiled Chromosomal DNA
RE Digestion of 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 FragmentsGelParticle
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 sameelectrophoresis 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
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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 (-)
A (+)B (+)
B (-)
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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 usedform of PFGE
Applies multiple pulsesfrom varying sources tocreate additionalvectors
Varying angles result in
increased accurateseparation and moreclear 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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