PFGE Theory

8/13/2019 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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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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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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PFGE Theory

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