In numerous areas of industry, technology, and research there is a need to reliably and reproducibly subject relatively small reactions to thermal cycling. The need to subject a sample to repeated temperature cycles is particularly acute in biotechnology applications. In the biotechnology field, it is often desirable to repeatedly heat and cool small samples of materials over a short period of time. One such biological process that is regularly carried out is cyclic DNA amplification.
Cyclic DNA amplification, using a thermostable DNA polymerase, allows automated amplification of primer specific DNA, widely known as PCR, or the polymerase chain reaction. It is well accepted that automation of this process requires controlled and precise thermal cycling of reaction mixtures.
PCR is a technique involving multiple cycles that results in the geometric amplification of certain polynucleotide sequence each time a cycle is completed. The technique of PCR is well known to the person of average skill in the art of molecular biology and has been described in many books, including, PCR: A Practical Approach, M. J. McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications, by Innis, et al., Academic Press (1990), and PCR Technology: Principals and Applications for DNA Amplification, H. A. Erlich, Stockton Press (1989). PCR has also been described in many U.S. patents, including U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; and 5,066,584, which are hereby incorporated by reference.
The PCR technique typically involves first denaturing a polynucleotide, followed by annealing at least a pair of primer oligonucleotides to the denatured polynucleotide, i.e., hybridizing the primer to the denatured polynucleotide template. After the annealing step, an amplicon extension step is completed using an enzyme with polymerase activity that catalyzes synthesis of a new polynucleotide strand that incorporates the primer oligonucleotide and uses the original denatured polynucleotide as a synthesis template. This series of steps (denaturation, primer annealing, and primer extension) constitutes a PCR cycle. As cycles are repeated, the amount of newly synthesized polynucleotide increases geometrically because the newly synthesized polynucleotides from an earlier cycle can serve as templates for synthesis in subsequent cycles. Primer oligonucleotides are typically selected in pairs that can anneal to opposite strands of a given double-stranded polynucleotide sequence so that the region between the two annealing sites is amplified.
One cycle of standard PCR is usually performed in 2 to 8 min requiring 1 to 4 hours for a 30-cycle amplification. The sample temperature response in most PCR instrumentation is very slow compared to the optimal durations required for denaturation, annealing, and extension. The physical (denaturation and annealing) and enzymatic (extension) reactions in PCR occur very quickly. Amplification times for PCR can be reduced from hours to less than 10 minutes. This can be accomplished by: (1) reducing the ramping time for the thermal cycler to change temperatures between the annealing, extension and denaturation steps; (2) reducing the run times for each of the 3 steps; and (3) reducing the temperature cycling profile from three different temperatures to two temperatures. This is accomplished by completing a two-step PCR cycling profile in which the annealing and extension steps are completed at the same temperatures.
Commercial programmable metal heat blocks have been used in the past to affect the temperature cycling of samples in microfuge tubes through the desired temperature versus time profile. Peltier heating and cooling are usually utilized in changing temperatures at a rate of approximately 0.5-4 degrees Celsius per second. However, the inability to quickly and accurately adjust the temperature of the heat blocks through a large temperature range over a short time period, has rendered the use of heat block type devices undesirable as a heat control system when carrying out the polymerase chain reaction in a rapid fashion.
Furthermore, devices using water baths with fluidic switching, (or mechanical transfer) have also been used as thermal cyclers for PCR. Although water baths have been used in cycling a PCR mixture through a desired temperature versus time profile, the high thermal mass and relatively low boiling point of water are significantly limiting factors in terms of rapid temperature time gradients. The 3-dimensional mechanical requirements of a water bath apparatus are also an impediment in terms of performance, accuracy and cost.
Devices using water baths are limited in their performance. This is because the water's thermal mass significantly restricts the maximum temperature versus time gradient which can be achieved thereby. Also, the water bath apparatus has been found to be very cumbersome due to the size and number of water carrying hoses and external temperature controlling devices for the water. Further, the need for excessive periodic maintenance and inspection of the water fittings for the purpose of detecting leaks in a water bath apparatus is tedious and time consuming. Finally, it is difficult with the water bath apparatus to control the temperature in the sample tubes with the desired accuracy.
A wide variety of instrumentation has been developed for carrying out nucleic acid amplifications, particularly PCR, e.g. Johnson et al, U.S. Pat. No. 5,038,852 (computer-controlled thermal cycler); Wittwer et al, Nucleic Acids Research, 17: 4353-4357 (1989)(capillary tube PCR); Hallsby, U.S. Pat. No. 5,187,084 (air-based temperature control); Garner et al, Biotechniques, 14: 112-115 (1993)(high-throughput PCR in 864-well plates); Wilding et al, International application No. PCT/US93/04039 (PCR in micro-machined structures); Schnipelsky et al, European patent application No. 0 381 501 A2 (disposable, single use PCR device), and the like. Important design goals fundamental to PCR instrument development have included fine temperature control, minimization of sample-to-sample variability in multi-sample thermal cycling, automation of pre- and post-PCR processing steps, high speed cycling, minimization of sample volumes, real-time measurement of amplification products, minimization of cross-contamination, or sample carryover, and the like.
Another prior art system is represented by a temperature cycler in which multiple temperature controlled blocks with vertical reaction vessel wells are maintained at different desired temperatures (U.S. Pat. Nos. 5,525,300, 5,779,981 and 6,054,263). A robotic arm is utilized by move reaction mixtures from block to block. The reaction vessels are lifted vertically from out of the heat block, transported to another heating block, and placed vertically down into said heating block. However, this system requires precision movement, pressurized thermal contact and expensive microprocessor controlled robotics. This robotic movement system also impedes a real-time fluorescent detection system of the nucleic acid amplification product during and after temperature cycling has completed.
Rapid cycling has been described before (e.g. U.S. Pat. No. 6,174,670, and U.S. Pat. No. 5,455,175). According to this prior art, rapid cycling techniques are made possible by the rapid temperature response and temperature homogeneity possible for samples in high surface area-to-volume sample containers such as capillary tubes. For further information, see also: C. T. Wittwer, G. B. Reed, and K. M. Ririe, Rapid cycle DNA amplification, in K. B. Mullis, F. Ferre, and R. A. Gibbs, The polymerase chain reaction, Birkhauser, Boston, 174-181, (1994). According to this prior art, improved temperature homogeneity allows the time and temperature requirements of PCR to be better defined and understood, while improved temperature homogeneity also increases the precision of any analytical technique used to monitor PCR during amplification.
The design of instruments that permit PCR to be carried out in closed reaction chambers and monitored in real time is highly desirable. Closed reaction chambers are desirable for preventing cross-contamination, e.g. Higuchi et al, Biotechnology, 10: 413-417 (1992) and 11: 1026-1030 (1993); and Holland et al, Proc. Natl. Acad. Sci., 88: 7276-7280 (1991). Clearly, the successful realization of such a design goal would be especially desirable in the analysis of diagnostic samples, where a high frequency of false positives and false negatives would severely reduce the value of the PCR-based procedure. Real-time monitoring allows the coupling of amplification and detection, thus decreasing contamination risks and labour time. As well, real-time monitoring of a PCR permits far more accurate measurement of starting target DNA concentrations in multiple-target amplifications, as the relative values of close concentrations can be resolved by taking into account the history of the relative concentration values during the PCR. Real-time monitoring also permits the efficiency of the PCR to be evaluated, which can indicate whether PCR inhibitors are present in a sample.
Holland et al (cited above) and others have proposed fluorescence-based approaches to provide real-time measurements of amplification products during a PCR. Such approaches have either employed intercalating dyes (such as ethidium bromide) to indicate the amount of double stranded DNA present, others have employed probes containing fluorescent-quencher pairs (the so-called “Taq-Man™” approach) that are cleaved during amplification to release a fluorescent product whose concentration is proportional to the amount of double stranded DNA present. Other fluorescent probe technologies have also been used in real-time PCR, including Fluorescent Resonance Energy Transfer (FRET) probes (U.S. Pat. Nos. 6,174,670 and 6,569,627), linear probes in which one probe stimulates and adjacent probes to fluoresce, and molecular beacons in which a hairpin loop is formed within the probe to quench the florescence when the probe is not hybridized to the target nucleic acid.
Fluorimetry is a sensitive and versatile technique with many applications in molecular biology. Ethidium bromide has been used for many years to visualize the size distribution of nucleic acids separated by gel electrophoresis. The gel is usually trans-illuminated with ultraviolet light with a peak wavelength of 340 nm and the resultant fluorescence of double stranded nucleic acid observed at a peak wavelength of 610 nm. Specifically, ethidium bromide is commonly used to analyze the products of PCR after amplification is completed. Furthermore, EP 0 640 828 A1 to Higuchi & Watson, hereby incorporated by reference, discloses using ethidium bromide during amplification to monitor the amount of double stranded DNA by measuring the fluorescence each cycle. The fluorescence intensity was noted to rise and fall inversely with temperature. The greatest fluorescence occurred at the annealing/extension temperature (50° C.). The least fluorescence occurred at the denaturation temperature (94° C.). Maximal fluorescence acquired after each cycle correlated to the amount of nucleic acid amplification product.
The Higuchi & Watson application, however, does mention using other fluorophores, including dual-labeled probe systems that generate fluorescence when hydrolyzed by the 5′-exonuclease activity of certain DNA polymerases, as disclosed in U.S. Pat. No. 5,210,015 to Gelfand et al. The fluorescence observed from these probes primarily depends on hydrolysis of the probe between its two fluorophores. The amount of PCR product is estimated by acquiring fluorescence once each cycle.
The specific hybridization of nucleic acid to a complementary strand for identification has been exploited in many different formats. For example, after restriction enzyme digestion, genomic DNA can be size fractionated and hybridized to probes by Southern blotting. As another example, single base mutations can be detected by “dot blots” with allele-specific oligonucleotides. Usually, hybridization is performed for minutes to hours at a single temperature to achieve the necessary discrimination. Alternately, the extent of hybridization can be dynamically monitored while the temperature is changing by using fluorescence techniques. For example, fluorescence melting curves have been used to monitor hybridization. L. E. Morrison & L. M. Stols, Sensitive fluorescence-based thermodynamic and kinetic measurements of DNA hybridization in solution, 32 Biochemistry 3095-3104, 1993). The temperature scan rates are usually 10° C./hour or less, partly because of the high thermal mass of the fluorimeter cuvette. The temperature scan, or melting analysis, was applied to real-time PCR by Wittwer C T et al. in U.S. Pat. No. 6,174,670.
The prior art in thermal cyclers, as explained above, carries out temperature cycling slowly or uses costly apparatus and unconventional reaction vessels. In the laboratory, there remains a need for a rapid, robust and cost-effective thermal cycler, especially one that can be easily coupled with a fluorescence detection system. Thus, it would be a great advance in the art to provide a system that is able to complete the amplification of nucleic acids with minimal ramp time and a robust design. It would also be beneficial for such a system to permit real-time analysis of the reaction without manipulation of the sample.
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