28. februar 2014 pulsfeldgelelektrophorese.1 february 28, 2014 pulsed field gel electrophoresis 1...

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+

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A-

A+

B-

B+

OFAGE – Electric Field

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OFAGE – Electric Field

A-

A+

B-

B+

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OFAGE – Electric Field

A-

A+

B-

B+

>>90°

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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

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Separation Principle < 90°

Field Orientation

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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

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Separation Principle < 90°

Field Orientation

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Separation Principle PFGE (>90°)

Field Orientation

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Separation Principle PFGE (>90°)

Field Orientation

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Entscheidungsschwierigkeiten

What do you mean ?

You are the head now ?

You have been the head last time !

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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Trennprinzip PFGE (>90°)

Field Orientation

Field Orientation

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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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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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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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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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Realization

CHEFRGE

TAFE

RFE

Biorad

Biometra

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Rotaphor Electrodes

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Rotordesign

Orientation of Pulse

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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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Salmonella Typing

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Salmonella Typing