System for real time detection of nucleic acid amplification products

• US 5,928,907 A
• Filed: 12/02/1996
• Issued: 07/27/1999
• Est. Priority Date: 04/29/1994
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: The Perkin-Elmer Corporation Applied Biosystems Division
• Inventors: Woudenberg, Timothy M., McBride, Lincoln J., Lee, Linda G., Tracy, David H., Young, Eugene F., Shigeura, John, Bodner, Kevin S., Connell, Charles R., Ganz, Alan M., Saviano, Paul G.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/752,973
• Time in Patent Office: 967 Days
• Field of Search: 422/82.05, 422/82.06, 422/82.07, 422/82.08, 422/82.11, 435/6, 435/91.1, 435/91.2
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: G01N 2021/6484 Optical fibres
• 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 21/6428 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

Abstract

A system is provided for carrying out real time fluorescence-based measurements of nucleic acid amplification products. In a preferred embodiment of the invention, an excitation beam is focused into a reaction mixture through a surface, the reaction mixture containing (i) a first fluorescent indicator capable of generating a first fluorescent signal whose intensity is proportional to the amount of an amplification product in the volume of the reaction mixture illuminated by the excitation beam and (ii) a second fluorescent indicator homogeneously distributed throughout the reaction mixture capable of generating a second fluorescent signal proportional to the volume of reaction mixture illuminated by the excitation beam. Preferably, the excitation beam is focused into the reaction mixture by a lens through a portion of a wall of a closed reaction chamber containing the reaction mixture. The same lens is used to collect the first and second fluorescent signals generated by the first and second fluorescent indicators, respectively, in response to the excitation beam. The ratio of the fluorescent intensities of the first and second fluorescent signals provides a stable quantitative indicator of the amount of amplification product synthesized in the course of the amplification reaction.

Claims

1. An apparatus for monitoring the formation of a nucleic acid amplification reaction product in real time, the apparatus comprising:

a sample holder for holding a sample of nucleic acids to be amplified;

a fiber optic cable for illuminating a volume of the sample with an excitation beam;

a lens co-axially disposed with the fiber optic cable for focusing the excitation beam into the volume of the sample and for collecting from the sample and transmitting to the fiber optic cable a first fluorescent signal whose intensity is proportional to the concentration of the amplification reaction product and a second fluorescent signal whose intensity is proportional to the volume of the sample illuminated by the excitation beam; and

a detection and analysis mechanism for receiving the first and second fluorescent signals from the fiber optic cable at a plurality of times during a nucleic acid amplification, the detection and analysis mechanism measuring the intensities of the first and second fluorescent signals at the plurality of times and producing a plurality of corrected intensity signals, each corrected intensity signal corresponding to a relationship between the intensities of the first and second fluorescent signals at a given time.

2. The apparatus according to claim 1 wherein the detection and analysis mechanism provides a readout corresponding to the plurality of corrected intensity signals as a function of time.

3. The apparatus according to claim 1 wherein the apparatus includes

a plurality of sample holders for holding a plurality of samples,

a plurality of fiber optic cables for illuminating volumes of the plurality of samples,

a plurality of lenses, each co-axially disposed with a first end of a fiber optic cable for focusing an excitation beam into a sample, and

a fiber optic multiplexer which couples the detection and analysis mechanism to a second end of each of the plurality of fiber optic cables.

4. The apparatus according to claim 1 wherein the sample holder includes a removable reaction chamber for holding the sample.

5. The apparatus according to claim 4 wherein the removable reaction chamber is sealable.

6. The apparatus according to claim 1 wherein the sample holder includes a sealable reaction chamber for holding the sample.

7. The apparatus according to claim 1 wherein the sample holder includes an optical interface through which the excitation beam is transmitted from the lens into the sample.

8. The apparatus according to claim 7 wherein the sample holder includes a sealable reaction chamber for holding the sample, the optical interface forming a wall of the reaction chamber.

9. The apparatus according to claim 7 wherein the apparatus further includes a mechanism for heating the optical interface to prevent condensation of the sample on the optical interface.

10. The apparatus according to claim 9 wherein the sample holder includes a sealable reaction chamber for holding the sample, the optical interface forming a wall of the reaction chamber.

11. The apparatus according to claim 7 wherein the sample holder includes a removable reaction chamber for holding the sample, the optical interface forming a wall of the reaction chamber which covers at least a portion of the sample and which is separated from the sample by an air gap.

25. The apparatus according to claim 1 wherein

the nucleic acid amplification includes a plurality of amplification cycles;

and

the detection and analysis mechanism receives the first and second fluorescent signals from the fiber optic cable at least once per amplification cycle and measures the intensities of the first and second fluorescent signals at least once per amplification cycle.

26. The apparatus according to claim 25 wherein the detection and analysis mechanism produces at least one corrected intensity signal per amplification cycle.

12. A method for monitoring the formation of a nucleic acid amplification reaction product in real time comprising:

taking a sample holder containing a nucleic acid sequence to be amplified to form a nucleic acid amplification reaction product, a first fluorescent indicator which produces a first fluorescent signal when illuminated by an excitation beam whose intensity is proportional to a concentration of the amplification reaction product in the sample, and a second fluorescent indicator which produces a second fluorescent signal when illuminated by the excitation beam whose intensity is proportional to a volume of sample illuminated by the excitation beam;

performing an amplification of the nucleic acid sequence in the sample holder;

transmitting an excitation beam into the sample holder at a plurality of times during the amplification and measuring the intensities of the first and second fluorescent signals at the plurality of times; and

monitoring the formation of the nucleic acid amplification reaction product in real time by calculating a plurality of corrected intensity signals, each corrected intensity signal corresponding to a relationship between the intensity of the first and second fluorescent signals measured at the plurality of times, a change in the corrected intensity signal over time indicating the formation of the nucleic acid amplification reaction product.

13. The method according to claim 12 wherein the first fluorescent indicator is a complex-forming dye.

14. The method according to claim 12, further including the step of sealing the sample within the sample holder prior to transmitting an excitation beam into the sample holder.

15. The method according to claim 12 wherein the sample holder includes an optical interface through which the excitation beam is transmitted to the sample, the sample holder also including an air gap separating the optical interface from the sample, the method further including the step of heating the optical interface to prevent condensation of the sample on the optical interface.

16. The method according to claim 15, further including the step of sealing the sample within the sample holder prior to transmitting an excitation beam into the sample.

17. The method according to claim 12 wherein the step of taking a sample holder includes

adding a sample to a reaction chamber which is removable from the sample holder;

and

adding the removable reaction chamber to the sample holder.

18. The method according to claim 17, further including the step of sealing the sample within the removable reaction chamber.

19. The method according to claim 17 wherein the removable reaction chamber includes an optical interface through which the excitation beam is transmitted from the lens to the sample and an air gap separating the optical interface from the sample, the method further including the step of heating the optical interface to prevent condensation of the sample on the optical interface.

20. The method according to claim 12 wherein performing the amplification includes performing at least one cycle of a polymerase chain reaction.

21. The method according to claim 12 wherein performing the amplification includes performing at least one cycle of a ligase chain reaction.

27. The method according to claim 12 wherein

performing the amplification includes performing a plurality of cycles of an amplification reaction;

and

transmitting an excitation beam into the sample holder at a plurality of times during the amplification includes transmitting the excitation beam at least once per amplification cycle.

28. The method according to claim 27 wherein

monitoring the formation of the nucleic acid amplification reaction product includes calculating at least one corrected intensity signal per amplification cycle.

22. A method for monitoring the formation of multiple nucleic acid amplification reaction products in real time comprising:

taking multiple sample holders, each sample holder containing a nucleic acid sequence to be amplified to form a nucleic acid amplification reaction product, a first fluorescent indicator which produces a first fluorescent signal when illuminated by an excitation beam whose intensity is proportional to a concentration of the amplification reaction product in the sample, and a second fluorescent indicator which produces a second fluorescent signal when illuminated by the excitation beam whose intensity is proportional to a volume of sample illuminated by the excitation beam;

performing an amplification of the nucleic acid sequences in the multiple sample holders;

transmitting an excitation beam into the multiple sample holders at a plurality of times during the amplification and measuring the intensities of the first and second fluorescent signals at the plurality of times; and

monitoring the formation of nucleic acid amplification reaction products in the multiple sample holders in real time by calculating a plurality of corrected intensity signals, each corrected intensity signal corresponding to a relationship between the intensity of the first and second fluorescent signals measured at the plurality of times, a change in the corrected intensity signal over time indicating the formation of the nucleic acid amplification reaction product.

23. A method for monitoring the formation of multiple nucleic acid amplification reaction products in real time comprising:

taking a sample holder containing a plurality of nucleic acid sequences to be amplified to form a plurality of nucleic acid amplification reaction products, a plurality of first fluorescent indicators which produce a first fluorescent signal when illuminated by an excitation beam whose intensity is proportional to a concentration of the amplification reaction product in the sample, and a second fluorescent indicator which produces a second fluorescent signal when illuminated by the excitation beam whose intensity is proportional to a volume of sample illuminated by the excitation beam;

performing an amplification of the nucleic acid sequence in the sample holder;

transmitting an excitation beam into the sample holder at a plurality of times during the amplification and measuring the intensities of the first and second fluorescent signals at the plurality of times; and

monitoring the formation of the plurality of nucleic acid amplification reaction products in the sample holder in real time by calculating a plurality of corrected intensity signals for each of the plurality of nucleic acid sequences in the sample holder at the plurality of times, each corrected intensity signal corresponding to a relationship between the intensity of the first and second fluorescent signals measured at the plurality of times, a change in the corrected intensity signal over time indicating the formation of the nucleic acid amplification reaction product.

24. A method for monitoring the formation of a nucleic acid amplification reaction product in real time comprising:

taking a sample holder containing a nucleic acid sequence to be amplified to form a nucleic acid amplification reaction product, and first and second fluorescent indicators covalently attached to an oligonucleotide capable of hybridizing to the amplification reaction product, the first fluorescent indicator producing a first fluorescent signal when illuminated by the excitation beam whose intensity is proportional to the concentration of amplification reaction product in the sample, the second fluorescent indicator producing a second fluorescent signal when illuminated by the excitation beam whose intensity is proportional to the volume of the sample illuminated by the excitation beam, the second fluorescent indicator also quenching the fluorescence of the first fluorescent indicator;

performing an amplification of the nucleic acid sequence in the sample holder;

transmitting an excitation beam into the sample holder at a plurality of times during the amplification and measuring the intensities of the first and second fluorescent signals at the plurality of times; and

monitoring the formation of the nucleic acid amplification reaction product in real time by calculating a plurality of corrected intensity signals, each corrected intensity signal corresponding to a relationship between the intensity of the first and second fluorescent signals at the plurality of times, a change in the corrected intensity signal over time indicating the formation of the nucleic acid amplification reaction product.