Instrument for monitoring polymerase chain reaction of DNA

• US 20070154939A1
• Filed: 02/26/2007
• Published: 07/05/2007
• Est. Priority Date: 05/16/1998
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Young, Eugene, Atwood, John, Lakatos, Edward, Stone, Susan, Cerrone, Anthony, Gambini, Michael, Atwood, Judith
• Application Number:
  US11/711,416
• Publication Number:
  US 20070154939A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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. A method of monitoring replication of DNA in a reaction apparatus, comprising:

generating an excitation beam with a light source;

directing the excitation beam through a beam splitter and simultaneously into a plurality of vials;

directing emission beams from two or more of the plurality of vials through the beam splitter and onto a detector;

generating primary data signals representative of the emission beams, each primary data signal corresponding to a respective concentration of DNA in a respective one of the plurality of vials;

emitting reference light from a reference emitter in response to the excitation beam;

focusing at least a portion of the reference light as a reference beam onto the detector;

generating a reference signal based on the reference beam received by the detector;

processing the reference signal to compute reference data; and

normalizing the primary data signals with the reference data for a chosen point in the replication of DNA, to correct for instrument drift during the monitoring.

2. The method of claim 1, wherein the reference emitter comprises a plurality of fluorescent reference emitters each emitting a reference beam of different intensity in response to the excitation beam.

3. The method of claim 2, further comprising focusing the reference beams of different intensities onto the detector.

4. The method of claim 2, wherein the reference data is computed from a ratio of a first reference data signal corresponding to the reference emitter having the highest signal intensity that is less than a predetermined maximum, and from a second reference data signal corresponding to the reference emitter having the next highest signal intensity.

5. The method of claim 1, wherein the generated reference signal is converted to the reference data by an analog/digital device.

6. The method of claim 1, further comprising:

generating a source beam comprising a secondary excitation frequency; and

directing the source beam at a standard concentration of a passive dye that emits light substantially without influence from DNA to cause the passive dye to emit light at a secondary emission frequency and to emit a secondary emission beam focused onto the detector.

7. The method of claim 6, further comprising generating secondary data signals based on the secondary emission beam.

8. The method of claim 7, further comprising:

processing the secondary data signals to compute secondary data; and

normalizing the primary data, whereby the computed concentration of DNA is normalized to the standard concentration of the passive dye.

9. The method of claim 6, wherein the standard concentration of passive dye is included in at least one vial of the plurality of vials.

10. The method of claim 6, wherein the standard concentration of passive dye is included in each vial of the plurality of vials.

11. The method of claim 6, wherein the standard concentration of passive dye comprises a nucleic acid sequence labeled with Rhodamine and fluorescein dye derivatives.

12. The method of claim 6, wherein the standard concentration of passive dye comprises Rox dye.

13. The method of claim 1, wherein the processing comprises processing with a computer processor.

14. The method of claim 1, wherein the plurality of vials comprises a plurality of wells.

15. The method of claim 1, wherein the plurality of vials comprises 96 wells in a plastic unitary tray.

16. The method of claim 1, further comprising replicating DNA in cycles, wherein the normalizing comprises normalizing the primary data in real time as a ratio to the reference data from an initial cycle of the DNA replication.

17. The method of claim 1, further comprising replicating DNA in cycles, wherein the normalizing comprises normalizing the primary data in real time as a ratio to the reference data from a previous cycle of the DNA replication.

18. The method of claim 1, wherein the replication of DNA comprises a polymerase chain reaction.

19. A method of monitoring replication of DNA in a reaction apparatus, comprising:

generating an excitation beam with a light source;

directing the excitation beam into a plurality of vials;

directing emission beams from two or more of the plurality of vials onto a detector;

generating primary data signals representative of the emission beams, each primary data signal corresponding to a respective concentration of DNA in a respective one of the plurality of vials;

generating dark signal data; and

correcting the primary data signals with the dark signal data.

20. The method of claim 19, wherein the correcting comprises subtracting the dark signal data from the primary data signals to yield dark-corrected data.

21. The method of claim 19, wherein the detector comprises pixels, and the generating primary data signals comprises collecting multiple normal exposures for each pixel.

22. The method of claim 19, further comprising converting both the primary data signals and the dark signal data from analog signals to digital signals using an analog/digital device.

23. The method of claim 19, further comprising:

generating a source beam comprising a secondary excitation frequency; and

directing the source beam at a standard concentration of a passive dye that emits light substantially without influence from DNA, to cause the passive dye to emit light at a secondary emission frequency and to emit a secondary emission beam focused onto the detector.

24. The method of claim 23, further comprising generating secondary data signals based on the secondary emission beam.

25. The method of claim 24, further comprising:

processing the secondary data signals to compute secondary data; and

normalizing the primary data, whereby the computed concentration of DNA is normalized to the standard concentration of the passive dye.

26. The method of claim 19, further comprising replicating DNA in cycles, wherein the normalizing comprises normalizing the primary data in real time from an initial cycle of the DNA replication.