Integrated polymerase chain reaction/electrophoresis instrument

• US 6,126,804 A
• Filed: 09/23/1997
• Issued: 10/03/2000
• Est. Priority Date: 09/23/1997
• Status: Expired due to Fees

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Andresen, Brian D.
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US08/935,936
• Time in Patent Office: 1,106 Days
• Field of Search: 204/601, 204/602, 204/603, 204/604, 204/605, 204/451, 204/452, 204/453, 204/454, 204/455, 204/450, 204/600, 435/287.2, 435/287.1, 435/287.3, 435/288.5
• US Class Current: 204/601
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

A new approach and instrument for field identification of micro-organisms and DNA fragments using a small and disposable device containing integrated polymerase chain reaction (PCR) enzymatic reaction wells, attached capillary electrophoresis (CE) channels, detectors, and read-out all on/in a small hand-held package. The analysis instrument may be made inexpensively, for example, of plastic, and thus is disposable, which minimizes cross contamination and the potential for false positive identification between samples. In addition, it is designed for multiple users with individual applications. The integrated PCR/CE is manufactured by the PCR well and CE channels are "stamped" into plastic depressions where conductive coatings are made in the wells and ends of the CE microchannels to carry voltage and current to heat the PCR reaction mixtures and simultaneously draw DNA bands up the CE channels. Light is transmitted through the instrument at appropriate points and detects PCR bands and identifies DNA fragments by size (retention time) and quantifies each by the amount of light generated as each phototransistor positioned below each CE channel detects a passing band. The instrument is so compact that at least 100 PCR/CE reactions/analyses can be performed easily on one detection device.

Claims

1. An integrated polymerase chain reaction/capillary electrophoresis instrument, comprising:

a substrate;

said substrate containing at least one capillary column formed therein;

said at least one capillary column including end sections having a cross-section smaller than a main section thereof;

said substrate including at least one well for polymerase chain reactions located adjacent one end of said capillary column;

said substrate including at least one well for capillary electrophoresis buffer material located adjacent said one end of said capillary column;

said substrate including a passageway interconnecting said wells and said one end of said capillary column located intermediate said wells;

means for providing electrical power to said wells and said capillary column; and

means for detecting material passing through said at least one capillary column.

2. The instrument of claim 1, additionally including means for covering the capillary column and the wells.

3. The instrument of claim 2, wherein said covering means comprises a glass plate.

4. The instrument of claim 1, wherein said means for providing electrical power includes a coating of electrically conductive material on surfaces forming said wells, the passageway, and end sections of said capillary column.

5. The instrument of claim 1, additionally including another well for containing buffer material located at an end of said capillary column opposite said one end connected to said wells for polymerase chain reactions and for capillary electrophoresis buffer material.

6. The instrument of claim 1, wherein said substrate includes a multiplicity of capillary columns, a well for polymerase chain reactions for each capillary column, and at least one well for capillary electrophoresis buffer material for each capillary column, said wells being connected to ends of said capillary columns.

7. The instrument of claim 6, wherein each of said substrate includes at least one well for buffer material located at an opposite end of said capillary columns.

8. The instrument of claim 6, wherein each capillary column terminates in a common buffer well.

9. The instrument of claim 1, wherein said substrate has a configuration selected from the group consisting of rectangular and circular.

10. The instrument of claim 1, wherein at least said wells and said passageway includes an electrically conductive coating, and wherein said means for providing electrical power includes means for activating and deactivating said conductive coating for controlling passage of material from said well for polymerase chain reactions into said capillary column.

11. The instrument of claim 10, wherein said electrically conductive coating is selected from the group consisting of Pt/Pd, Ag, and Sn.

12. The instrument of claim 1, wherein said detecting means includes at least one photodiode located adjacent said at least one capillary column, and means for directing light onto at least said capillary column.

13. The instrument of claim 1, wherein at least said well for polymerase chain reactions is provided with means for supplying said well with reaction solution material.

14. A system which detects and identifies organisms by polymerase chain reactions followed by capillary electrophoresis, comprising:

an integrated polymerase chain reaction/capillary electrophoresis instrument which includes at least one well for polymerase chain reactions, at least one capillary column, at least one well for capillary electrophoresis buffer material, and means for detecting material passing through said at least one capillary column, wherein polymerase chain reactions from said at least one well for polymerase chain reactions are passed directly into one end of said capillary column along with buffer material from said at least one well for capillary electrophoresis buffer material for electrophoresis identification,

said capillary column including end sections having a cross-section smaller than a main section thereof,

said means for detecting material passing through said at least one capillary column being located in said main section of said at least one capillary column.

15. The system of claim 14, wherein said instrument comprises more than one capillary column, a polymerase chain reaction chamber located adjacent an end of each capillary column, and at least one chamber for containing buffer material located adjacent said end of each capillary column.

16. The system of claim 15, wherein opposite ends of said capillary columns terminate in a common chamber for containing buffer material.

17. The system of claim 15, wherein said capillary columns, said reaction chambers, and said chamber for containing buffer material are located on a single substrate.

18. The system of claim 17, additionally including a coating of electrically conductive material on the reaction chambers, the buffer material chambers, and at least one end of said capillary column.