28. Februar 2014 Pulsfeldgelelektrophorese.1 February 28, 2014 Pulsed Field Gel Electrophoresis 1...
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10. April 2023 Pulsfeldgelelektrophorese.1 April 10, 2023 Pulsed Field Gel Electrophoresis 1
Pulsed Field Gel Electrophoresis
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10. April 2023 Pulsfeldgelelektrophorese.2 April 10, 2023 Pulsed Field Gel Electrophoresis 2
Overview
• History How does it work• Theory of PFGE
– Parameters and their effects
• Realization of PFGE with the Rotaphor• Comparison with the Chef-Mapper
• Discussion
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10. April 2023 Pulsfeldgelelektrophorese.3 April 10, 2023 Pulsed Field Gel Electrophoresis 3
Original Design
Orthogonal field alternation electrophoresis
Carle &OlsonNAR 1984
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10. April 2023 Pulsfeldgelelektrophorese.4 April 10, 2023 Pulsed Field Gel Electrophoresis 4
OFAGE – Electric Field
A-
A+
B-
B+
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10. April 2023 Pulsfeldgelelektrophorese.5 April 10, 2023 Pulsed Field Gel Electrophoresis 5
A-
A+
B-
B+
OFAGE – Electric Field
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10. April 2023 Pulsfeldgelelektrophorese.6 April 10, 2023 Pulsed Field Gel Electrophoresis 6
OFAGE – Electric Field
A-
A+
B-
B+
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10. April 2023 Pulsfeldgelelektrophorese.7 April 10, 2023 Pulsed Field Gel Electrophoresis 7
OFAGE – Electric Field
A-
A+
B-
B+
>>90°
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10. April 2023 Pulsfeldgelelektrophorese.8 April 10, 2023 Pulsed Field Gel Electrophoresis 8
OFAGE – Electric Field
A-
A+
B-
B+
>>90°
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10. April 2023 Pulsfeldgelelektrophorese.9 April 10, 2023 Pulsed Field Gel Electrophoresis 9
Electrode Array
Zhang et al, NAR 1991
Programmable, autonomously controlled electrode array
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10. April 2023 Pulsfeldgelelektrophorese.10 April 10, 2023 Pulsed Field Gel Electrophoresis 10
Angle Dependency
Birren et al, Electrophoresis 1989
Volz, UniTübingen, 1988
RotaphorCHEF
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10. April 2023 Pulsfeldgelelektrophorese.11 April 10, 2023 Pulsed Field Gel Electrophoresis 11
Separation Principle < 90°
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.12 April 10, 2023 Pulsed Field Gel Electrophoresis 12
Separation Principle < 90°
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.13 April 10, 2023 Pulsed Field Gel Electrophoresis 13
Separation Principle < 90°
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.14 April 10, 2023 Pulsed Field Gel Electrophoresis 14
Separation Principle < 90°
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.15 April 10, 2023 Pulsed Field Gel Electrophoresis 15
Separation Principle < 90°
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.16 April 10, 2023 Pulsed Field Gel Electrophoresis 16
Separation Principle < 90°
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.17 April 10, 2023 Pulsed Field Gel Electrophoresis 17
Separation Principle < 90°
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.18 April 10, 2023 Pulsed Field Gel Electrophoresis 18
Separation Principle < 90°
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.19 April 10, 2023 Pulsed Field Gel Electrophoresis 19
Separation Principle PFGE (>90°)
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.20 April 10, 2023 Pulsed Field Gel Electrophoresis 20
Separation Principle PFGE (>90°)
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.21 April 10, 2023 Pulsed Field Gel Electrophoresis 21
Entscheidungsschwierigkeiten
What do you mean ?
You are the head now ?
You have been the head last time !
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10. April 2023 Pulsfeldgelelektrophorese.22 April 10, 2023 Pulsed Field Gel Electrophoresis 22
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.23 April 10, 2023 Pulsed Field Gel Electrophoresis 23
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.24 April 10, 2023 Pulsed Field Gel Electrophoresis 24
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.25 April 10, 2023 Pulsed Field Gel Electrophoresis 25
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.26 April 10, 2023 Pulsed Field Gel Electrophoresis 26
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.27 April 10, 2023 Pulsed Field Gel Electrophoresis 27
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.28 April 10, 2023 Pulsed Field Gel Electrophoresis 28
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.29 April 10, 2023 Pulsed Field Gel Electrophoresis 29
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.30 April 10, 2023 Pulsed Field Gel Electrophoresis 30
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.31 April 10, 2023 Pulsed Field Gel Electrophoresis 31
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.32 April 10, 2023 Pulsed Field Gel Electrophoresis 32
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.33 April 10, 2023 Pulsed Field Gel Electrophoresis 33
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.34 April 10, 2023 Pulsed Field Gel Electrophoresis 34
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.35 April 10, 2023 Pulsed Field Gel Electrophoresis 35
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.36 April 10, 2023 Pulsed Field Gel Electrophoresis 36
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.37 April 10, 2023 Pulsed Field Gel Electrophoresis 37
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.38 April 10, 2023 Pulsed Field Gel Electrophoresis 38
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.39 April 10, 2023 Pulsed Field Gel Electrophoresis 39
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.40 April 10, 2023 Pulsed Field Gel Electrophoresis 40
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.41 April 10, 2023 Pulsed Field Gel Electrophoresis 41
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.42 April 10, 2023 Pulsed Field Gel Electrophoresis 42
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.43 April 10, 2023 Pulsed Field Gel Electrophoresis 43
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.44 April 10, 2023 Pulsed Field Gel Electrophoresis 44
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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10. April 2023 Pulsfeldgelelektrophorese.45 April 10, 2023 Pulsed Field Gel Electrophoresis 45
Parameter
• Interval• Voltage
• Agarose concentration
• Temperature
• Angle
• Duration
Upper Limit of SeparationSeparation distance changes by modified number of pulses
Sharpness of bandsDuration of Electrophoresis
Distance of bands from starting pointDistance between different sized
bands
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10. April 2023 Pulsfeldgelelektrophorese.46 April 10, 2023 Pulsed Field Gel Electrophoresis 46
Theory
Bustamante, Tibtech, 1993
DNA of a T2 Phage (164kb)
Conventional Electrophoresis visualized with a Fluorescence microskope
1% Agarose, 8V/cm (= 240V with Rotaphor)
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10. April 2023 Pulsfeldgelelektrophorese.47 April 10, 2023 Pulsed Field Gel Electrophoresis 47
Trapping
Molecules > 1000 kbp result increasingly in additional loops
the larger the DNA the more loops
the faster (high voltage) the stronger
increases with higher agarose concentrationhandling limit 0,5%
Yeast chromosome ~1Mbp
direction
Break through
Blocking Spiral
Dead end
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10. April 2023 Pulsfeldgelelektrophorese.48 April 10, 2023 Pulsed Field Gel Electrophoresis 48
Trapping Example
Duration 252h = 10,5 Tage
Trapping inceases with voltage exponentially
Separation of the smallest human Chromosome takes 1,5 years
Homo
sapi
ens
S. p
ombe
S. cer
evisia
e
5,7 Mbp
3,0 Mbp
1,5 Mbp
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10. April 2023 Pulsfeldgelelektrophorese.49 April 10, 2023 Pulsed Field Gel Electrophoresis 49
Realization
CHEFRGE
TAFE
RFE
Biorad
Biometra
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10. April 2023 Pulsfeldgelelektrophorese.50 April 10, 2023 Pulsed Field Gel Electrophoresis 50
Rotaphor Electrodes
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10. April 2023 Pulsfeldgelelektrophorese.51 April 10, 2023 Pulsed Field Gel Electrophoresis 51
Rotordesign
Orientation of Pulse
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10. April 2023 Pulsfeldgelelektrophorese.52 April 10, 2023 Pulsed Field Gel Electrophoresis 52
Comparison
Size of gel:
Slots
Moving parts
Buffer
Refrigeration cascadable
Very short pulses
Dropout memory
20 x 20 cm
18 (– 50)
yes, stepper motor
Internal cooling
Yes
Not viable
Month
13x14cm
15
No
External cooling
No
Not usefull
3 h
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10. April 2023 Pulsfeldgelelektrophorese.53 April 10, 2023 Pulsed Field Gel Electrophoresis 53
Salmonella Typing
![Page 54: 28. Februar 2014 Pulsfeldgelelektrophorese.1 February 28, 2014 Pulsed Field Gel Electrophoresis 1 Pulsed Field Gel Electrophoresis.](https://reader036.fdocuments.in/reader036/viewer/2022081511/55155850550346486b8b45aa/html5/thumbnails/54.jpg)
10. April 2023 Pulsfeldgelelektrophorese.54 April 10, 2023 Pulsed Field Gel Electrophoresis 54
Salmonella Typing