WO1998025136A1 - Procede et dispositif d'electrophorese - Google Patents

Procede et dispositif d'electrophorese Download PDF

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Publication number
WO1998025136A1
WO1998025136A1 PCT/AU1997/000828 AU9700828W WO9825136A1 WO 1998025136 A1 WO1998025136 A1 WO 1998025136A1 AU 9700828 W AU9700828 W AU 9700828W WO 9825136 A1 WO9825136 A1 WO 9825136A1
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WO
WIPO (PCT)
Prior art keywords
gel
wells
sample
comb
well
Prior art date
Application number
PCT/AU1997/000828
Other languages
English (en)
Inventor
Robert Dixon Teasdale
Original Assignee
Forbio Research Pty. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Forbio Research Pty. Ltd. filed Critical Forbio Research Pty. Ltd.
Priority to AU51851/98A priority Critical patent/AU5185198A/en
Publication of WO1998025136A1 publication Critical patent/WO1998025136A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44743—Introducing samples

Definitions

  • This invention relates to an electrophoretic method and apparatus therefor.
  • This invention has particular but not exclusive application to methods and apparatus for use in gel electrophoresis of DNA and RNA samples, and for illustrative purposes reference will be made to such application. However, it is to be understood that this invention could be used in other applications, such as electrophoretic determinations of proteins and other charged molecules.
  • Comparative electrophoresis requires that multiple lanes of a gel are loaded with samples which are then run under the same condition variables to provide a calibrated, qualitative or quantitative result comparing respective lanes.
  • a limitation on the experimental and productive use of electrophoretic methods has been limitations on available resolution of electrophoresis using automated loading processes.
  • Gel combs are toothed forms, usually of TEFLON or other polymer, which are positioned in a tray into which an agarose or other gel material is cast, so that after the gel has set, the comb can be removed to leave a set of cavities, or wells, corresponding to the shape and position of the teeth on the comb.
  • Sample solutions are then placed in these wells using a pipette dispenser, after which the sample components are separated by electrophoresis . This process is often assisted by increasing the density of the sample solution so that it is higher than a "running buffer" with which the wells are normally filled.
  • This general arrangement is used for separation of biological macromolecules such as DNA, RNA, enzymes and other proteins, and some carbohydrates, among others.
  • the thickness of the well can be a limiting factor.
  • the loading of sample is correspondingly more difficult, requiring precise positioning of the pipette tip into the well. This task is more difficult when loading occurs with a multi-channel pipettor, such as various commonly used octi-pipettes, where any slight deflection or rotation of the comb or the octi-pipette can result in the misloading of some samples.
  • the present invention aims to substantially alleviate at least one of the above disadvantages and to provide an electrophoretic method and apparatus which will be reliable and efficient in use. Other objects and advantages of this invention will hereinafter become apparent.
  • this invention resides broadly in an electrophoretic method including providing a gel having a plurality of sample wells integrally formed in the gel, said sample wells each having a substantially flat front face of width sufficient to provide an appropriate lane width, and a lower rear face spaced apart therefrom by a sample width selected to provide a selected resolution, and an upper rear face diverging from said front wall to provide a loading opening for said well larger than said sample width to facilitate automated loading of said wells.
  • the wells may be formed by any suitable means and are preferably formed by a gel comb.
  • this invention resides in a gel comb comprising a body member adapted to support a plurality of spaced well forming members substantially vertically in a gel to be cast, each well forming member having a substantially flat front surface of width sufficient to provide an selected lane width, and a lower rear surface spaced apart therefrom by a dimension selected to form sample well of selected resolution by width, and an upper rear face diverging from said front surface.
  • the lower portion of the gel comb well forming members includes a lower rear surface, the thickness of the well forming member being selected to provide wells having the appropriate dimension in the direction of electrophoresis to provide a selected resolution in the gel to be cast.
  • the exact thickness of the well forming member at this point will be determined by the samples to run, the gel type, and many other variables.
  • the thickness of the well forming member at this point will be of the order of the thickness of a high resolution, manually loaded well.
  • the thickness may be in the region of 0.5mm, which is generally regarded as too small for automated loading.
  • the vertical length of the well forming member lower portion will be selected to provide an appropriate sample volume for a selected gel thickness.
  • wells formed are in the region of 3.3mm wide and 3.5mm high to the filling region.
  • the upper portion of the well forming member includes an upper rear surface portion extending upward and away from the front surface at about 45°.
  • the resulting well may present an upper opening that is of a plan dimension adequate for use of automated and multiple loading such as by means of an octipipette.
  • the divergent upper rear surface portion is preferably adapted to be inserted into the gel until the opening at the surface is just sufficient for gel loading, although it is envisaged that the well forming member may be inserted further into the gel since the filling height is determined by the dispensed volume.
  • Preferably methods in accordance with the present invention include forming the wells in the gel, filling the wells with a displaceable buffer or other liquid and loading the samples in dilute gel into the wells, whereby the buffer or liquid is displaced from the wells.
  • Gel loaded samples are preferably loaded such that the sample meniscus is located at the lower region of the divergent portion of the well.
  • the wells of the present invention include an upper divergent portion which also acts as a funnel so that the entire sample aliquot flows into the narrow part of the well.
  • the well forming members are preferably integrally formed with the body member of suitable material to produce a gel tooth comb.
  • the spacing of the gel teeth may be selected to be any suitable spacing and can be such as to correspond to standard distances encountered in multi-channel dispensers.
  • the mean spacing of teeth may be 4.5mm, which is twice the separation of tips in standard octi-pipette dispensers, which in turn corresponds to the 9mm separation of wells in standard 96-well multi-well trays for many biochemical and molecular-biological reactions.
  • each second well formed in the gel may correspond to a row of wells in one tray, with the intermediate gel samples corresponding to the second row; with appropriate placement of samples in the sample tray any required order of loading on gels can be attained.
  • the use of wider teeth to match precisely the spacing of multi- tip dispensers is also envisaged.
  • the preferred thickness of the teeth of 0.5mm provides excellent resolution for DNA samples in the range 400 to 15000bp. Thinner teeth can provide narrower bands, but a limit to improved resolution is reached due to diffusion, so that thinner bands will lead to lower signals and signal to noise ratios. With gels of low density, some distortion of well walls can lead to actual well thickness being less than that of the comb, and optima are best empirically determined for particular systems.
  • the combs can be constructed of any machinable or castable material compatible with the gel buffers, and may include brass, stainless steel, TEFLON, or other polymers.
  • FIG. 1 is a front elevation of apparatus in accordance with the present invention.
  • FIG.2 is an end elevation of the apparatus of FIG 1.
  • a gel tooth comb 10 having a brass body portion 11 extending between supporting lugs 12 of standard dimensions.
  • a plurality of gel teeth 13 are formed integrally with the body portion 11 .
  • mean spacing 4.5mm is twice the separation of tips in standard octi-pipette dispensers, which in turn corresponds to the 9mm separation of wells in standard 96-well multi-well trays for many biochemical and molecular-biological reactions.
  • each second well formed in the gel corresponds to a row of wells in one tray, with the intermediate gel samples corresponding to the second row; with appropriate placement of samples in the sample tray any required order of loading on gels can be attained.
  • the teeth 13 comprise a lower portion 14 corresponding to the substantive well portion, and an upper portion 15.
  • the lower portion in this example of the improved comb is 0.5mm thick, which provides excellent resolution for DNA samples in the range 400 to 15000bp.
  • the lower portion 14 is 3.5mm long and 3.3mm wide.
  • the upper portion 15 is 3 mm thick.
  • the transition in thickness from the lower tooth portion 14 to the upper tooth portion 15 is provided by a sloping portion 16 disposed to the back of the comb, that is, in the direction away from the electrophoresis direction.
  • the sloping portion 16 is disposed at 45° to the horizontal plane.
  • RAPD reactions were set up in a double volume, to enable the same reaction mix to be loaded on gels using the wedge comb and conventional comb.
  • the four primers used were chosen for high levels of polymorphism.
  • the primers were run on DNA samples from P. radiata and E. globulus. All RAPD fragments were separated using agarose gel electrophoresis using 1.5% gels run at 180V in 1 X TAE buffer for 2.5 hours. The comparison was made by finding the resolutions of gels using both combs. Resolution of a gel is defined as the minimum band separation between two RAPD fragments from the same lane detectable by the naked eye. This is expressed as a percentage of the band size.
  • bandl and band2 are the closest separated bands in the gel and that bandl has a smaller molecular weight than band2. Testing data is as following:
  • Standard Comb Wedge Comb bandl (bp) 1649 1225 band2 (bp) 1689 1243 size(band2)- 40 18 size( bandl ) resolution ( % ) 2.4 1 . 5
  • Standard Comb Wedge Comb bandl ( p ) 928 781 band2 (bp) 949 793 size(band2)- 21 12 size( bandl ) resolution ( % ) 2.2 1.5
  • wedge comb produces 26.4% more detectable bands on average.
  • a simple gel comb has been designed which improves both resolution and ease of loading.
  • the comb is particularly suited to automated robotic loading, but is also advantageous for manual loading by either single tip or multi-tip dispensers.

Abstract

L'invention concerne un procédé d'électrophorèse consistant à créer des puits dans un gel au moyen d'un peigne (10) possédant une partie corps (11) s'étendant entre des saillies de support (12) et une pluralité de dents (13) composées d'une partie inférieure (14) correspondant à la partie puits effective et d'une partie supérieure (15), la transition d'épaisseur depuis la partie inférieure (14) de la dent vers sa partie supérieure (15) étant constituée par une partie (16) inclinée à 45° et située dans un sens s'éloignant du sens de l'électrophorèse.
PCT/AU1997/000828 1996-12-05 1997-12-04 Procede et dispositif d'electrophorese WO1998025136A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU51851/98A AU5185198A (en) 1996-12-05 1997-12-04 Electrophoretic method and apparatus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPO4038 1996-12-05
AUPO4038A AUPO403896A0 (en) 1996-12-05 1996-12-05 Electrophoretic method and apparatus

Publications (1)

Publication Number Publication Date
WO1998025136A1 true WO1998025136A1 (fr) 1998-06-11

Family

ID=3798347

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1997/000828 WO1998025136A1 (fr) 1996-12-05 1997-12-04 Procede et dispositif d'electrophorese

Country Status (2)

Country Link
AU (1) AUPO403896A0 (fr)
WO (1) WO1998025136A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000002039A1 (fr) * 1998-07-03 2000-01-13 Wita Proteomics Ag Procede et dispositif pour separer des biomolecules
WO2000047984A1 (fr) * 1999-02-12 2000-08-17 Bio-Rad Laboratories, Inc. Systeme de coulage d'ensemble rapide pour gels en plaque
US6905584B2 (en) 2000-09-29 2005-06-14 Proteome Systems Limited Electrophoresis system and method therefor
WO2011028532A1 (fr) 2009-08-24 2011-03-10 Life Technologies Corporation Cassette et peigne d'électrophorèse sur gel
USD794823S1 (en) 2010-08-24 2017-08-15 Life Technologies Corporation Electrophoresis tank with a base and cassette inserted in
USD795449S1 (en) 2012-06-01 2017-08-22 Life Technologies Corporation Electrophoresis wedge-well retainer cassette
USD806894S1 (en) 2015-03-19 2018-01-02 Life Technologies Corporation Electrophoresis comb with wedge-shaped teeth

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4294684A (en) * 1980-06-30 1981-10-13 Board Of Reagents, University Of Texas Template for forming multiple gel tracks within a single electrophoretic gel frame
DD251829A1 (de) * 1986-08-01 1987-11-25 Akad Wissenschaften Ddr Vorrichtung und verfahren zur isolierung von nukleinsaeuren durch horizontale gel-elektrophorese
US5164065A (en) * 1991-12-13 1992-11-17 Bio-Rad Laboratories, Inc. Non-breakable, electrically insulating sample well inserts for slab electrophoresis
US5284565A (en) * 1992-12-23 1994-02-08 Bio-Rad Laboratories, Inc. Sample well insert with wedge-shaped profile for ultra-thin slab gels in electrophoresis
US5318682A (en) * 1993-09-08 1994-06-07 Gensura Laboratories, Inc. Sample-well forming comb for horizontal gel electrophoresis device
GB2284484A (en) * 1993-12-03 1995-06-07 Univ London Electrophoresis gel-matrix layer

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4294684A (en) * 1980-06-30 1981-10-13 Board Of Reagents, University Of Texas Template for forming multiple gel tracks within a single electrophoretic gel frame
DD251829A1 (de) * 1986-08-01 1987-11-25 Akad Wissenschaften Ddr Vorrichtung und verfahren zur isolierung von nukleinsaeuren durch horizontale gel-elektrophorese
US5164065A (en) * 1991-12-13 1992-11-17 Bio-Rad Laboratories, Inc. Non-breakable, electrically insulating sample well inserts for slab electrophoresis
US5284565A (en) * 1992-12-23 1994-02-08 Bio-Rad Laboratories, Inc. Sample well insert with wedge-shaped profile for ultra-thin slab gels in electrophoresis
US5318682A (en) * 1993-09-08 1994-06-07 Gensura Laboratories, Inc. Sample-well forming comb for horizontal gel electrophoresis device
GB2284484A (en) * 1993-12-03 1995-06-07 Univ London Electrophoresis gel-matrix layer

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000002039A1 (fr) * 1998-07-03 2000-01-13 Wita Proteomics Ag Procede et dispositif pour separer des biomolecules
WO2000047984A1 (fr) * 1999-02-12 2000-08-17 Bio-Rad Laboratories, Inc. Systeme de coulage d'ensemble rapide pour gels en plaque
US6162342A (en) * 1999-02-12 2000-12-19 Bio-Rad Laboratories, Inc. Rapid assembly casting system for slab gels
US6869514B1 (en) 1999-02-12 2005-03-22 Bio-Rad Laboratories, Inc. Rapid assembly casting system for slab gels
US7128819B2 (en) 1999-02-12 2006-10-31 Bio-Rad Laboratories, Inc. Rapid assembly casting system for slab gels
EP1731904A3 (fr) * 1999-02-12 2007-06-27 Bio-Rad Laboratories, Inc. Système de moulage pour assemblage rapide de gels visqueux
EP1722220A3 (fr) * 1999-02-12 2009-12-02 Bio-Rad Laboratories, Inc. Système de moulage pour assemblage rapide de gels visqueux
US7658827B2 (en) 1999-02-12 2010-02-09 Bio-Rad Laboratories, Inc. Rapid assembly casting system for slab gels
US6905584B2 (en) 2000-09-29 2005-06-14 Proteome Systems Limited Electrophoresis system and method therefor
EP2853891A1 (fr) * 2009-08-24 2015-04-01 Life Technologies Corporation Peigne et cassette de gel d'électrophorèse
EP3399307A1 (fr) * 2009-08-24 2018-11-07 Life Technologies Corporation Peigne et cassette de gel d'électrophorèse
EP2470691A4 (fr) * 2009-08-24 2013-03-06 Life Technologies Corp Cassette et peigne d'électrophorèse sur gel
WO2011028532A1 (fr) 2009-08-24 2011-03-10 Life Technologies Corporation Cassette et peigne d'électrophorèse sur gel
US9097663B2 (en) 2009-08-24 2015-08-04 Life Technologies Corporation Gel electrophoresis, imaging, and analysis methods, devices, systems, and materials
EP3168610A1 (fr) * 2009-08-24 2017-05-17 Life Technologies Corporation Électrophorese gel cassette et comb
US9714918B2 (en) 2009-08-24 2017-07-25 Life Technologies Corporation Electrophoresis gel cassette and comb
EP2470691A1 (fr) * 2009-08-24 2012-07-04 Life Technologies Corporation Cassette et peigne d'électrophorèse sur gel
US10613052B2 (en) 2009-08-24 2020-04-07 Life Technologies Corporation Electrophoresis gel cassette and comb
USD794823S1 (en) 2010-08-24 2017-08-15 Life Technologies Corporation Electrophoresis tank with a base and cassette inserted in
USD831847S1 (en) 2010-08-24 2018-10-23 Life Technologies Corporation Electrophoresis wedge-well divider cassette
USD832458S1 (en) 2010-08-24 2018-10-30 Life Technologies Corporation Electrophoresis cassette base
USD890363S1 (en) 2010-08-24 2020-07-14 Life Technologies Corporation Lid of an electrophoresis tank
USD795449S1 (en) 2012-06-01 2017-08-22 Life Technologies Corporation Electrophoresis wedge-well retainer cassette
USD806894S1 (en) 2015-03-19 2018-01-02 Life Technologies Corporation Electrophoresis comb with wedge-shaped teeth
USD858793S1 (en) 2015-03-19 2019-09-03 Life Technologies Corporation Electrophoresis comb with wedge-shaped teeth

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