Instrument for monitoring polymerase chain reaction of DNA

• US 8,557,566 B2
• Filed: 01/17/2006
• Issued: 10/15/2013
• Est. Priority Date: 05/16/1998
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Gambini, Michael R., Atwood, John G., Young, Eugene F., Lakatos, Edward J., Cerrone, Anthony L.
• Primary Examiner(s): HOBBS, MICHAEL L
• Application Number:
  US11/333,483
• Publication Number:
  US 20060128009A1
• Time in Patent Office: 2,828 Days
• Field of Search: 378/42, 378/45, 435/283.1, 435/285.2, 435/286.1, 435/446, 118/620
• US Class Current: 435/287.2
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0663 Whole sensors
• CPC Class Codes: B01L 2300/1811 using electromagnetic induction heating
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• 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: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6463 Optics
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 2201/0245 with insertable-removable part
• CPC Class Codes: G01N 2201/06113 : Coherent sources; lasers
• CPC Class Codes: G01N 2201/0628 : Organic LED [OLED]
• CPC Class Codes: G01N 2201/0635 : Structured illumination, e.g. with grating
• CPC Class Codes: G01N 2201/0636 : Reflectors
• CPC Class Codes: G01N 2201/068 : Optics, miscellaneous

Abstract

An optical instrument monitors PCR replication of DNA in a reaction apparatus having a temperature cycled block with vials of reaction ingredients including dye that fluoresces in presence of double-stranded DNA. A beam splitter passes an excitation beam to the vials to fluoresce the dye. An emission beam from the dye is passed by the beam splitter to a CCD detector from which a processor computes DNA concentration. A reference strip with a plurality of reference emitters emit reference beams of different intensity, from which the processor selects an optimum emitter for compensating for drift. Exposure time is automatically adjusted for keeping within optimum dynamic ranges of the CCD and processor. A module of the beam splitter and associated optical filters is associated with selected dye, and is replaceable for different dyes.

Claims

1. An instrument comprising:

a plurality of spaced-apart reaction ingredient containers;

a light source adapted to direct an excitation beam toward the plurality of containers;

a focusing optic disposed such that the excitation beam passes through the focusing optic to simultaneously illuminate two or more containers of the plurality of spaced-apart reaction ingredient containers;

a detector disposed to receive emission beams from the two or more containers that also pass through the focusing optic.

2. The instrument of claim 1, wherein the detector generates primary data signals representative of the emission beams.

3. The instrument of claim 2, further comprising a processor to receive the primary data signals and compute corresponding concentrations in the respective containers.

4. The instrument of claim 2, further comprising a detector lens disposed along an emission beam path, between the focusing optic and the detector.

5. The instrument of claim 1, further comprising a plurality of focusing lenses disposed along an excitation beam path between the focusing optic and the plurality of containers.

6. The instrument of claim 1, wherein excitation beam comprises a plurality of illumination beams configured to be simultaneously directed toward the plurality of containers.

7. The instrument of claim 1, wherein the plurality of containers comprises 96 containers.

20. The instrument of claim 1, further comprising a beam splitter disposed along the excitation beam path between the light source and the focusing optic.

21. The instrument of claim 1, wherein the focusing optic is a Fresnel lens.

24. The instrument of claim 1, further comprising a first reflective element and a second reflective element disposed along the excitation beam path, the excitation beam path sequentially containing the plurality of spaced-apart reaction ingredient containers the first reflective element, the second reflective element, and the detector.

25. The instrument of claim 24, wherein the second reflective element is a beam splitter.

30. The instrument of claim 1, further comprising a second lens disposed along the emission beam path between the focusing lens and the detector.

8. An instrument comprising:

a plurality of spaced-apart reaction ingredient containers;

a light source adapted to provide an excitation beam;

a lens disposed along an excitation beam path between the light source and the plurality of containers;

a detector disposed to receive emission beams from at least two containers of the plurality of containers when the emission beams pass through the lens;

wherein the lens is positioned relative to the detector such that the excitation beam passes through the lens and simultaneously illuminates the at least two containers.

9. The instrument of claim 8, wherein the detector generates primary data signals representative of the emission beams.

10. The instrument of claim 9, further comprising a processor to receive the primary data signals and compute corresponding concentrations in the respective containers.

11. The instrument of claim 9, further comprising a detector lens disposed along an emission beam path, between the lens and the detector.

12. The instrument of claim 8, further comprising a plurality of focusing lenses disposed along an excitation beam path between the lens and the plurality of containers.

13. The instrument of claim 8, wherein the instrument is adapted to simultaneously direct a plurality of excitation beams respectively toward the plurality of containers.

22. The instrument of claim 8, wherein the focusing optic is a Fresnel lens.

26. The instrument of claim 8, further comprising a first reflective element and a second reflective element disposed along an optical path, the optical path sequentially containing the plurality of spaced-apart reaction ingredient containers the first reflective element, the second reflective element, and the detector.

27. The instrument of claim 26, wherein the second reflective element is a beam splitter.

14. An instrument comprising:

a plurality of spaced-apart reaction ingredient containers;

a light source providing an excitation beam disposed along an excitation beam path comprising an optical path from the light source to the plurality of spaced-apart reaction ingredient containers;

a thermal cycler for holding at least one of the plurality of spaced-apart reaction ingredient containers;

a detector disposed to receive emission beams emitted from at least one of the plurality of spaced-apart reaction ingredient containers;

an emission beam path disposed comprising an optical path from the detector to the at least one of the plurality of spaced-apart reaction ingredient containers; and

an optic disposed along both the excitation beam path and the emission beam path;

wherein the excitation beam transmits through the optic and simultaneously illuminates at least two of the plurality of spaced-apart reaction ingredient containers;

wherein the detector generates primary data signals representative of the emission beams.

15. The instrument of claim 14, wherein the excitation beam comprises a plurality of illumination beams, the instrument being configured to simultaneously direct a plurality of illumination beams respectively toward the plurality of spaced-apart reaction ingredient containers.

16. The instrument of claim 14, wherein the excitation beam comprises a plurality of illumination beams, the instrument being configured to simultaneously direct a plurality of emission beams toward the detector.

17. The instrument of claim 14, wherein the thermal cycler comprises a block, and the plurality of spaced-apart reaction ingredient containers comprises a plurality of wells arranged in the block.

18. The instrument of claim 14, further comprising a thermal cycler controller programmed to cycle the thermal cycler through a specified temperature program.

19. The instrument of claim 14, further comprising:

caps disposed on the plurality of spaced-apart reaction ingredient containers, and

a second heater in thermal contact with the caps.

23. The instrument of claim 14, wherein the optic is a Fresnel lens.

28. The instrument of claim 14, further comprising a first reflective element and a second reflective element disposed along an optical path, the optical path sequentially containing the plurality of spaced-apart reaction ingredient containers the first reflective element, the second reflective element, and the detector.

29. The instrument of claim 28, wherein the second reflective element is a beam splitter.