Instrument for monitoring polymerase chain reaction of DNA

• US 20070238161A1
• Filed: 05/17/2007
• Published: 10/11/2007
• Est. Priority Date: 05/16/1998
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Young, Eugene, Lakatos, Edward, Stone, Susan, Cerrone, Anthony, Gambini, Michael, Atwood, Judith
• Granted Patent: US 9,671,342 B2
• Time in Patent Office: Days
• US Class Current: 435/286.100
• 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 21/0303 Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment G01N21/031 - G01N21/15 take precedence
• 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 optical instrument for monitoring a reaction in a reaction apparatus that includes a thermal cycler for holding at least one vial containing ingredients for the reaction, the ingredients including a fluorescent dye that fluoresces proportionately in the presence of a target DNA, the instrument comprising:

a light source for emitting a source beam having at least a primary excitation frequency;

an excitation beam focusing lens disposed to focus the source beam into the ingredients such that the fluorescent dye emits an emission beam having an emission frequency, the emission beam having an intensity representative of a concentration of the target DNA in the ingredients, the excitation beam focusing lens being receptive of and passing the emission beam;

an emission focusing lens for focusing the emission beam;

a detector disposed to be receptive of the emission beam from the emission focusing lens such that the emission beam is focused onto the detector, the detector generating primary data signals representative of the emission beam and thereby a corresponding concentration of target DNA in each vial;

a processor receptive of the primary data signals for computing primary signal data and the corresponding concentration of target DNA; and

a housing containing the light source, the detector, the excitation beam focusing lens, and a filter module, wherein an excitation beam filter and an emission beam filter are affixed in the filter module and are associated with the fluorescent dye, the excitation beam filter is disposed between the light source and the excitation beam focusing lens, and the emission beam filter is disposed between the emission beam focusing lens and the detector.

2. The instrument of claim 1, wherein the excitation beam filter passes light having the excitation frequency and substantially blocks light having the emission frequency, and the emission beam filter passes light having the emission frequency and substantially blocks light having the excitation frequency.

3. The instrument of claim 1, wherein the light source comprises a halogen lamp.

4. The instrument of claim 1, wherein the light source comprises an ellipsoid reflector.

5. The instrument of claim 1, wherein the light source comprises a projection lamp.

6. The instrument of claim 1, wherein the light source comprises a laser.

7. The instrument of claim 1 further comprising a fluorescent reference emitter that emits reference light in response to the excitation beam, the reference emitter being disposed for the emission focusing lens to focus the at least a portion of the reference light as a reference beam onto the detector, the detector being further receptive of the reference beam to generate a reference signal, and the processor being receptive of the reference signal for computing reference data and normalizing the primary data with the reference data for a chosen point in the reaction replication of DNA, thereby correcting for instrument drift during monitoring.

8. The instrument of claim 7, wherein the reference emitter comprises a plurality of reference emitters each emitting a reference beam of different intensity in response to the excitation beam, the reference emitters being disposed for the emission focusing lens to focus each reference beam onto the detector, the detector being further receptive of each reference beam to generate a set of reference signals for each reference emitter, and the processor being receptive of the reference signals for computing corresponding sets of reference data, selecting from the sets the selected reference data that has the highest signal data less than a predetermined maximum, and using the selected reference data for normalizing the primary data.

9. The instrument of claim 1, further comprising a beam splitter disposed to be receptive of the source beam to effect the excitation beam, and receptive of the emission beam to pass the emission beam at the emission frequency to the detector.

10. The instrument of claim 9, wherein the beam splitter is contained in the filter module.

11. The instrument of claim 9, wherein the beam splitter is disposed to reflect light having the excitation frequency and pass light having the emission frequency.

12. The instrument of claim 1, wherein the detector comprises a charge coupled device.

13. The instrument of claim 1, wherein the detector comprises a digital imaging device.

14. The instrument of claim 1, wherein the filter module is removable from the housing for replacement with another filter module associated with a different fluorescent dye.

15. The instrument of claim 14, further comprising:

a second filter module that is removed from the housing, wherein a second excitation beam filter and a second emission beam filter are affixed in the second filter module and are associated with a second fluorescent dye that differs from the first-mentioned fluorescent dye.

16. The instrument of claim 15, wherein the second fluorescent dye has a second excitation frequency and a second emission frequency, the second excitation beam filter passes light having the second excitation frequency and substantially blocks light having the second emission frequency, and the second emission beam filter passes light having the second emission frequency and substantially blocks light having the second excitation frequency.

17. The instrument of claim 15, wherein the second filter module further comprises a second beam splitter contained therein.