Gel electrophoresis system including optical stage, sample applicator and sample retriever

• US 5,275,710 A
• Filed: 02/14/1992
• Issued: 01/04/1994
• Est. Priority Date: 05/14/1990
• Status: Expired due to Term

• Current Assignee: Labintelligence Incorporated
• Original Assignee: Labintelligence Incorporated
• Inventors: Roth, Alex T., Gombocz, Erich A.
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): Starsiak, Jr., John S.
• Application Number:
  US07/837,067
• Time in Patent Office: 690 Days
• Field of Search: 204/299 R, 204/182.8, 204/182.7, 204/182.9, 204/180.1
• US Class Current: 204/461
• CPC Class Codes: G01N 27/44704 Details; Accessories
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44782 of a plurality of samples

Abstract

An electrophoretic system is provided which allows for carrying out electrophoresis while monitoring and regulating the temperature and the electrical field gradient in the gel. In addition, photometric monitoring, including use of an optical stage, is provided so as to monitor the progress of the electrophoretic separation and vary conditions to change the progress as desired. A computer is employed which receives the signals from the electrophoretic and photometric apparatuses and regulates temperature and voltage to either maintain conditions, or change the conditions to vary the progress of the electrophoresis and to store the separation results. A sample applicator and a sample retriever are also provided, to provide even and consistent application of sample to the gel and to allow recovery of portions of the sample without destroying or removing pieces from the gel. Gel molds are provided for forming the lanes in a gel plate, as well as a light module, for reading the bands present in the gel lanes with the photometer.

Claims

1. A gel electrophoresis system for separating components of a mixture by their physical and chemical characteristics by passage through a gel under a voltage gradient, said system comprising:

a. a housing comprising a gel electrophoresis module, and electrical circuitry for external connection, sample applicator and retrieval means, and a gel plate;

b. said gel electrophoresis module comprising a temperature control platform, a removable optical stage reflective for light in the ultraviolet range and transparent for light in the visible range, supported by said platform, buffer wells on opposite sides of said platform, power electrodes for creating a voltage gradient in said gel, and sensing electrodes for reading the voltage gradient for feedback to the power electrodes;

c. a gel plate for mounting on said platform and supporting a gel, said plate comprising aligned orifices proximal to opposite ends of a horizontal gel support, and first and second vertical support means for extending into said buffer wells and comprising channels communicating with said orifices, a separation gel on said horizontal support and a stacking gel in said channels of said first vertical support, wherein said stacking gel and said separation gel abut at said orifices or on said horizontal gel support in the direction of migration;

d. a sample applicator assembly for application of sample to said gel, said applicator assembly comprising a housing having at least one passageway, with an upper opening for receiving liquid sample and a lower opening covered with an absorbent material for absorbing said sample and delivering said sample to said gel; and

e. a sample retriever assembly for removing portions of sample from said gel after at least partial separation, said retriever assembly comprising a housing having at least one passageway for receiving an electrically conductive medium, with an upper opening for withdrawing sample and a lower opening covered with an absorbent material to contact said gel over said portion, said electrically conductive medium in said passageway for electrical contact with said absorbent material; and

an electrode positioned for contact with said medium.

2. A gel plate comprising:

a flat horizontal support for a gel comprising aligned orifices at opposite ends of said support;

a separation gel in a plurality of lanes recessed in said horizontal support; and

vertical support means at opposite ends of said horizontal support, said vertical support means comprising aligned channels filled with a stacking gel in contact with said separation gel at said orifice or on said horizontal support.

3. A gel plate of claim 2, wherein said channels terminate in a beveled bottom at an acute angle, and wherein said horizontal support has a side wall with at least two spaced apart apertures for receiving sensing electrodes.

4. A sample application device comprising:

a housing;

at least one channel extending through said housing and having an upper sample receiving opening and a lower sample component dispensing opening;

a gel in said channel extending from said lower opening at least a portion of the length of said channel; and

an absorbent wick layer covering said lower opening and in contact with said gel.

5. A sample application device of claim 4, wherein said housing has a plurality of channels and wicks, with each wick separated from the other wicks.

6. A sample application device of claim 5, wherein said plurality of channels are spaced apart in relation to the spacing of lanes in a gel for electrophoresis.

7. A sample application device of claim 4, wherein said housing is transparent.

8. A sample application device of claim 4, wherein said wick is composed of spun fabric.

9. A sample retriever device comprising:

a housing;

at least one channel extending through said housing to provide upper and lower openings in said housing;

material in said housing extending from said lower opening at least a portion of the length of said channel, said material absorbing or comprising an electrically conducting medium in said channel;

an absorbent material wick covering said lower opening and in contact with said material; and

an electrode in contact with said material distal from said lower opening.

10. A sample retriever of claim 9, comprising a plurality of channels, and wherein said electrode is formed of individually chargeable sections.

11. A sample retriever of claim 9, comprising a plurality of channels and wherein said electrode is a singly chargeable electrode.

12. A sample retriever of claim 9, comprising a plurality of channels, wherein said channels are spaced apart in relation to the spacing of lanes in a gel for electrophoresis.

13. A method for applying, separating, retrieving and reapplying a component of a sample from a gel, said method comprising;

a. applying sample to be electrophoresed to a gel by means of a sample applicator, comprising a container which includes said sample and an absorbent wick in contact with said sample for contacting said gel, said applying comprising contacting said gel with said wick at a sample receiving site under the influence of an electrical field in said gel;

b. applying current across said gel between first and second electrodes such that components of said sample migrate at different rates through said gel;

c. identifying at least one individual component in said gel;

d. applying an absorbent wick to said gel at said identified component, wherein said wick is in electrical communication with a retrieval electrode through an electrically conducting medium and covers only a small area including said component;

e. passing a current through said retrieval electrode and an electrode in electrical communication with said gel and on the opposite side of said component from said retrieval electrode,

whereby said component migrates from said gel into said electrically conducting medium and is available for reapplication to a gel.

14. A method of claim 13, wherein said electrically conducting medium is an absorbent material and an electrically conductive liquid.

15. A method of claim 13, wherein said component is fluorescent or is labeled with a fluorescent molecule and said identifying comprises:

irradiating said gel with UV light, wherein said UV light passes through said gel and is reflected back through said gel; and

detecting the presence of fluorescence from a position of said gel as indicative of the presence of a component of said sample at said position.

16. A method of claim 13, wherein said component is light absorbent or is labeled with a light absorbent molecule, said method comprising:

irradiating a position of said gel with visible light on one side of said gel; and

detecting the absorption of light at said position as indicative of the presence of said component of said sample at said position.

17. A method of separating temperature denaturable components in a sample, said method comprising:

transferring said sample to an electrophoresis gel at a sample receiving site at a temperature below denaturing of said components;

applying an electrical field to said gel while raising the temperature of said gel in accordance with a predetermined regimen; and

identifying the position in said gel of said components in relation to the temperature at the time said component migrated to said position;

whereby said components are identified by their rate of migration and denaturation temperature.

18. A method of claim 17, wherein said electrophoresis gel is a soft gel.

19. A method of claim 17, wherein said temperature regimen is controlled by controlling the rate of cooling of said gel, wherein said gel is heated as a result of said electrical field.

20. A method of claim 17, wherein said components are proteins.

21. A method of claim 17, wherein said components are first partially separated by an electrical field at a fixed temperature below a denaturation temperature.

22. A kit comprising in combination,

a sample applicator assembly for application of sample to said gel, said applicator assembly comprising a housing having at least one passageway, with an upper opening for receiving liquid sample and a lower opening covered with an absorbent material for absorbing said sample and delivering said sample to said gel;

and

a sample retriever assembly for removing portions of sample from said gel after at least partial separation, said retriever assembly comprising a housing having at least one passageway for receiving an electrically conductive medium, with an upper opening for withdrawing sample and a lower opening covered with an absorbent material to contact said gel over said portion, said electrically conductive medium in said passageway for electrical contact with said absorbent material; and

an electrode positioned for contact with said medium.

23. A kit of claim 22, wherein said kit further comprises an optical plate having a coating reflective below about 500-550 nm and transmissive above about 500-550 nm.

24. A method for detecting the presence of a component of a sample, said method comprising:

(a) applying said sample to be electrophoresed to a gel;

(b) applying current across said gel between first and second electrodes such that components of said sample migrate at different rates through said gel;

(c) identifying the presence of at least one component in said gel;

(d) applying an absorbent wick to said gel at said identified component, wherein said wick is in electrical communication with the retrieval electrode through an electrically-conducting medium and covers only a small area including said component;

(e) passing a current through said retrieval electrode and an electrode in electrical communication with said gel and on the opposite side of said component from said retrieval electrode,

whereby said component migrates from said gel into said electrically conducting medium.

25. A method according to claim 24, wherein said identified component in said electrically-conducting medim is further characterized.

26. A method according to claim 24, wherein said identified component is identified by means of fluorescence.