Pulsed Field Gel Electrophoresis 16 September 2020 Pulsed
Pulsed Field Gel Electrophoresis 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 1 Field Gel Electrophoresis 1
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 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 2 Field Gel Electrophoresis 2
Original Design Carle &Olson NAR 1984 Orthogonal field alternation electrophoresis 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 3 Field Gel Electrophoresis 3
OFAGE – Electric Field 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 4 Field Gel Electrophoresis 4
OFAGE – Electric Field 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 5 Field Gel Electrophoresis 5
OFAGE – Electric Field 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 6 Field Gel Electrophoresis 6
OFAGE – Electric Field 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 7 Field Gel Electrophoresis 7
OFAGE – Electric Field >>90° 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 8 Field Gel Electrophoresis 8
Electrode Array Programmable, autonomously controlled electrode array Zhang et al, NAR 1991 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 9 Field Gel Electrophoresis 9
Angle Dependency CHEF Rotaphor Birren et al, Electrophoresis 1989 16 September 2020 Volz, Uni. Tübingen, 1988 Pulsed. Pulsfeldgelelektrophorese. 10 Field Gel Electrophoresis 10
Separation Principle < 90° Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 11 Field Gel Electrophoresis 11
Separation Principle < 90° Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 12 Field Gel Electrophoresis 12
Separation Principle < 90° Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 13 Field Gel Electrophoresis 13
Separation Principle < 90° Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 14 Field Gel Electrophoresis 14
Separation Principle < 90° Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 15 Field Gel Electrophoresis 15
Separation Principle < 90° Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 16 Field Gel Electrophoresis 16
Separation Principle < 90° Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 17 Field Gel Electrophoresis 17
Separation Principle < 90° Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 18 Field Gel Electrophoresis 18
Separation Principle PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 19 Field Gel Electrophoresis 19
Separation Principle PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 20 Field Gel Electrophoresis 20
Entscheidungsschwierigkeiten What do you mean ? You are the head now ? You have been the head last time ! 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 21 Field Gel Electrophoresis 21
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 22 Field Gel Electrophoresis 22
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 23 Field Gel Electrophoresis 23
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 24 Field Gel Electrophoresis 24
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 25 Field Gel Electrophoresis 25
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 26 Field Gel Electrophoresis 26
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 27 Field Gel Electrophoresis 27
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 28 Field Gel Electrophoresis 28
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 29 Field Gel Electrophoresis 29
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 30 Field Gel Electrophoresis 30
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 31 Field Gel Electrophoresis 31
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 32 Field Gel Electrophoresis 32
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 33 Field Gel Electrophoresis 33
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 34 Field Gel Electrophoresis 34
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 35 Field Gel Electrophoresis 35
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 36 Field Gel Electrophoresis 36
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 37 Field Gel Electrophoresis 37
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 38 Field Gel Electrophoresis 38
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 39 Field Gel Electrophoresis 39
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 40 Field Gel Electrophoresis 40
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 41 Field Gel Electrophoresis 41
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 42 Field Gel Electrophoresis 42
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 43 Field Gel Electrophoresis 43
Trennprinzip PFGE (>90°) Field Orientation 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 44 Field Gel Electrophoresis 44
Parameter • • Interval Voltage Upper Limit of Separation distance changes by modified number of pulses • • Agarose concentration Temperature • Angle Sharpness of bands Duration of Electrophoresis • Duration Distance of bands from starting point Distance between different sized bands 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 45 Field Gel Electrophoresis 45
Theory DNA of a T 2 Phage (164 kb) Conventional Electrophoresis visualized with a Fluorescence microskope 1% Agarose, 8 V/cm (= 240 V with Rotaphor) Bustamante, Tibtech, 1993 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 46 Field Gel Electrophoresis 46
Trapping Yeast chromosome ~1 Mbp Molecules > 1000 kbp result increasingly in additional loops the larger the DNA the more loops direction the faster (high voltage) the stronger Dead end Break through increases with higher agarose concentration handling limit 0, 5% Blocking Spiral 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 47 Field Gel Electrophoresis 47
e ia vis re S. ce be m po Ho Duration 252 h = 10, 5 Tage S. m o sa pi en s Trapping Example Trapping inceases with voltage exponentially Separation of the smallest human Chromosome takes 1, 5 years 5, 7 Mbp 3, 0 Mbp 1, 5 Mbp 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 48 Field Gel Electrophoresis 48
Realization TAFE Biorad Biometra RFE RGE 16 September 2020 CHEF Pulsed. Pulsfeldgelelektrophorese. 49 Field Gel Electrophoresis 49
Rotaphor Electrodes 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 50 Field Gel Electrophoresis 50
Rotordesign Orientation of Pulse 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 51 Field Gel Electrophoresis 51
Comparison 13 x 14 cm Size of gel: 15 Slots No Moving parts External cooling No Buffer Refrigeration cascadable 20 x 20 cm 18 (– 50) yes, stepper motor Internal cooling Yes Not usefull Very short pulses Not viable 3 h Dropout memory Month 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 52 Field Gel Electrophoresis 52
Salmonella Typing 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 53 Field Gel Electrophoresis 53
Salmonella Typing 16 September 2020 Pulsed. Pulsfeldgelelektrophorese. 54 Field Gel Electrophoresis 54
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