WO1995011450A1 - Electrophorese amelioree - Google Patents

Electrophorese amelioree Download PDF

Info

Publication number
WO1995011450A1
WO1995011450A1 PCT/GB1994/002297 GB9402297W WO9511450A1 WO 1995011450 A1 WO1995011450 A1 WO 1995011450A1 GB 9402297 W GB9402297 W GB 9402297W WO 9511450 A1 WO9511450 A1 WO 9511450A1
Authority
WO
WIPO (PCT)
Prior art keywords
tubes
sample
gel
samples
array
Prior art date
Application number
PCT/GB1994/002297
Other languages
English (en)
Inventor
Andrew Robert Watson
Andrew Robert Smith
Original Assignee
Medical Research Council
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 Medical Research Council filed Critical Medical Research Council
Publication of WO1995011450A1 publication Critical patent/WO1995011450A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • 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 electrophoresis, and concerns a method and apparatus for supplying samples to an electrophoretic gel.
  • Electrophoresis is a well known analytical technique in which samples in solution are applied to wells in an electrophoretic gel, typically comprising polyacrylamide or agarose, and are exposed to an electric field which causes differential migration of molecules on the basis of charge, size and shape. Electrophoresis is used widely in the separation of proteins, DNA and RNA, finding application, e.g., in DNA sequencing.
  • Typical electrophoretic separation apparatus comprises a thin slab of gel (0.3 to 0.4 mm thick) in vertical orientation, confined between parallel plates held a suitable distance apart by spacers at the sides.
  • a series of sample-receiving wells is formed in the upper face of the gel.
  • this is conveniently achieved by use of a suitably shaped piece of rigid plastics material (known as a shark's tooth comb) comprising a plurality of pointed teeth the sides of which define well walls.
  • samples are supplied manually to a gel, typically using a pipette.
  • samples are loaded individually to each well, although multiple pipettes are used in some cases, e.g. for simultaneous loading of 4 wells.
  • Sample loading is a physically difficult, time consuming, labour intensive step in which errors can easily be made, requiring the services of relatively skilled personnel.
  • DNA sequencing operation can now be performed in a fully automated manner, using commercially available automated sequencing apparatus, the need for a skilled operative to perform the loading step provides an undesirable constraint on the overall ease and speed with which sequencing operations can be performed.
  • apparatus for simultaneously supplying a plurality of samples to an electrophoretic gel comprising an array of a plurality of tubes, each tube having an inlet end for receiving a sample from a receptacle and an outlet end for supplying a sample to a well in an electrophoretic gel; and a multichannel pump through which the array of tubes passes for simultaneously pumping samples through individual tubes in the array from respective sample receptacles to respective wells of the gel.
  • the invention also provides a method of simultaneously supplying a plurality of samples to a plurality of wells in an electrophoretic gel from a plurality of sample receptacles, comprising simultaneously pumping samples through a plurality of tubes in an array, each tube leading from a respective sample receptacle to a respective well.
  • the invention finds particular, but not exclusive, application in connection with sequencing of DNA, where large numbers of samples require electrophoretic treatment.
  • the tubes conveniently have a bore diameter of about 0.2 mm, and are preferably in the form of ribbon tubing.
  • the number of tubes in the array can be selected to suit requirements, being infinitely variable and expansible within the constraints of the gel.
  • the array conveniently comprises, say, 36 or 96 tubes.
  • the pump conveniently comprises a multichannel peristaltic pump, preferably having a variable speed drive.
  • a multichannel peristaltic pump By suitable selection of pump speed and tube bore, the rate of delivery of samples can be accurately controlled.
  • the pump can either be run at constant (slow) speed, or can be run slowly as samples are being picked up and dispensed and more quickly as samples are passing through the bulk of the tubes in order to speed up the process.
  • Suitable peristaltic pumps adapted for use with one or more multichannel cartridge blocks each adapted to receive a plurality of individual tubes, are commercially available.
  • Ismatec MV-MS/CA8 variable speed peristaltic pump from Ismatec UK Limited is designed for use with up to 5 snap-in cartridge blocks each having 8 channels and so is suitable for use with an array of up to 40 tubes.
  • a similar arrangement designed for use with ribbon tubing of desired size could be readily constructed.
  • the apparatus preferably includes control means, e.g. computer control means, for controlling operation of the pump in appropriate manner to achieve desired sample delivery.
  • control means e.g. computer control means, for controlling operation of the pump in appropriate manner to achieve desired sample delivery.
  • the inlet ends of the tubes are conveniently held in an arrangement matching the arrangement of sample receptacles, e.g. with the ends passing through a suitably apertured holder.
  • the sample receptacles will typically be Eppendorf tubes in racks, usually in an array of 36, or a 96 well microtitre plate.
  • a respective fine bore needle is preferably fitted in each tube inlet end to facilitate location in sample receptacles.
  • the outlet ends of the tubes may be secured to a shark's tooth comb, with a respective tube terminating in or above a respective gap between well-defining teeth side walls.
  • the tube outlet ends may be otherwise suspended above or within pre-formed wells within a gel.
  • a respective fine bore needle is preferably fitted in each tube outlet end, for accurate delivery of samples into the wells.
  • the shark's tooth comb with attached tubes is fitted in conventional manner into the top of a gel, e.g. in automated DNA sequencing apparatus, so that the outlet ends of the tubes (or associated needles) open in well-defining gaps between teeth side walls.
  • the inlet ends of the tubes are located in sample receptacles, e.g. with the aid of an apertured holder.
  • the pump is then operated, possibly under control of computer control means, simultaneously to deliver a desired quantity of sample to each well.
  • the gel is then run in conventional manner, with the comb either being removed or left in place as appropriate.
  • Fitting of a comb to a gel will generally be performed manually, although this is not a particularly skilled task and can be done by relatively unskilled personnel, unlike current sample loading procedures.
  • the remaining steps including sample supply and gel running will generally be fully automated so operator intervention is reduced to an unskilled minimum.
  • the sample loading operation can thus be performed with the assistance of relatively unskilled personnel and can be quicker, more accurate and more reliable than is currently possible.
  • the invention is also suitable for use with agarose gels which are generally in horizontal orientation in an open-topped trough, with open-topped sample-receiving wells formed at one end of the gel.
  • the present invention provides automated DNA sequencing apparatus in combination with sample supply apparatus of the invention.
  • Figure 1 is a schematic illustration of one embodiment of apparatus in accordance with the invention.
  • Figure 2 is a perspective view illustrating a further embodiment of apparatus in accordance with the invention.
  • Figure 3 shows a detail of the arrangement of the inlet ends of the tubes of the Figure
  • FIG 4 illustrates schematically the inlet transfer arrangement of the embodiment of Figure 2;
  • Figure 5 shows, on an enlarged scale, part of the outlet arrangement of the embodiment of Figure 2;
  • Figure 6 shows a modified version of Figure 5.
  • the apparatus illustrated in Figure 1 comprises a length of ribbon tubing 10.
  • the arrangement shown comprises an array of 20 tubes, but it is clear that other numbers (smaller or larger) of tubes may be included in the array, and an array of, say, 36 or 96 tubes may be desired for use in connection with DNA sequencing operations as discussed above.
  • the tubing is of flexible, plastics material, possibly transparent, and each tube conveniently has a bore diameter of about 0.2mm.
  • the inlet ends 12 of the tubes are held in array, e.g. by means of an apertured holder shown schematically at 13, to match the positions of sample receptacles such as that shown schematically at 15, e.g. in the form of an array of 36 Eppendorf tubes in racks or a 96 well microtitre plate.
  • a respective fine bore needle 14 is fitted in the inlet end of each tube to facilitate location in the sample receptacles.
  • the outlet ends 16 of the tubes are secured, e.g. glued, to a shark's tooth comb 18 of rigid plastics material, with each tube terminating above a gap between well defining teeth 20 of the comb.
  • a respective needle 22 is fitted in the outlet end of each tube, terminating in the associated gap between the well defining side walls of adjacent teeth 20, for delivery of sample to a well.
  • the ribbon tubing passes through a peristaltic pump, shown schematically at 24.
  • the shark's tooth comb 18 is fitted in conventional manner by an operator to the top of an electrophoretic gel shown schematically at 26, e.g. forming part of an automated DNA sequencer such as an ABI 373A sequencer.
  • the needles 14 in the inlet ends 12 of the tubes are located in sample receptacles, with the samples typically comprising DNA for analysis.
  • the pump 24 is operated, e.g. under control of suitable control means shown schematically at 28, simultaneously to supply a desired quantity, e.g. 4-5 ⁇ l, of sample to each well.
  • the gel is then run in conventional manner, with the comb either remaining in position or being removed after sample loading, as desired.
  • the illustrated arrangement comprises a length of ribbon tubing 50 having 96 side-by-side tubes leading from an inlet unit 52 to an outlet bracket 54.
  • the tubing passes through a peristaltic pump, shown schematically at 55.
  • the components are mounted on a base 56, which also carries a housing 58 for a drive unit and control means.
  • a bracket 60 which includes two recesses or holders 62 and 64 for receiving respective standard 96 well microtitre plates, such as plate 66 shown schematically in recess 64. In the interests of clarity a plate is not shown in recess 62.
  • Bracket 60 also carries a transfer arrangement for moving the inlet unit between positions above the two plate-receiving recesses 62 and 64.
  • FIG. 3 illustrates schematically the arrangement within unit 52.
  • a pair of apertured plates 80, 82 are held a fixed distance apart by spacers 84, with each plate including an array of holes corresponding to the array of wells in a standard 96 well microtitre plate. For clarity, only 4 holes are shown.
  • Tubes 85 of the ribbon tubing 50 pass through holes 86 in the upper metal plate 80, the hole diameter being slightly smaller than the tube diameter so the tube is gripped and retained in position.
  • a fine bore (hypodermic) needle 88 is fitted into the end of each tube, with a weight 90 attached to an upper region of each needle, between the plates.
  • the needles pass through apertures 92 in the lower plastics plate, the hole diameter being slightly larger than the needle diameter to permit movement of the needle relative to the lower plate 82.
  • the portions of tube between the plates are longer than the distance between the plates so that a kink or bend 93 is formed in each tube.
  • the kink in combination with the effect of the weight, acts to bias the associated needle downwardly, so that in use the needles are urged into engagement with the bottoms of wells 94 in a microtitre plate 96 therebelow, for efficient sample collection.
  • the weights also act as stops, to limit downwards movement of the needles relative to the lower plate, while the arrangement permits the needles to move easily upwards relative to the lower plate by a few milimetres.
  • the inlet transfer arrangement comprises a pair of generally U-shaped brackets 68 and 70 pivotally mounted, at the right hand ends thereof as shown in the Figures, within bracket 60 at 72 and 74, respectively.
  • the left hand ends of the brackets are pivotally attached at 76 and 78 to the base of unit 52.
  • unit 52 initially in the left hand position as shown in Figure 2, over a microtitre plate 79 ( Figure 4), simultaneous pivoting of both brackets in a clockwise direction causes the unit 52 (represented schematically by a rectangle in Figure 4) to be moved by a parallel linkage motion up and across to the right hand side of the bracket 56, to overlie microtitre plate 66.
  • the transfer arrangement enables the input unit to be manoeuvred in such a way that the array of needles is protected, and so as to ensure consistent and reliable positioning of the needles within the wells of the microtitre plates.
  • Bracket 54 is shown in greater detail in Figure 5, and comprises a vacuum brazed metal extrusion shaped to be clipped to the top edge of one of the vertical gel-supporting plates shown schematically 2 at 105.
  • the needles are spaced to be located, in use, above or within pre-formed wells 104 in the gel represented schematically at 106.
  • the bracket 54 is shown mounted on a storage bracket 102.
  • Figure 6 illustrates an alternative version of outlet bracket 108 in the form of a plastics fabrication.
  • the operator first prepares a microtitre plate containing the samples to be run on the gel.
  • the plate is placed in recess or holder 62 or 64 as appropriate, and the inlet unit 52 positioned over the plate with the inlet needles located in the plate wells.
  • Outlet bracket 54 is clipped to the gel plate 105 with the associated needles either just above or just within pre-cast wells in the gel, depending on the method to be used.
  • the pump is then turned on and left running until all the samples have been dispensed into the wells.
  • the pump can either run at a constant (slow) speed, or to speed things up the pump can be run slowly as samples are being picked up and dispensed and quickly as the samples are passing through the bulk of the tubing.
  • Samples can be loaded with the gel wet or dry. To dry load, the gel is washed and then any liquid in the wells is aspirated off. The dispensing needles are positioned just inside the wells so that each sample falls straight into the bottom of the associated well. In wet loading the gel is washed as before. The gel is then placed against a tank which is filled with a buffer solution. This could be done either in a sequencing machine used to run the gel, or off line in a separate tank. In this case the dispensing needles are positioned slightly above the well openings. The samples have a slightly higher density than the buffer solution, so the samples fall into the wells. Any air pumped through the system is able to bubble up to the surface, and so does not affect the loading operation. With this method, an electric field can be applied vertically across the gel. This field attracts the sample down into the well more rapidly than if it were to fall on its own.
  • any electrophoresis power is turned off and the dispensing needles are removed from the gel.
  • the inlet unit 52 is transferred to be above the other recess or holder 64 or 62 as appropriate, in which is located a microtitre plate with deionized water in the wells.
  • the pump is operated to pump water through the tubes to remove any remaining sample.
  • the tubes are usually left full of water to prevent salt crystals from forming inside the needles and blocking them. If loaded off line, the gel can then be connected to an electrophoresis power supply (not shown) with the gel then being placed on a sequencing machine (not shown).
  • the apparatus can also be used as a means of loading agarose gels. These are thick horizontal slabs of gel that have been cast in a plastics trough. Wells are formed in situ using a square tooth plastics comb that stands perpendicular to the surface of the gel. Samples are loaded from above.
  • the apparatus of the invention would be used generally as described above, with the outlet needles suitably suspended above the wells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Dispositif servant à placer simultanément une pluralité d'échantillons dans un gel électrophorétique et comprenant un ensemble constitué par une pluralité de tubes; chaque tube possède une extrémité d'entrée servant à recevoir un échantillon depuis un réceptacle et une extrémité de sortie servant à alimenter un puits situé dans un gel électrophorétique en un échantillon; une pompe à canaux multiples à travers laquelle passe l'ensemble de tubes, afin de pomper simultanément des échantillons à travers des tubes individuels de l'ensemble depuis des réceptacles respectifs vers des puits respectifs du gel. L'invention concerne également un procédé servant à alimenter simultanément en une pluralité d'échantillons une pluralité de puits situés dans un gel électrophorétique depuis une pluralité de réceptacles d'échantillon. Ledit procédé consiste en le pompage simultané d'échantillons à travers une pluralité de tubes d'un ensemble, chaque tube s'étendant depuis un réceptacle d'échantillon respectif vers un puits respectif. L'invention s'adresse particulièrement, mais non exclusivement, à la mise en séquence d'ADN, dans laquelle on doit soumettre des quantités élevées d'échantillons à un traitement électrophorétique.
PCT/GB1994/002297 1993-10-20 1994-10-20 Electrophorese amelioree WO1995011450A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9321650.5 1993-10-20
GB939321650A GB9321650D0 (en) 1993-10-20 1993-10-20 Improvements in or relating to electrophoresis

Publications (1)

Publication Number Publication Date
WO1995011450A1 true WO1995011450A1 (fr) 1995-04-27

Family

ID=10743857

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1994/002297 WO1995011450A1 (fr) 1993-10-20 1994-10-20 Electrophorese amelioree

Country Status (2)

Country Link
GB (1) GB9321650D0 (fr)
WO (1) WO1995011450A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021309A1 (fr) * 1999-09-23 2001-03-29 Genset Dispositif de stockage et de distribution de fluides notamment de reactifs
FR2798867A1 (fr) * 1999-09-23 2001-03-30 Commissariat Energie Atomique Dispositif de distribution de fluides dans un microsysteme fluidique
EP1249705A2 (fr) * 1996-12-31 2002-10-16 Genometrix Genomics Incorporated Dispositif d'analyse moléculaire multiplexé et procédé de sa fabrication
US6652809B1 (en) 1997-09-17 2003-11-25 Glaxo Research And Development Limited Apparatus for performing photometric assays
AU771520B2 (en) * 1998-09-08 2004-03-25 Tibotec N.V. Method for the rapid screening of analytes
EP1411340A2 (fr) * 1999-02-26 2004-04-21 EXACT Sciences Corporation Dispositifs biochimiques de purification au moyen de sondes de capture et leurs utilisations

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1320290A (fr) * 1961-03-13 1963-03-08 Technicon Instr Appareil et procédé d'analyse automatique par électrophorèse d'un ampholyte
DE2445409A1 (de) * 1974-09-23 1976-04-08 Cubana Export Import Vertikale elektrophoresevorrichtung
FR2628215A1 (fr) * 1988-03-02 1989-09-08 Helena Lab Corp Procede et appareil automatique d'electrophorese

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1320290A (fr) * 1961-03-13 1963-03-08 Technicon Instr Appareil et procédé d'analyse automatique par électrophorèse d'un ampholyte
DE2445409A1 (de) * 1974-09-23 1976-04-08 Cubana Export Import Vertikale elektrophoresevorrichtung
FR2628215A1 (fr) * 1988-03-02 1989-09-08 Helena Lab Corp Procede et appareil automatique d'electrophorese

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
X. C. HUANG: "DNA SEQUENCING USING CAPILLARY ARRAY ELECTROPHORESIS", ANALYTICAL CHEMISTRY, vol. 64, no. 18, 15 September 1992 (1992-09-15), pages 2149 - 2154, XP000319973 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1249705A2 (fr) * 1996-12-31 2002-10-16 Genometrix Genomics Incorporated Dispositif d'analyse moléculaire multiplexé et procédé de sa fabrication
JP2003107097A (ja) * 1996-12-31 2003-04-09 Genometrix Inc 多重化分子分析装置および方法
EP1249705A3 (fr) * 1996-12-31 2003-11-05 Genometrix Genomics Incorporated Dispositif d'analyse moléculaire multiplexé et procédé de sa fabrication
US6652809B1 (en) 1997-09-17 2003-11-25 Glaxo Research And Development Limited Apparatus for performing photometric assays
AU771520B2 (en) * 1998-09-08 2004-03-25 Tibotec N.V. Method for the rapid screening of analytes
EP1411340A2 (fr) * 1999-02-26 2004-04-21 EXACT Sciences Corporation Dispositifs biochimiques de purification au moyen de sondes de capture et leurs utilisations
EP1411340A3 (fr) * 1999-02-26 2004-05-19 EXACT Sciences Corporation Dispositifs biochimiques de purification au moyen de sondes de capture et leurs utilisations
WO2001021309A1 (fr) * 1999-09-23 2001-03-29 Genset Dispositif de stockage et de distribution de fluides notamment de reactifs
FR2798867A1 (fr) * 1999-09-23 2001-03-30 Commissariat Energie Atomique Dispositif de distribution de fluides dans un microsysteme fluidique
FR2807952A1 (fr) * 1999-09-23 2001-10-26 Genset Sa Dispositif de stockage et de distribution de fluides notamment de reactifs

Also Published As

Publication number Publication date
GB9321650D0 (en) 1993-12-08

Similar Documents

Publication Publication Date Title
US6136173A (en) Automated system for two-dimensional electrophoresis
US6656724B1 (en) Apparatus for automatic implementation of chemical or biological methods
US5217593A (en) Nucleic acid purification system and method
AU755104B2 (en) Sample handling system for a multi-channel capillary electrophoresis device
EP2059799B1 (fr) Procédé et dispositif d'électrophorèse libre sans vecteur
US6171463B1 (en) Electrophoresis method and apparatus for separating bio-organic molecules
US20010048897A1 (en) Gel loading adapter
EP0500506A1 (fr) Appareil pour immunoessais
US4726889A (en) Process and apparatus for conducting electrophoresis and transfer
WO1995011450A1 (fr) Electrophorese amelioree
WO1994026396A1 (fr) Electrophorese capillaire pour des applications specifiques
EP0236153B1 (fr) Méthode et appareil pour exécuter l'électrophorèse et le transfert suivant
EP0198767A2 (fr) Procédé et dispositif de fractionnement séquentiel
JP2002048765A (ja) 水平面型ゲル電気泳動装置用試料導入装置、水平面型ゲル電気泳動システム、その試料導入方法およびその試料採取方法
EP0256552A2 (fr) Procédé et appareil de focalisation isoélectrique par recyclage et isotachophorésis
Fawcett et al. A motor‐driven syringe‐type gradient maker for forming immobilized pH gradient gels
WO2020259816A1 (fr) Cartouche, système de traitement d'échantillon par électromouillage et son alimentation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase