On-line DNA analysis system with rapid thermal cycling

• US 5,935,522 A
• Filed: 06/30/1997
• Issued: 08/10/1999
• Est. Priority Date: 06/04/1990
• Status: Expired due to Fees

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Swerdlow, Harold P., Wittwer, Carl T.
• Primary Examiner(s): Le, Long V.
• Application Number:
  US08/885,625
• Time in Patent Office: 771 Days
• Field of Search: 422/67, 422/70, 422/81, 422/82.05, 422/82.08, 422/100, 422/101, 422/68.1, 436/43, 436/53, 436/164, 436/165, 436/174, 436/177, 436/178, 436/180, 436/161, 436/162, 436/807, 204/180.1, 204/299 R, 204/601, 204/604, 210/198.2, 210/656, 210/142, 73/61.52, 73/61.55, 73/61.56, 435/4, 435/6, 435/286.1, 435/287.2
• US Class Current: 422/70
• CPC Class Codes: B01D 15/34 Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
• CPC Class Codes: B01L 2400/0644 rotary valves
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: G01N 2030/027 Liquid chromatography
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 30/02 Column chromatography
• CPC Class Codes: G01N 30/06 Preparation
• CPC Class Codes: G01N 30/463 for multidimensional chromatography
• CPC Class Codes: G01N 30/466 with separation columns in parallel
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10T 436/11 Automated chemical analysis
• CPC Class Codes: Y10T 436/118339 with formation of a segmented stream
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

An apparatus particularly suited for subjecting biological samples to any necessary sample preparation tasks, subjecting the sample to rapid thermal cycling, and then subjecting the sample to subsequent on-line analysis using one or more of a number of analytical techniques. The apparatus includes a chromatography device including an injection means, a chromatography pump, and a chromatography column. In addition, the apparatus also contains a capillary electrophoresis device consisting of a capillary electrophoresis column with an inlet and outlet end, a means of injection, and means of applying a high voltage to cause the differential migration of species of interest through the capillary column. Effluent from the liquid chromatography column passes over the inlet end of the capillary electrophoresis column through a tee structure and when the loading of the capillary electrophoresis column is desired, a voltage supply is activated at a precise voltage and polarity over a specific duration to cause sample species to be diverted from the flowing stream to the capillary electrophoresis column. A laser induced fluorescence detector preferably is used to analyze the products separated while in the electrophoresis column.

Claims

1. An apparatus for subjecting a biological sample to rapid thermal cycling and subsequent on-line analysis of DNA products derived therefrom, the apparatus comprising:

means for holding a liquid biological sample;

first means for controlling and rapidly varying a temperature of the means for holding a liquid biological sample at a rate at least as great as one degree centigrade per second repeatedly through a predetermined temperature cycle such that the temperature repeatedly ramps from a first temperature to at least a second higher temperature and ramps from at least the second temperature to a lower temperature where said predetermined temperature cycle facilitates DNA amplification;

a primary separation means for separating one or more components of the liquid biological sample, the primary separation means being spaced from the first means for controlling and rapidly varying a temperature of the means for holding a sample;

means for fluidic transferring of the liquid biological sample from the means for holding the liquid biological sample to the primary separation means;

a secondary separation means for separating the components of the liquid biological sample;

means for fluidic transferring of at least some of the liquid biological sample from the primary separation means and injecting at least some of the liquid biological sample components into the secondary separation means;

means for detecting one or more separated components of interest in the secondary separation means; and

means for controlling the first means for controlling and rapidly varying the temperature of the liquid biological sample such that thermal cycling of the biological sample and the desired DNA amplification is carried out, and for controlling the primary separation means such that primary separation is carried out, and for controlling the secondary separation means such that secondary separation is carried out, and for controlling the means for detection such that detection is carried out on the liquid biological sample and the liquid biological sample is analyzed without manual intervention.

2. The apparatus in claim 1 wherein said primary separation means is a liquid chromatograph apparatus.

3. The apparatus in claim 1 wherein said secondary separation means is an electromigration apparatus.

4. The apparatus in claim 1 wherein said sample holding means is a liquid chromatography injection valve.

5. The apparatus in claim 1 wherein the primary separation means is an electromigration apparatus.

6. The apparatus in claim 1, further comprising second means for controlling and rapidly varying the temperature of the means for holding a sample repeatedly through a predetermined temperature cycle.

7. An apparatus for subjecting a liquid biological sample to rapid thermal cycling and subsequent on-line analysis of DNA products derived therefrom, the apparatus comprising:

means for holding a liquid biological sample;

first means for controlling and rapidly varying a temperature of said liquid biological sample at a rate at least as great as one degree centigrade per second repeatedly through a predetermined temperature cycle such that the temperature repeatedly ramps from a first temperature to at least a second temperature and ramps from the second temperature to a lower temperature and where said predetermined temperature cycle facilitates DNA amplification of said liquid biological sample;

a primary separation means for separating one or more components of the liquid biological sample, the primary separation means being spaced from the first means for controlling and rapidly varying a temperature of the liquid biological sample;

means for fluidic transferring of said liquid biological sample from the means for holding said liquid biological sample to the primary separation means;

a secondary separation means for separating the components of the liquid biological sample;

means for fluidic transferring of at least some of the liquid biological sample from the primary separation means and injecting at least some of the liquid biological sample components into the secondary separation means, the fluidic transfer to the secondary separation means being continuous;

temperature control means for maintaining the temperature of the primary separation means and the secondary separation means;

means for removing salt from the liquid biological sample;

means for detecting one or more separated components of interest in the secondary separation means; and

means for controlling the first means for controlling and rapidly varying the temperature of the liquid biological sample such that thermal cycling of the biological sample and DNA amplification is carried out, and for controlling the primary separation means such that primary separation of DNA derivatives is carried out, and for controlling the secondary separation means such that secondary separation of DNA derivatives is carried out, and for controlling the means for detection such that detection of DNA derivatives is carried out on the sample and the sample is analyzed without manual intervention.

8. The apparatus in claim 7 wherein said primary separation means is a liquid chromatograph apparatus.

9. The apparatus in claim 7 wherein said secondary separation means is an electromigration apparatus.

10. The apparatus in claim 7 wherein said sample holding means is a liquid chromatography injection valve.

11. The apparatus in claim 7 wherein the primary separation means is an electromigration apparatus.

12. The apparatus in claim 7 wherein the secondary separation means comprises means for collecting fractions from the biological sample.

13. The apparatus in claim 7, further comprising second means for controlling and rapidly varying the temperature of the liquid biological sample repeatedly through a predetermined temperature cycle.