Instrument for monitoring polymerase chain reaction of DNA

• US 6,818,437 B1
• Filed: 11/29/2001
• Issued: 11/16/2004
• Est. Priority Date: 05/16/1998
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Young, Eugene F., Gambini, Michael R., Atwood, John G., Cerrone, Anthony L., Lakatos, Edward J.
• Primary Examiner(s): Redding, David A.
• Application Number:
  US09/700,536
• Time in Patent Office: 1,083 Days
• Field of Search: 435/287.2, 435/288.4, 435/288.7, 422/82.08, 378/42, 378/45, 378/48
• US Class Current: 435/288.7
• 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 optical instrument for monitoring polymerase chain reaction replication of DNA in a reaction apparatus that includes a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent primary dye that fluoresces proportionately in presence of DNA, the instrument comprising:

a light source for emitting a source beam having at least a primary excitation frequency that causes the primary dye to fluoresce at an emission frequency;

first means disposed to be receptive of the source beam to effect an excitation bean having the excitation frequency;

primary focusing means disposed to focus the excitation beam into each suspension such that the primary dye emits an emission beam having the emission frequency, the emission beam having an intensity representative of concentration of DNA in each suspension, the focusing means being receptive of and passing the emission beam;

second means disposed to be receptive of the emission beam from the focusing means so as to further pass the emission bean at the emission frequency;

emission focusing means for focusing the emission beam;

a detector disposed to be receptive of the emission beam from the second means and the emission focusing means 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 DNA in each vial; and

processing means receptive of the primary data signals for computing primary signal data and the corresponding concentration of DNA;

wherein the block is configured to hold a plurality of vials, the focusing means comprises a corresponding plurality of vial lenses each being disposed for positioning over a vial such that the emission beam comprises individual beams each associated with a vial, and the detector comprises an array of photoreceptors receptive of the individual beams and capable of generating corresponding data signals such that the processing means is capable of computing a respective concentration of DNA in each vial.

2. The instrument of claim 1 wherein the first means and the second means together comprise a beam splitter that is 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.

3. The instrument of claim 2 wherein the beam splitter is disposed to reflect light having the excitation frequency and pass light having the emission frequency.

4. The instrument of claim 1 wherein the vials have transparent vial caps, the instrument further comprises a platen having holes therein aligned with the vial lenses so as to pass the individual beams and associated portions of the excitation bear therethrough, the platen being disposed for the holes to fit over the caps in contact therewith, and further comprises heating means for heating the platen sufficiently to prevent condensation under the caps without interfering with DNA replication in the vials.

10. The instrument of claim 1 wherein the first means further comprises an excitation filter, the second means further comprises an emission filter, and the first means and the second means together comprise a beam splitter, the excitation filter being disposed between the light source and the beam splitter, the emission filter being disposed between the beam splitter and the detector, the excitation filter passing light having the excitation frequency and substantially blocking light having the emission frequency, and the emission filter passing light having the emission frequency and substantially blocking light having the excitation frequency, the excitation filter and the beam splitter being cooperatively receptive of the source beam to effect the excitation beam, and the emission filter and the beam splitter being cooperatively receptive of the emission beam to pass the emission beam having the emission frequency to the detector.

12. The instrument of claim 1 wherein the light source comprises a halogen lamp and an ellipsoid reflector disposed proximate to the lamp oppositely from the first means, the lamp being disposed at a focal distance from the ellipsoid reflector to effect the source beam with light reflected from the ellipsoid reflector, and the ellipsoid reflector substantially reflecting visible light and transmitting infrared light.

13. 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 means to focus 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 processing means comprises means receptive of the reference signal for computing reference data, and means for normalizing the primary data with the reference data for a chosen point in the reaction replication of DNA, thereby correcting for instrument drift during the monitoring.

14. The instrument of claim 13 wherein the emitter member 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 means 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 processing means comprises means receptive of the reference signals for computing corresponding sets of reference data, means for selecting from the sets the selected reference data that has the highest signal data less than a predetermined maximum, the selected reference data being used for normalizing the primary data.

18. The instrument of claim 1 further comprising a plurality of fluorescent 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 means 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 processing means comprises means receptive of the reference signals for computing corresponding sets of reference data, means for selecting from the sets the selected reference data that has the highest signal data less than a predetermined maximum, and means for normalizing the primary data with corresponding selected reference data for a chosen point in the reaction replication of DNA, thereby correcting for instrument drift during the monitoring.

19. The instrument of claim 1 wherein sets of data signals are generated sequentially in a replication sequence, the processing means or the detector or a combination thereof have a saturation limit for the data signals in each set, the detector is operatively connected to the processing means for the detector to integrate emission beam input over a preselected exposure time for generating each set of data signals, and the processing means comprises adjustment means for automatically effecting adjustments in exposure time to maintain the primary data within a predetermined operating range for maintaining corresponding data signals less than the saturation limit, and means for correcting the primary data in proportion to the adjustments in exposure time.

20. The instrument of claim 19 wherein the detector comprises an array of photoreceptors receptive of the emission beam for generating corresponding data signals in an associated exposure time, the predetermined operating range is for each photoreceptor, and the processing means further comprises means for computing photoreceptor data from the data signals for each photoreceptor, and the adjustment means comprises means for ascertaining highest photoreceptor data, means for determination of whether the highest photoreceptor data are less than, within or higher than the predetermined operating range, and means based on such determination for respectively increasing, retaining or reducing the exposure time so as to effect a subsequent exposure time for maintaining subsequent photoreceptor data within the predetermined operating range.

21. The instrument of claim 1 further comprising a plurality of fluorescent 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 means 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 processing means comprises means receptive of the reference signals for computing corresponding sets of reference data, means for selecting from the sets the selected reference data that has the highest signal data less than a predetermined maximum, and means for normalizing the primary data with corresponding selected reference data for a chosen point in the reaction replication of DNA, thereby correcting for instrument drift during the monitoring.

22. The instrument of claim 19 wherein the ingredients for the at least one vial further include a standard concentration of fluorescent passive dye that fluoresces substantially without influence from DNA, the source beam includes a secondary excitation frequency that causes the passive dye to fluoresce at a secondary emission frequency and thereby emit a secondary emission beam passed by the second means and focused onto the detector to generate corresponding secondary data signals, the processing means further comprises means receptive of the secondary data signals for computing secondary data, and means for dye normalizing the primary data, whereby the computed concentration of DNA is normalized to the standard concentration of passive dye.

23. The instrument of claim 22 wherein the secondary excitation frequency is identical to the primary excitation frequency, the passive dye fluoresces such that the secondary beam is substantially at the emission frequency, the primary data signals are generated during an extension phase of cycling of the thermal cycler block when DNA is recombined and correspondingly primary dye emission is maximized, and the secondary data signals are generated during a denature phase of cycling of the thermal cycler block when DNA is denatured and correspondingly primary dye emission is minimized, whereby data signals for the extension phase are substantially representative of DNA concentration and data signals for the denature phase are substantially representative of the standard concentration of passive dye.

24. The instrument of claim 1 wherein the ingredients for a vial further include a standard concentration of fluorescent passive dye that fluoresces substantially without influence from DNA, the source beam includes a secondary excitation frequency that causes the passive dye to fluoresce at a secondary emission frequency and thereby emit a secondary emission bean passed by the second means and focused onto the detector to generate corresponding secondary data signals, the processing means comprises means receptive of the secondary data signals for computing secondary data, and means for dye normalizing the primary data, whereby the computed concentration of DNA is normalized to the standard concentration of passive dye.

25. The instrument of claim 24 wherein the secondary excitation frequency is identical to the primary excitation frequency, the passive dye fluoresces such that the secondary beam is substantially at the emission frequency, the primary data signals are generated during an extension phase of cycling of the thermal cycler block when DNA is recombined and correspondingly primary dye emission is minimized, and the secondary data signals are generated during a denature phase of cycling of the thermal cycler block when DNA is denatured and correspondingly primary dye emission is minimized, whereby data signals for the extension phase are substantially representative of DNA concentration and data signals for the denature phase are substantially representative of the standard concentration of passive dye.

5. An optical instrument for monitoring polymerase chain reaction replication of DNA in a reaction apparatus that includes a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent primary dye that fluoresces proportionately in presence of DNA, the instrument comprising:

a light source for emitting a source bean having at least a primary excitation frequency that causes the primary dye to fluoresce at an emission frequency;

first means disposed to be receptive of the source beam to effect an excitation beam having the excitation frequency;

primary focusing means disposed to focus the excitation beam into each suspension such that the primary dye emits an emission beam having the emission frequency, the emission beam having an intensity representative of concentration of DNA in each suspension, the focusing means being receptive of and passing the emission beam;

second means disposed to be receptive of the emission beam from the focusing means so as to further pass the emission beam at the emission frequency;

emission focusing means for focusing the emission beam;

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

processing means receptive of the primary data signals for computing primary signal data and the corresponding concentration of DNA;

wherein the block is configured to hold a plurality of vials, the focusing means comprises a corresponding plurality of vial lenses each being disposed for positioning over a vial such that the emission beam comprises individual beams each associated with a vial, and a field lens disposed cooperatively with the vial lenses to effect focusing of the excitation bean into each suspension, and to pass the individual beams to the second means, and the detector comprises an array of photoreceptors receptive of the individual beams to generate corresponding data signals such that the processing means computes concentration of DNA in each vial.

6. The instrument of claim 5 wherein the field lens is an aspherically corrected Fresnel lens.

7. The instrument of claim 5 wherein the emission focusing means comprises a detector lens disposed between the second means and the detector, the detector lens being cooperative with the vial lenses and the field lens to focus the individual beams on the detector.

8. The instrument of claim 7 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 means to focus 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 processing means comprises means receptive of the reference signal for computing reference data, and means for normalizing the primary data with the reference data for a chosen point in the reaction replication of DNA, thereby correcting for instrument drift during the monitoring.

9. The instrument of claim 8 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 means 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 processing means comprises means receptive of the reference signals for computing corresponding sets of reference data, means for selecting from the sets the selected reference data that has the highest signal data less than a predetermined maximum, the selected reference data being used for normalizing the primary data.

11. An optical instrument for monitoring polymerase chain reaction replication of DNA in a reaction apparatus that includes a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent primary that fluoresces proportionately in the presence of DNA, the instrument comprising:

a light source for emitting a source beam having at least a primary excitation frequency that causes the primary dye to fluoresce at an emission frequency;

first means disposed to be receptive of the source beam to effect an excitation beam having the excitation frequency;

primary focusing means disposed to focus the excitation beam into each suspension such that the primary dye emits an emission beam having the emission frequency, the emission beam having an intensity representative of concentration of DNA in each suspension the focusing means being receptive of and passing the emission beam;

second means disposed to be receptive of the emission beam from the focusing means so as to further pass the emission beam at the emission frequency;

emission focusing means for focusing the emission beam;

a detector disposed to be receptive of the emission beam from the second means and the emission focusing means 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 DNA in each vial;

processing means receptive of the primary data signals for computing primary signal data and the corresponding concentration of DNA; and

a housing containing the light source, the detector, the focusing means and a filter module, wherein the beam splitter, the excitation filter and the emission filter are affixed in the module and are associated with a selected primary dye for the suspension, and the module is removable from the housing for replacement with another module associated with another selected primary dye;

wherein the first means further comprises an excitation filter, the second means further comprises an emission filter, and the first means and the second means together comprise a beam splitter, the excitation filter being disposed between the light source and the bean splitter, the emission filter being disposed between the beam splitter and the detector, the excitation filter passing light having the excitation frequency and substantially blocking light having the emission frequency, and the emission filter passing light having the emission frequency and substantially blocking light having the excitation frequency, the excitation filter and the beam splitter being cooperatively receptive of the source beam to effect the excitation beam, and the emission filter and the beam splitter being cooperatively receptive of the emission beam to pass the emission beam having the emission frequency to the detector.

15. An optical instrument for monitoring polymerase chain reaction replication of DNA in a reaction apparatus that includes a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent primary dye that fluoresces proportionately in presence of DNA, the instrument comprising:

a light source for emitting a source beam having at least a primary excitation frequency that causes the primary dye to fluoresce at an emission frequency;

first means disposed to be receptive of the source beam to effect an excitation beam having the excitation frequency;

primary focusing means disposed to focus the excitation beam into each suspension such that the primary dye emits an emission beam having the emission frequency, the emission beam having an intensity representative of concentration of DNA in each suspension, the focusing means being receptive of and passing the emission beam;

second means disposed to be receptive of the emission beam from the focusing means so as to further pass the emission beam at the emission frequency;

emission focusing means for focusing the emission beam;

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

processing means receptive of the primary data signals for computing primary signal data and the corresponding concentration of DNA; and

a fluorescent reference emitter that emits reference light in response to the excitation beam, the reference emitter being disposed for the emission focusing means to focus 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 processing means comprises means respective of the reference signal for computing reference data, and means for normalizing the primary data with the reference data for a chosen in the reaction replication of DNA, thereby correcting for instrument drift during the monitoring;

wherein the reference emitter comprises a plurality of reference emitters each emitting a reference bean of different intensity in response to the excitation beam, the reference emitters being disposed for the emission focusing means to focus each reference beam onto the detector, the reference member further comprises a plastic fluorescent strip and a neutral density filter mounted on the fluorescent strip such that the reference beam and a portion of the excitation beam are attenuated by the neutral density filter, the neutral density filter having a series of densities to effect the plurality of reference emitters each emitting a reference beam, the detector being further receptive of each reference beam to generate a set of reference signals for each reference emitter and the processing means comprises means receptive of the reference signals for computing corresponding sets of reference data, means for selecting from the sets the selected reference data that has the highest signal data less than a predetermined maximum, the selected reference data being used for normalizing the primary data.

16. The instrument of claim 15 further comprising temperature means for maintaining the reference member at substantially constant temperature.

17. The instrument of claim 16 wherein the temperature means comprises a heating strip mounted under the fluorescent strip, and means for controllably heating the heating strip.

26. A system for replication of DNA and monitoring thereof, comprising a reaction apparatus for polymerase chain reaction replication of DNA, and an optical instrument for monitoring presence of DNA during such replication, wherein the apparatus comprises a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent dye that fluoresces proportionately in presence of DNA, and further comprises means for thermal cycling the block and thereby the suspension so as to effect the polymerase chain reaction;

wherein the instrument comprises;

a light source for emitting a source beam having at least an excitation frequency that causes the dye to fluoresce at an emission frequency;

first means disposed to be receptive of the source beam to effect an excitation beam having the excitation frequency and to pass the excitation beam to a focusing means, the focusing means being for focusing the exitation beam into each suspension such that the dye emits an emission beam having the emission frequency, and for passing the emission beam to a second means, the second means being for further passing the emission beam to a detector, the detector being disposed to be receptive of the emission beam from the second means so as to generate data signals representative of the emission beam and thereby concentration of DNA; and

processing means receptive of the data signals for computing and displaying the concentration of DNA;

wherein the block is configured to hold a plurality of vials the focusing means comprises a corresponding plurality of vial lenses each being disposed for positioning over a vial such that the emission beam comprises individual beams each associated with a vial, and the detector comprises an array of photoreceptors receptive of the individual beams and capable of generating corresponding data signals such that the processing means is capable of computing a respective concentration of DNA in each vial.

27. The system of claim 26 wherein the first means and the second means together comprise a beam splitter receptive of the source beam to effect the excitation beam, and receptive of the emission bean to pass the emission beam to the detector.

28. A filter module for an optical instrument that monitors polymerase chain reaction replication of DNA in a reaction apparatus that includes a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent dye that fluoresces proportionately in presence of DNA, the instrument including a housing, a light source disposed in the housing for emitting a source beam having at least an excitation frequency that causes the dye to fluoresce at an emission frequency, focusing means disposed in the housing for focusing an excitation beam having the excitation frequency into each suspension such that the dye emits an emission beam having the emission frequency, a detector disposed in the housing to be receptive of the emission beam so as to generate data signals representative of the emission beam and thereby concentration of DNA, and processing means receptive of the data signals for computing and displaying the concentration of DNA, wherein the module comprises:

a support frame, the instrument being receptive of the support frame into the instrument;

a beam splitter affixed in the support frame so that, with the module inserted, the beam splitter is receptive of the source beam so as to effect the excitation beam, and receptive of the emission beam to pass the emission beam to the detector;

an excitation filter that passes light having the excitation frequency and substantially blocks light having the emission frequency, the exitation filter being affixed in the support frame so that, with the module inserted, the excitation filter is disposed between the light source and the beam splitter; and

an emission filter that passes light having the emission frequency and substantially blocks light having the excitation frequency, the emission filter being affixed in the support frame so that, with the module inserted, the emission filter is disposed between the detector and the beam splitter;

the beam splitter, the excitation filter and the emission filter, and thereby the module, being associated with a selected dye for the suspension, and the module being removable from the housing for replacement with another module associated with another selected dye.

29. The module of claim 28 wherein the beam splitter reflects light having an excitation frequency, and passes light having the emission frequency.