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
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
10. April 2023 Pulsfeldgelelektrophorese.3 April 10, 2023 Pulsed Field Gel Electrophoresis 3
Original Design
Orthogonal field alternation electrophoresis
Carle &OlsonNAR 1984
10. April 2023 Pulsfeldgelelektrophorese.4 April 10, 2023 Pulsed Field Gel Electrophoresis 4
OFAGE – Electric Field
A-
A+
B-
B+
10. April 2023 Pulsfeldgelelektrophorese.5 April 10, 2023 Pulsed Field Gel Electrophoresis 5
A-
A+
B-
B+
OFAGE – Electric Field
10. April 2023 Pulsfeldgelelektrophorese.6 April 10, 2023 Pulsed Field Gel Electrophoresis 6
OFAGE – Electric Field
A-
A+
B-
B+
10. April 2023 Pulsfeldgelelektrophorese.7 April 10, 2023 Pulsed Field Gel Electrophoresis 7
OFAGE – Electric Field
A-
A+
B-
B+
>>90°
10. April 2023 Pulsfeldgelelektrophorese.8 April 10, 2023 Pulsed Field Gel Electrophoresis 8
OFAGE – Electric Field
A-
A+
B-
B+
>>90°
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
10. April 2023 Pulsfeldgelelektrophorese.10 April 10, 2023 Pulsed Field Gel Electrophoresis 10
Angle Dependency
Birren et al, Electrophoresis 1989
Volz, UniTübingen, 1988
RotaphorCHEF
10. April 2023 Pulsfeldgelelektrophorese.11 April 10, 2023 Pulsed Field Gel Electrophoresis 11
Separation Principle < 90°
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.12 April 10, 2023 Pulsed Field Gel Electrophoresis 12
Separation Principle < 90°
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.13 April 10, 2023 Pulsed Field Gel Electrophoresis 13
Separation Principle < 90°
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.14 April 10, 2023 Pulsed Field Gel Electrophoresis 14
Separation Principle < 90°
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.15 April 10, 2023 Pulsed Field Gel Electrophoresis 15
Separation Principle < 90°
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.16 April 10, 2023 Pulsed Field Gel Electrophoresis 16
Separation Principle < 90°
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.17 April 10, 2023 Pulsed Field Gel Electrophoresis 17
Separation Principle < 90°
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.18 April 10, 2023 Pulsed Field Gel Electrophoresis 18
Separation Principle < 90°
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.19 April 10, 2023 Pulsed Field Gel Electrophoresis 19
Separation Principle PFGE (>90°)
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.20 April 10, 2023 Pulsed Field Gel Electrophoresis 20
Separation Principle PFGE (>90°)
Field Orientation
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 !
10. April 2023 Pulsfeldgelelektrophorese.22 April 10, 2023 Pulsed Field Gel Electrophoresis 22
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.23 April 10, 2023 Pulsed Field Gel Electrophoresis 23
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.24 April 10, 2023 Pulsed Field Gel Electrophoresis 24
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.25 April 10, 2023 Pulsed Field Gel Electrophoresis 25
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.26 April 10, 2023 Pulsed Field Gel Electrophoresis 26
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.27 April 10, 2023 Pulsed Field Gel Electrophoresis 27
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.28 April 10, 2023 Pulsed Field Gel Electrophoresis 28
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.29 April 10, 2023 Pulsed Field Gel Electrophoresis 29
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.30 April 10, 2023 Pulsed Field Gel Electrophoresis 30
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.31 April 10, 2023 Pulsed Field Gel Electrophoresis 31
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.32 April 10, 2023 Pulsed Field Gel Electrophoresis 32
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.33 April 10, 2023 Pulsed Field Gel Electrophoresis 33
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.34 April 10, 2023 Pulsed Field Gel Electrophoresis 34
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.35 April 10, 2023 Pulsed Field Gel Electrophoresis 35
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.36 April 10, 2023 Pulsed Field Gel Electrophoresis 36
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.37 April 10, 2023 Pulsed Field Gel Electrophoresis 37
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.38 April 10, 2023 Pulsed Field Gel Electrophoresis 38
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.39 April 10, 2023 Pulsed Field Gel Electrophoresis 39
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.40 April 10, 2023 Pulsed Field Gel Electrophoresis 40
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.41 April 10, 2023 Pulsed Field Gel Electrophoresis 41
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.42 April 10, 2023 Pulsed Field Gel Electrophoresis 42
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.43 April 10, 2023 Pulsed Field Gel Electrophoresis 43
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
10. April 2023 Pulsfeldgelelektrophorese.44 April 10, 2023 Pulsed Field Gel Electrophoresis 44
Trennprinzip PFGE (>90°)
Field Orientation
Field Orientation
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
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)
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
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
10. April 2023 Pulsfeldgelelektrophorese.49 April 10, 2023 Pulsed Field Gel Electrophoresis 49
Realization
CHEFRGE
TAFE
RFE
Biorad
Biometra
10. April 2023 Pulsfeldgelelektrophorese.50 April 10, 2023 Pulsed Field Gel Electrophoresis 50
Rotaphor Electrodes
10. April 2023 Pulsfeldgelelektrophorese.51 April 10, 2023 Pulsed Field Gel Electrophoresis 51
Rotordesign
Orientation of Pulse
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
10. April 2023 Pulsfeldgelelektrophorese.53 April 10, 2023 Pulsed Field Gel Electrophoresis 53
Salmonella Typing
10. April 2023 Pulsfeldgelelektrophorese.54 April 10, 2023 Pulsed Field Gel Electrophoresis 54
Salmonella Typing