Instrument for monitoring polymerase chain reaction of DNA

• US 7,183,103 B2
• Filed: 02/20/2003
• Issued: 02/27/2007
• Est. Priority Date: 05/16/1998
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Gambini, Michael R., Young, Eugene F., Cerrone, Anthony L., Stone, legal representative, Atwood, legal representative, Atwood, deceased, Lakatos, Edward J.
• Primary Examiner(s): REDDING, DAVID A
• Application Number:
  US10/370,846
• Publication Number:
  US 20030148505A1
• Time in Patent Office: 1,468 Days
• Field of Search: 435/287.2,288.4,288.7 422/82.08 378/42,45,48 250/458.1,483.1,459.1,462.1,461.2 356/73,141,146
• 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 block with a plurality of spaced-apart sample wells;

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

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

a detector disposed along an emission beam path and arranged to receive emission beams from the Fresnel lens;

wherein the Fresnel lens is disposed along an emission beam path between the plurality of wells and the detector, and the Fresnel lens is adapted to direct emission beams from two or more of the plurality of spaced-apart sample wells to the detector.

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 wells.

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

5. The instrument of claim 1, further comprising an excitation beam filter disposed along an excitation beam path between the light source and the Fresnel lens.

6. The instrument of claim 1, further comprising an emission beam filter disposed along an emission beam path between the detector and the Fresnel lens.

7. The instrument of claim 1, further comprising a fold mirror disposed along an excitation beam path, between the light source and the plurality of sample wells.

8. The instrument of claim 1, further comprising a beam splitter disposed along an excitation beam path, between the light source and the Fresnel lens.

9. The instrument of claim 1, further comprising a plurality of focusing lenses disposed along excitation beam paths between the Fresnel lens and the plurality of sample wells.

10. The instrument of claim 1, wherein the instrument is adapted to simultaneously direct a plurality of excitation beams respectively toward the plurality of sample wells.

11. The instrument of claim 1, further comprising reaction ingredients disposed in each of the plurality of sample wells.

12. The instrument of claim 11, wherein the reaction ingredients include a concentration of a marker dye that emits emission beams when illuminated with an excitation beam.

13. The instrument of claim 11, wherein the reaction ingredients include components for polymerase chain reaction.

14. The instrument of claim 1, wherein the plurality of sample wells comprises 96 sample wells.

34. The instrument of claim 1, wherein the Fresnel lens comprises two or more lenses.

15. 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 Fresnel lens disposed along an excitation beam path, between the light source and the plurality of containers; and

a beam splitter disposed along an excitation beam path, between the light source and the Fresnel lens;

wherein the Fresnel lens is disposed along an emission beam path between the plurality of containers and the beam splitter, and the Fresnel lens is adapted to direct emission beams from two or more of the plurality of spaced-apart reaction ingredient containers to the beam splitter.

16. The instrument of claim 15, further comprising a detector arranged to receive emission beams emitted from the plurality of containers and to generate primary data signals representative of the emission beams.

17. The instrument of claim 16, further comprising a detector lens disposed along an emission beam path, between the Fresnel lens and the detector.

18. The instrument of claim 15, further comprising an excitation beam filter disposed along an excitation beam path between the light source and the beam splitter.

19. The instrument of claim 16, further comprising an emission beam filter disposed along an emission beam path between the detector and the beam splitter.

20. The instrument of claim 15, further comprising a fold mirror disposed along an excitation beam path, between the light source and the Fresnel lens.

35. The instrument of claim 15, wherein the Fresnel lens comprises two or more lenses.

21. An instrument comprising:

a block with a plurality of spaced-apart sample wells;

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

a Fresnel lens disposed along an excitation beam path, between the light source and the plurality of sample wells; and

a beam splitter disposed along an excitation beam path, between the light source and the Fresnel lens;

wherein the Fresnel lens is disposed along an emission beam path between the plurality of sample wells and the beam splitter, and the Fresnel lens is adapted to direct emission beams from two or more of the plurality of spaced sample wells to the beam splitter.

22. The instrument of claim 21, further comprising a detector arranged to receive emission beams emitted from the plurality of wells and to generate primary data signals representative of the emission beams.

23. The instrument of claim 22, further comprising a detector lens disposed along an emission beam path, between the Fresnel lens and the detector.

24. The instrument of claim 21, further comprising an excitation beam filter disposed along an excitation beam path between the light source and the beam splitter.

25. The instrument of claim 22, further comprising an emission beam filter disposed along an emission beam path between the detector and the beam splitter.

26. The instrument of claim 21, further comprising a fold mirror disposed along an excitation beam path, between the light source and the Fresnel lens.

36. The instrument of claim 21, wherein the Fresnel lens comprises two or more lenses.

27. A method of directing an excitation beam toward a plurality of wells in a block, the method comprising:

providing an instrument that includes in series a light source, a beam splitter, a Fresnel lens, and a block with a plurality of wells;

generating an excitation beam with the light source; and

focusing the excitation beam with the Fresnel lens into the plurality of wells, wherein the Fresnel lens is adapted to direct emission beams from two or more of the plurality of wells to the beam splitter.

28. The method of claim 27, wherein the wells contain respective samples.

29. The method of claim 28, wherein the respective samples each include a concentration of a marker dye that emits emission beams in response to being illuminated by the excitation beam, and wherein the method further includes:

transmitting emission beams emitted from the respective samples through the Fresnel lens;

detecting the emission beams transmitted through the Fresnel lens; and

generating primary data signals representative of the detected emission beams.

30. The method of claim 29, further comprising processing the primary data signals to compute corresponding concentrations of marker dye in the respective wells.

37. The method of claim 27, wherein the Fresnel lens comprises two or more lenses.

31. A method of illuminating a plurality of spaced-apart reaction ingredient containers, the method comprising:

providing an instrument that includes in series a light source, a beam splitter, a Fresnel lens, and a plurality of spaced-apart reaction ingredient containers containing respective samples;

generating an excitation beam with the light source; and

focusing the excitation beam with the Fresnel lens into the samples in the respective plurality of spaced-apart reaction ingredient containers, wherein the Fresnel lens is adapted to direct emission beams from two or more of the plurality of spaced-apart reaction ingredient containers to the beam splitter.

32. The method of claim 31, wherein the respective samples each include a concentration of a marker dye that emits emission beams in response to being illuminated by the excitation beam, and wherein the method further includes:

transmitting emission beams emitted from the marker dye in the respective samples, through the Fresnel lens;

detecting the emission beams transmitted through the Fresnel lens; and

generating primary data signals representative of the detected emission beams.

33. The method of claim 32, further comprising processing the primary data signals to compute corresponding concentrations of marker dye in the respective samples.

38. The method of claim 31, wherein the Fresnel lens comprises two or more lenses.

39. An instrument comprising:

a block with a plurality of spaced-apart sample wells;

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

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

a detector disposed along an emission beam path and arranged to receive emission beams emitted from the Fresnel lens;

wherein the Fresnel lens is disposed along an emission beam path between the plurality of wells and the detector, and the Fresnel lens is adapted to direct emission beams from more than one of the plurality of sample wells, simultaneously.

40. 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 Fresnel lens disposed along an excitation beam path, between the light source and the plurality of containers; and

a beam splitter disposed along an excitation beam path, between the light source and the Fresnel lens;

wherein the Fresnel lens is disposed along an emission beam path between the plurality of containers and the beam splitter, and the Fresnel lens is adapted to direct emission beams from more than one of the plurality of containers, simultaneously.

41. An instrument comprising:

a block with a plurality of spaced-apart sample wells;

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

a Fresnel lens disposed along an excitation beam path, between the light source and the plurality of sample wells; and

a beam splitter disposed along an excitation beam path, between the light source and the Fresnel lens;

wherein the Fresnel lens is disposed along an emission beam path between the plurality of sample wells and the beam splitter, and the Fresnel lens is adapted to direct emission beams from more than one of the plurality of sample wells, simultaneously.

42. A method of directing an excitation beam toward a plurality of wells in a block, the method comprising:

providing an instrument that includes in series a light source, a beam splitter, a Fresnel lens, and a block with a plurality of wells;

generating an excitation beam with the light source; and

focusing the excitation beam with the Fresnel lens into the plurality of wells;

wherein the Fresnel lens is adapted to direct emission beams from more than one of the plurality of wells, simultaneously.

43. A method of illuminating a plurality of spaced-apart reaction ingredient containers, the method comprising:

providing an instrument that includes in series a light source, a beam splitter, a Fresnel lens, and a plurality of spaced-apart reaction ingredient containers containing respective samples;

generating an excitation beam with the light source; and

focusing the excitation beam with the Fresnel lens into the samples in the respective plurality of spaced-apart reaction ingredient containers, wherein the Fresnel lens is capable of directing emission beams from more than one of the plurality of reaction ingredient containers, simultaneously.