Extreme PCR

Abstract

Provide methods, apparatus, and kits for PCR with improved efficiency and yield in <20-second cycles.

Description

[0001] This application is a divisional application of the invention patent application filed on May 23, 2013, with application number 201380039367.8 (PCT / US2013 / 042473) and entitled "Extreme PCR". Background of the Invention

[0002] Polymerase chain reaction (PCR) is a widely used technique in molecular biology. Its name comes from one of its key components, a DNA polymerase that amplifies a segment of DNA through in vitro enzymatic replication. As PCR proceeds, the resulting DNA (amplifier) ​​itself serves as a template for replication. This keeps the chain reaction running, where the DNA template amplifies exponentially. With PCR, it is possible to amplify single copies or even just a few copies of a DNA segment by orders of magnitude, producing millions or more copies of the DNA fragment. PCR utilizes a thermostable polymerase, dNTPs, and a pair of primers.

[0003] PCR is conceptually divided into three reactions, each typically assumed to occur over time at three different temperatures. This "equilibrium paradigm" of PCR is easily understood based on the three reactions (denaturation, annealing, and extension) occurring in three time periods at three different temperatures per cycle. However, this equilibrium paradigm does not perfectly align with physical reality. Instantaneous temperature changes do not occur; it takes time to change the sample temperature. Furthermore, the rates of each reaction vary with temperature, and polymerase extension follows immediately after primer annealing. Especially for rapid PCR, a more accurate approach is the kinetic paradigm, where reaction rates and temperatures are constantly changing. Maintaining a constant temperature during PCR is not necessary, as long as product denaturation and primer annealing occur. In the kinetic paradigm of PCR, product denaturation, primer annealing, and polymerase extension may overlap briefly, and their rates continuously change with temperature. In the equilibrium paradigm, cycles are defined by three temperatures, each lasting for a specific period, while the kinetic paradigm requires a gradual denaturation rate and a target temperature. Figures 15a-15b Illustrative time / temperature plots are shown for equilibrium and kinetic paradigms. However, it should be understood that these temperature plots are illustrative only, and in some PCR implementations, the annealing and extension steps are combined, making only two temperatures necessary.

[0004] There are no right or wrong paradigms, but they differ in their practicality. The equilibrium paradigm is easy to understand and well-suited for engineering concepts and instrumentation. The kinetic paradigm is more relevant to biochemistry, rapid cycling PCR, and melting curve analysis.

[0005] When PCR was first popularized in the late 1980s, the process was slow. A typical protocol was denaturation at 94°C for 1 minute, annealing at 55°C for 2 minutes, and extension at 72°C for 3 minutes. Including time for temperature transitions, an 8-minute cycle was typical, resulting in 30 cycles completed in 4 hours. 25% of the cycle time was spent on temperature transitions. As cycle speeds increased, the proportion of time spent on temperature transitions also increased, and the kinetic paradigm became increasingly relevant. During rapid cycling PCR, the temperature frequently changes. For rapid cycling PCR of short products (<100 bp), 100% of the time may be spent on temperature transitions, with no holding time required. For rapid cycling PCR of longer products, temperatures may be maintained at the optimal extension temperature.

[0006] In isolation, the term “rapid PCR” is both relevant and ambiguous. A 1-hour PCR is rapid compared to 4 hours, but slow compared to 15 minutes. Furthermore, a shorter PCR protocol can be achieved by starting with a higher template concentration or using fewer cycles. A more specific metric is the time required for each cycle. Thus, “rapid cycling PCR” (or “rapid cycling”) was defined in 1994 as 30 cycles completed in 10–30 minutes (1), resulting in cycles of 20–60 seconds each. This actual time for each cycle is longer than the sum of the times typically programmed for denaturation, annealing, and extension, because time is needed for the temperature to gradually change between each of these stages. Preliminary studies in the early 1990s confirmed the feasibility of rapid cycling using capillaries and hot gas for temperature control. Over the years, systems have become faster, and the kinetic requirements for denaturation, annealing, and extension have become clearer.

[0007] In an early rapid system, the PCR sample in the heating element and dryer fan, thermocouple, and capillary was enclosed in a chamber (2). The fan generated a rapid flow of hot air through the thermocouple and capillary. By matching the thermal response of the thermocouple to the sample, the temperature of the thermocouple closely tracked the temperature of the sample, even during temperature changes. Despite the low thermal conductivity of air, the rapid movement of air along the large surface area exposed by the capillary was sufficient to cycle the sample between denaturation, annealing, and extension temperatures. An electronic controller monitored the temperature, regulated the power of the heating element, and provided the timing and number of cycles required. For cooling, the controller activated a solenoid, which opened an inlet to the outside air, introducing cooling air into the otherwise sealed chamber.

[0008] Temperature can be rapidly changed using a capillary / air system. Using samples in a low-thermal-mass chamber, circulating air, and glass capillaries, PCR products >500 bp were observed on ethidium bromide-stained gels after only 10 minutes of PCR (30 cycles of 20 seconds each) (3). Product yield was affected by extension time and polymerase concentration. With a cycle time of 30 seconds (approximately 10 seconds for extension between 70 and 80 °C), band intensity increased with polymerase concentration from 0.1 units / 10 µl reactant to 0.8 units / 10 µl reactant. Note that the definition of polymerase units can be confusing. For native Taq polymerase, under typical rapid cycling conditions, 0.4 U / 10 µl is approximately 1.5 nM (50).

[0009] At the denaturation and annealing temperatures, the rapid process utilizes an instantaneous or "0" second hold. That is, the temperature-time profile shows the temperature peaks of denaturation and annealing, without holding the top and bottom temperatures. Denaturation and annealing can occur very quickly.

[0010] Rapid and precise temperature control allows for the analysis of the temperature and time required for PCR studies. For the illustrative 536 bp fragment of human genomic DNA (β-globin), denaturation temperatures between 91°C and 97°C are equally effective, as are denaturation times from <1 second to 16 seconds. However, denaturation times longer than 16 seconds have been found to actually reduce product yield. Specific products with good yields are obtained using annealing temperatures of 50–60°C, provided the primer annealing time is limited. That is, optimal specificity is achieved through rapid cooling from denaturation to annealing and an annealing time of <1 second. Yields are optimal at extension temperatures of 75–79°C and increase with extension times of up to approximately 40 seconds.

[0011] The conclusions drawn from this early study are: 1) PCR products denature very rapidly, eliminating the need to maintain a denaturation temperature; 2) primer annealing occurs extremely quickly, and maintaining an annealing temperature may not be necessary; and 3) the required extension time depends on the length of the PCR product and the polymerase concentration. Furthermore, in terms of specificity and yield, rapid cycling PCR is not only faster but also better (4, 5), provided the temperature is precisely controlled. PCR speed is not limited by available biochemistry but by the instrumentation required to precisely or rapidly control sample temperature.

[0012] However, modern laboratory PCR instruments perform poorly due to their rapid denaturation and annealing times, with many not even allowing for programming of a "0" second hold time. The time delay of heat transfer through the conical tube walls, the low surface area-to-volume ratio, and the heating of large samples force most instruments to rely on extended time intervals during denaturation and annealing to ensure the sample reaches the desired temperature. With these time delays, the exact temperature and time intervals become ambiguous. The result is limited reproducibility within commercial products and high variability between commercial products (6). Many instruments exhibit significant temperature changes during temperature transitions (7, 8). Temperature undershoot and / or overshoot is a common problem that is difficult to address by attempting to predict with software that depends on sample volume. The thermal properties of the instrument, which can change with aging, further complicate these difficulties.

[0013] Over time, conventional heated block instruments have become faster, with improvements including thin-walled tubes, greater heat distribution through conduction between samples, low thermal mass blocks, and other "faster" improvements. However, systems that cycle quickly enough to complete a cycle in less than 60 seconds are uncommon. Few heated block systems achieve cycles of <60 seconds, typically limited to two temperature cycles within a finite temperature range. Rapid cycling can be achieved by flattening the sample container, either through resistance heating and air cooling (9), or by moving the sample within a flexible tube in the heated zone maintained at a constant temperature (US Patent No. 6,706,617).

[0014] Commercially available air / capillary systems have been available for PCR since 1991 (1) and for real-time PCR since 1996 (10, 11). The rapid cycling capabilities of other instruments are typically compared to the earliest air / capillary standards, which showed 20–60 seconds of cycling. Most notably, there has been a trend over the years to run capillary / air systems slower, perhaps reflecting discomfort among many users with “0”-second denaturation and annealing times. Furthermore, heat-activated enzymes require long activation periods, often doubling the run time, even when using “rapid” activating enzymes. Another compromise away from rapid cycling is the use of plastic capillaries. These capillaries are not thermally matched to the instrument, thus typically requiring a 20-second hold to reach the target temperature during denaturation and annealing (12).

[0015] Some advances in further reducing PCR cycle time have occurred in microsystems where small volumes are naturally handled (13, 14). However, even with sample chambers having a high surface area-to-volume ratio, cycles can still be long if the heating element has a high thermal mass and is located outside the chamber (15). With thin-film resistance heaters and temperature sensors close to the sample, amplification in 10–30 minutes is achievable (16, 17).

[0016] While cooling of low thermal mass systems is generally achieved through passive heat diffusion and / or forced ventilation, several valuable heating methods have been developed. Infrared radiation can be used for heating (18), with standardized infrared pyrometers used for temperature monitoring (19). Alternatively, a thin metal film on a glass capillary can be used as both a resistive heating element for rapid cycling and a temperature sensor (20). Finally, direct Joule heating and temperature monitoring of PCR solutions via electrolytic resistors is possible and has been implemented in capillaries (21). All of the above methods transfer heat back and forth between stationary samples.

[0017] Samples can be physically moved to different temperature baths or through channels with fixed temperature zones as an alternative to transferring heat back and forth between stationary samples. Microfluidic methods have become popular, where the PCR fluid passes through channels by maintaining different segments at denaturing, annealing, and extension temperatures. Continuous flow PCR has been demonstrated in meandering channels that pass through three temperature zones (22) and in loops with increasing or decreasing radii across three temperature sectors (23). Variants with meandering layouts use static thermal gradients instead of isothermal zones to more closely fit the kinetic paradigm of PCR (24). To limit the length of the microchannels necessary for PCR, some systems shuttle samples back and forth between temperature zones using bidirectional pressure-driven flow (25), gas dynamics (26), or kinetic electricity (27). Single loop channels can be used in conjunction with sample movement driven by magnetic ferrofluids (28) or by convection (29) to replace linear sample shuttle. A potential advantage of microsystem PCR (including continuous flow methods) is the circulation speed.

[0018] While some microsystems still require >60 seconds of cycling, many operate within the 20–60 second cycle range of rapid cycling PCR (13, 30). Minimum cycle times have been reported for infrared heating in the range of 16–37 seconds (18, 19). Metal-coated capillaries have achieved 40-second PCR cycles (20), while direct electrolytic heating extends this to 21-second cycles (20). Minimum cycle times for closed-loop convection PCR have been reported in the range of 24–42 seconds (29, 31). Several groups have worked to reduce PCR cycle times to <20 seconds, faster than the initial definition of rapid cycling PCR first indicated in 1990. Thin-film resistance heating of 25 µl samples reduces cycle times as low as 17 seconds (32) for stationary samples and as low as 8.5 seconds for 100 nl samples (17). With thermal gradient PCR (24) and sample shuttle (26), continuous flow systems have achieved 12–14 seconds of cycling, while ferrofluid loops have claimed successful PCR with 9 seconds of cycling (28). For different PCR product sizes, continuous flow systems with glass and plastic matrices have achieved cycling times of 6.9 seconds (22) and 5.2 seconds (23). A 1 µl droplet was amplified in oil via alternating hot and cold water conduction through an aluminum matrix (33). Similarly, a 5 µl sample was amplified in 4.6 seconds via water conduction through a porous copper block (34). A 3-second cycling time was achieved with a continuous flow device using a 1 µl reaction plug amplified by vapor pressure (35). Additionally, an 85 bp fragment of E. coli Stx phage has been reported to be amplified in capillary cycles of 2.7 seconds by immersing a capillary in gallium sandwiched between Peltier elements (36). Alternatively, PCR amplification in a capillary tube circulated with pressurized hot and cold gases was performed for 2.6 seconds (48).

[0019] Table 1 summarizes studies that minimize PCR cycle times to below 20 seconds, the initial definition of “rapid PCR.” Over the past 20 years, new prototype instruments have been developed with progressively increasing cycle speeds. However, actual PCR performance (efficiency and yield) is often low. Routinely, as cycles become shorter, claims of successful PCR are associated with lower complexity targets (bacteria, phages, multicopy plasmids, or even PCR products) used at higher starting concentrations (see, for example, US Patent No. 6,210,882, where 5 ng of amplicon is used as the starting sample). In fact, none of the studies listed in Table 1 with <20-second cycles used complex eukaryotic DNA such as human DNA. The starting copy number of the template molecule was often very high (e.g., 180,000,000 copies of λ phage / µl), making amplification almost unnecessary before claiming success. Furthermore, the lack of template-free controls in many studies raises questions about the validity of positive results, especially in environments with high template concentrations. One instrument-oriented report delves into the design and modeling of thermal cycling devices, whose final, concise PCR illustrations use high concentrations of low complexity targets. Based on modeling and measurements in the absence of PCR samples, heating and cooling rates (up to 175 °C / s) were reported (17).

[0020]

[0021]

[0022] One approach to reducing cycle time is to incorporate variations into the PCR protocol to reduce temperature cycling requirements. Longer primers with higher Tm allow for higher annealing temperatures. By limiting product length and its Tm, denaturation temperatures can be lowered to just above the product Tm. Higher annealing and lower denaturing temperatures combined reduce the temperature range required for successful amplification. Reducing three cycles (denaturation, annealing, and extension) to two (denaturation and combined annealing / extension steps) similarly simplifies temperature cycling requirements. Both the reduced temperature range and two-step cycling are typical for studies with cycle times <20 seconds in Table 1. However, two-step cycling may impair polymerase extension rates if the combined annealing / extension step is performed below the optimal 70–80 °C temperature (where polymerase is most active). With temperatures up to approximately 70–80 °C, polymerase extension rates are log-linear, with 60–120 bp / s being the reported maximum (50).

[0023] Even with protocol changes, amplification efficiency and yield are often lower when cycle times are <20 seconds compared to control reactions (22, 23). These efforts toward faster PCR appear to be engineering-driven, with little regard for biochemistry. As cycle times decrease from 20 seconds to 2 seconds, PCR yield decreases and eventually disappears, reflecting a lack of robustness even with simple targets of high copy numbers.

[0024] The instruments described in the references in Table 1 may be suitable for extremely fast PCR, provided the reaction conditions are compatible. As disclosed herein, efforts to increase the concentrations of primers, polymerase, and Mg++ allow for “extreme PCR” (PCR with <20-second cycles (30 cycles in <10 minutes)) while maintaining reaction robustness and yield. Invention Overview

[0025] In one aspect of the invention, a method is provided for amplifying a target nucleic acid sequence in a biological sample during amplification, the method comprising the step of adding a thermostable polymerase and primers configured for amplifying the target nucleic acid sequence to the biological sample, wherein the polymerase is provided at a concentration of at least 0.5 µM and each primer is provided at a concentration of at least 2 µM, and the target nucleic acid sequence is amplified by polymerase chain reaction by thermally cycling the biological sample through multiple amplification cycles between at least denaturation temperature and extension temperature, utilizing an extreme temperature cycling profile in which each cycle is completed in less than 20 seconds / cycle.

[0026] In another aspect of the invention, a method is provided for amplifying a target nucleic acid sequence in a biological sample during amplification, the method comprising the steps of adding a thermostable polymerase and primers configured for amplifying the target nucleic acid sequence to the biological sample, wherein the ratio of polymerase to primers is (about 0.03 to about 0.4 polymerase):(total primer concentration), and the polymerase concentration is at least 0.5 µM; and utilizing the extreme temperature cycling characteristic wherein each cycle is completed in less than 20 seconds, the target nucleic acid sequence is amplified by polymerase chain reaction by thermally cycling the biological sample through multiple amplification cycles between at least denaturation temperature and extension temperature.

[0027] In another aspect of the invention, an apparatus for performing PCR is provided, the apparatus comprising a sample container for containing a PCR sample, a tool for heating the sample, a tool for cooling the sample, and a control tool for repeatedly placing the sample container in the tool for heating the sample and the tool for cooling the sample to thermally cycle the sample at a slow rate of at least 200°C / s.

[0028] In another embodiment, a kit for PCR of a target nucleic acid is provided, the kit comprising: dNTPs, polymerase provided at a concentration of at least 0.5 µM, and a pair of primers configured for amplifying the target nucleic acid, wherein each primer is provided at a concentration of at least 2 µM.

[0029] Other features of the invention will be apparent to those skilled in the art from the following detailed description of preferred embodiments, which illustrate the best mode of carrying out the invention as currently understood. Brief description of the attached diagram

[0030] Figure 1a This shows a schematic diagram for performing extreme PCR.

[0031] Figure 1b It is an illustrative device for performing extreme PCR with the ability to monitor a sample tube in a water bath in real time.

[0032] Figure 1c It is an illustrative device for performing extreme PCR with three temperature cycles.

[0033] Figure 1d middle Figure 1b A close-up view of the optical components of the device described herein, which also shows a temperature reference capillary.

[0034] Figure 2a Is to make Figure 1b A curve showing the overlap between the position of the sample clamp (-----) and the temperature of the sample (——).

[0035] Figure 2b Is using Figure 1b Temperature curves for extreme PCR of the device shown.

[0036] Figure 2c It is aimed at Figure 2b The temperature profiles of rapid cyclic PCR using the LightCycler rotary conveyor are compared and shown.

[0037] Figure 3a Display uses Figure 2b Temperature curves show the derivative melting curves of the extreme PCR product (-----) and the rapid cycling PCR product (—··—), including negative controls with extreme (——) and rapid (— − —) cycles.

[0038] Figure 3b yes Figure 3a2% SeaKem LE agarose gel of the same sample, lanes 1 and 8 are size markers, lanes 2 and 3 are products generated by 30-second extreme PCR, lane 4 is a template-free control of 30-second extreme PCR, lanes 5 and 6 are products generated by 12-minute PCR, and lane 7 is a template-free control of 12-minute PCR.

[0039] Figure 3c Amplification Figure 3a and 3b The extreme PCR temperature traces of the same product shown are (-----), and the real-time monitoring of the reaction is shown (——).

[0040] Figure 4a This shows an extreme PCR temperature trace where temperature control improves the extension rate.

[0041] Figure 4b show Figure 4a The magnified portion makes Figure 1b The position of the sample clamp (——) overlaps with the temperature of the sample (-----).

[0042] Figure 4c The negative derivative melting curve (-dF / dT) of the 58 bp amplicon of IRL10RB shows the AA(——), AG(— − —) and GG(-----) genotypes.

[0043] Figure 5a This involves using extreme PCR to plot a three-dimensional graph of polymerase concentration relative to primer concentration relative to PCR product concentration.

[0044] Figure 5b yes Figure 5a The extreme PCR temperature trace used in the study.

[0045] Figure 5c Showing from Figure 5a Negative derivative melting curve of 4 µM Klentaq polymerase (KT POL) product.

[0046] Figure 5d It is displayed from Figure 5a Agarose gel images of extreme PCR results using different polymerase concentrations at a 10 µM primer concentration.

[0047] Figure 6a The temperature traces are from extreme PCR performed in a No. 19 stainless steel tube.

[0048] Figure 6b It is based on Figure 6a Gel of PCR products generated by extreme temperature cycling.

[0049] Figure 7a It is an extreme PCR temperature trace with a long (1 second) combined annealing / extension step.

[0050] Figure 7b The method employs extreme PCR targeting a 102 bp product to plot a three-dimensional graph of polymerase concentration relative to primer concentration relative to PCR product concentration.

[0051] Figure 8a This shows an extreme PCR temperature profile for amplifying a 226 bp product using a 1-second combined annealing / extension step.

[0052] Figure 8b This shows an extreme PCR temperature profile for amplifying a 428 bp product using a combined 4-second annealing / extension step.

[0053] Figure 8c Showing data obtained from Figure 8a Real-time results and similar temperature traces using a 2-second annealing / stretching step, including template-free controls for each.

[0054] Figure 8d Showing data obtained from Figure 8b Real-time results and similar temperature traces using a 5-second annealing / stretching step, including template-free controls for each.

[0055] Figure 9a The amplification curves of the 45 bp fragment of KCNE1 are shown at different initial concentrations.

[0056] Figure 9b It is Cq relative to the source Figure 9a data log 10 The curve of (initial template copy). The reaction was carried out in five copies. Figures 9c-9d Similar to Figures 9a-9b It only shows the amplification of a 102 bp fragment of NQO1.

[0057] Figure 10a The method employs extreme PCR targeting a 300 bp product (20 cycles, 4.9 seconds / cycle) to plot a three-dimensional graph of polymerase concentration relative to primer concentration relative to PCR product concentration.

[0058] Figure 10b The fluorescence versus cycle number curves for PCR amplification of a 500 bp synthetic template using KAPA2G FAST polymerase and an extension time of 1–5 seconds are shown.

[0059] Figure 10c This is a graph showing the extension length relative to the minimum extension time for several KlenTaq polymerase concentrations and KAPA2G FAST polymerase.

[0060] Figures 11a-11c The following fluorescence versus cycle number curves are shown for PCR amplification of products of the following sizes: 100 bp ( Figure 11a ), 200 bp ( Figure 11b ), 300 bp ( Figure 11c ), 400 bp ( Figure 11d ) and 500 bp ( Figure 11e ).

[0061] Figure 12a The negative derivative melting curves of a 60 bp fragment of AKAP10 are shown after 35 extreme PCR cycles using different magnesium concentrations.

[0062] Figure 12b yes Figure 12a The gel of PCR products is shown in the negative derivative melting curve.

[0063] Figure 13a The display shows the use of different cycle times and 5 mM Mg ++ The negative derivative unchaining curve of a 60 bp fragment of AKAP10 after 35 cycles. The cycle times were 0.32 seconds (—), 0.42 seconds (—··—), 0.52 seconds (— − —), and 0.62 seconds (-----). The cycle times included holding the sample in a 60° bath for 0.1–0.4 seconds.

[0064] Figure 13b yes Figure 13a The gel of PCR products is shown in the negative derivative melting curve.

[0065] Figure 14a Negative derivative melting curves of the 60 bp fragment of AKAP10 amplified on the following three different instruments: (1) Extreme PCR, (2) LightCycler (Roche), and (3) CFX96 (BioRad).

[0066] Figure 14b yes Figure 14a The gel of PCR products is shown in the negative derivative melting curve.

[0067] Figures 15a-15b This demonstrates a balanced paradigm for PCR. Figure 15a ) and dynamic paradigm ( Figure 15b This is an explanatory overview. Solid black indicates nucleic acid denaturation during thermal cycling, stripes indicate annealing, and solid white indicates extension. Invention Details

[0068] As used herein, the terms “a,” “an,” and “the” are defined to mean one or more, including plural, unless the context is inappropriate. A range herein may be expressed as from “about” one specific value and / or to “about” another specific value. The term “about” herein is used to mean approximately, roughly, substantially, or about. When the term “about” is used with a numerical range, the range is modified by extending the boundaries above and below the indicated value. Generally, the term “about” is used herein to modify differences of up to 5% between numerical values ​​and specified values. Another embodiment of expressing such a range includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation using the preceding “about,” it should be understood that the specific value forms another embodiment. It should also be understood that the endpoint of each range is valid both relative to and independent of another endpoint.

[0069] The word “or” as used in this article refers to any single member of a specific list, and also includes any combination of members of that list.

[0070] The term "sample" means an animal for which testing is performed as described herein; tissues or organs from an animal; cells (which may be in a subject, taken directly from the subject, or maintained in culture or derived from a cultured cell line); cell lysates (or lysate fractions) or cell extracts; solutions containing one or more molecules (e.g., polypeptides or nucleic acids) derived from cells, cellular material, or viral material; or solutions containing naturally occurring or non-naturally occurring nucleic acids. Samples may also be any bodily fluid or excretion containing cells, cellular components, or nucleic acids (e.g., but not limited to blood, urine, feces, saliva, tears, bile).

[0071] As used herein, the term "nucleic acid" refers to naturally occurring or synthetic oligonucleotides or polynucleotides, whether DNA or RNA or DNA-RNA hybrids, single-stranded or double-stranded, sense or antisense, capable of hybridizing with complementary nucleic acids via Watson-Crick base pairing. The nucleic acids of this invention may also include nucleotide analogs (e.g., BrdU, dUTP, 7-deazon-dGTP) and nonphosphodiester nucleoside bonds (e.g., peptide nucleic acid (PNA) or thiodiester bonds). Specifically, nucleic acids may include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0072] The terms "probe," "primer," or "oligonucleotide" refer to a single-stranded DNA or RNA molecule with a prescribed sequence that can pair with the bases of a second DNA or RNA molecule containing a complementary sequence ("target"). The stability of the resulting hybrid depends on length, GC content, nearest-neighbor stacking energy, and the degree of base pairing. The degree of base pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the stringency of the hybridization conditions. The stringency of the hybridization is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules (e.g., formamide), and is determined by methods known to those skilled in the art. Probes, primers, and oligonucleotides can be radioactively, fluorescently, or non-radioactively detectable by methods known to those skilled in the art. dsDNA-binding dyes (dyes that fluoresce more strongly when bound to double-stranded DNA than when bound to single-stranded DNA or free in solution) can be used to detect dsDNA. It should be understood that "primers" are specifically configured for extension by polymerase, while "probes" or "oligonucleotides" may or may not be configured in this way.

[0073] The term "specific hybridization" refers to the recognition of essentially complementary nucleic acids (such as sample nucleic acids) by probes, primers, or oligonucleotides, and their physical interaction (i.e., base pairing) with the essentially complementary nucleic acids under highly stringent conditions, while essentially not pairing with other nucleic acid bases.

[0074] The term "high-tightness conditions" refers to conditions that allow hybridization equivalent to that produced by using a DNA probe of at least 40 nucleotides in length at 65°C in a buffer containing 0.5 M NaHPO4 (pH 7.2), 7% SDS, 1 mM EDTA, and 1% BSA (component V), or at 42°C in a buffer containing 48% formamide, 4.8X SSC, 0.2 M Tris-Cl (pH 7.6), 1X Denhardt solution, 10% dextran sulfate, and 0.1% SDS. Other conditions for high-tightness hybridization, such as those for PCR, RNA blotting, DNA blotting, or in situ hybridization, DNA sequencing, etc., are well known to those skilled in the art in molecular biology (47).

[0075] In the illustrative embodiments, methods and kits for PCR with cycle times <20 seconds are provided, with some embodiments using cycle times of <10 seconds, <5 seconds, <2 seconds, <1 second, and <0.5 seconds. At these cycle times, 30 cycles of PCR are completed within <10 minutes, <5 minutes, <2.5 minutes, <1 minute, <30 seconds, and <15 seconds, respectively. As the PCR speed increases, primer and polymerase concentrations are increased, thereby maintaining PCR efficiency and yield.

[0076] Impaired PCR reactions (primer annealing, polymerase extension, and template denaturation) can limit PCR efficiency and yield. For example, if primers are annealed to only 95% of the template, the PCR efficiency will not exceed 95%, even with 100% template denaturation and 100% primer-template extension to full-length product. Similarly, if the extension efficiency is only 95%, the maximum possible PCR efficiency is only 95%. To double the PCR product concentration with each cycle, all components must be completed to 100%. Denaturation, annealing, and extension will be considered sequentially in the following paragraphs.

[0077] Inadequate denaturation is a common cause of PCR failure in slow (>60 sec cycles), fast (20–60 sec cycles), and extreme (<20 sec cycles) PCR temperature cycling. The goal is complete denaturation in each cycle to provide a quantitative template that can be used for primer annealing. Initial denaturation of the template, especially genomic DNA, before PCR often requires more stringent conditions than denaturation of the amplified products during PCR. Initial optimization of rapid cycling PCR is performed after boiling the template (a good way to ensure initial denaturation of genomic DNA) (4). Using high Tm targets, especially those with highly stable flanking regions, may result in incomplete initial denaturation (37). This can impair quantitative PCR, especially if small temperature differences during denaturation affect PCR efficiency (e.g., for genomic insertions or deletions) (37–39). If boiling or restriction digestion beforehand is not required (37), and higher denaturation temperatures impair polymerase, adjuvants that lower product Tm can be used to assist denaturation.

[0078] While 94°C is commonly used as the default target temperature for denaturation, it is not always optimal. PCR products denature within the 40°C range, primarily depending on GC content and length (43). Low denaturation target temperatures offer advantages in both speed and specificity when PCR product denaturation is low enough to allow for the use of even lower denaturation temperatures. The lower the denaturation temperature, the faster the sample reaches the denaturation temperature and the faster PCR can be performed. Additional specificity results in the self-elimination of all potential products with higher denaturation temperatures, as these potential products remain double-stranded and will not be usable for primer annealing. To amplify high-Tm products, the target temperature may need to be increased above 94°C. However, the most common thermostable polymerases begin to denature above 97°C, and PCR solutions can boil between 95 and 100°C, depending on altitude, so there is not much leeway in increasing the temperature. Lowering monovalent salts and Mg ++Concentration lowers the product Tm. Similarly, incorporation of dUTP and / or 7-deazon-dGTP also lowers the product Tm, but may reduce the polymerase extension rate. Most dedicated PCR “enhancers” are simple organic compounds that lower the product Tm, allowing high-Tm products to denature (and amplify). The most common of these are DMSO, betaine, glycerol, ethylene glycol, and formamide. In addition to lowering Tm, some of these additives also increase the boiling point of the PCR mixture (particularly useful at high altitudes). As the concentration of the enhancer increases, the product Tm decreases, but polymerase inhibition may increase.

[0079] However, denaturation need not be rate-limiting even under extreme cycling conditions, as DNA unwinding is first-order and very fast (10–100 msec), even when the temperature is only slightly above the product Tm. Denaturation occurs so rapidly at 2–3 °C above the amplified product Tm that it is difficult to measure, but complete denaturation of the amplicon may occur in less than 0.1 seconds. If the product unwinds in multiple domains, the target denaturation temperature should be 2–3 °C above the largest unwound domain. Denaturation is very rapid once the sample reaches this temperature, even for long products. Using capillary tubes and a water bath (40), complete denaturation of PCR products exceeding 20 kB occurs in less than 1 second (52). Product Tm and unwound domains are determined experimentally according to the instructions using DNA dyes and high-resolution unwinding (41). Although Tm estimation can be obtained through software prediction (42), its accuracy is limited. Furthermore, the observed Tm depends heavily on local reaction conditions, such as salt concentration and the presence of any dyes and auxiliary agents. Therefore, the observed Tm usually matches the reaction conditions better.

[0080] Without affecting efficiency, the rate of approach to denaturation can be as fast as possible, for example, 200-400℃ / s. Figure 2a and 6a As shown. At these rates, the denaturation temperature is reached in approximately 0.1–0.2 seconds. However, as the target temperature approaches, a slower rate reduces the risk of exceeding the target temperature and avoids potential polymerase inactivation or boiling of the solution. An illustrative method for reaching a slower approach temperature is to immerse the sample in a hot water bath 5–10 °C above the target temperature. The temperature difference between the target temperature and the bath temperature determines the exponential approach curve, which automatically slows down as the difference decreases. By continuously monitoring the temperature, the next stage (towards annealing cooling) is initiated when the denaturation target is reached. In general, complete product denaturation in PCR requires a temperature 2–3 °C above the temperature of the product's highest denaturing domain, <0.2 s, and approaches the denaturation temperature as quickly as possible, for example, 40–400 °C / s. Since denaturation is primary, its rate depends only on the product concentration, and the efficiency (or the percentage of denatured product) is independent of the product concentration.

[0081] Incomplete and / or misdirected primer annealing can lead to poor PCR. Inefficient PCR results are produced if not all template sites are initiated. Furthermore, if initiation occurs at undesired sites, substitute products may be produced. The goal is to anneal essentially the entire primer to the desired site each cycle, providing a quantitative primer template for polymerase extension.

[0082] Rapid PCR protocols with 20–60-second cycles indicate annealing times of <1 second at 5°C with primer Tm below 500 nM (52). For instruments attempting <20-second cycles, primer concentrations range from 200–1,000 nM (Table 1). These concentrations are similar to those used in conventional PCR (>60-second cycles) where long annealing times are employed. Lowering primer concentrations is often used to improve specificity, while increasing primer concentrations is rarely considered due to concerns about nonspecific amplification. However, with rapid cycling, the improved specificity is attributed to shorter annealing times (5). If this trend continues, it can be expected that extremely short annealing times for extreme PCR should tolerate high primer concentrations. To facilitate annealing, annealing temperatures below primer Tm by 5°C are recommended for 20–60-second cycles. Ideally, Tm should be measured experimentally using saturated DNA dyes and oligonucleotides via a melting assay under the same buffer conditions used for amplification. The primers were mixed with complementary targets having 5'-extensions as overhangs to maximize the stability of the primers annealed to their templates, and then subjected to stranding analysis.

[0083] In contrast to denaturation, annealing efficiency depends on primer concentration. Primer annealing can become restrictive at very fast cycling rates. Primer annealing is a second-order reaction dependent on both primer and target concentrations. However, during most PCR, primer concentrations are much higher than target concentrations, and annealing is practically quasi-first-order and depends only on primer concentration. In this case, the fraction of induced product (annealing efficiency) depends only on primer concentration, not product concentration, making higher primer concentrations allow for shorter annealing times. Furthermore, while not bound by theory, this relationship is considered linear. As annealing times become increasingly shorter, high primer concentrations become necessary to maintain PCR efficiency and yield. For example, at temperatures 5°C below primer Tm, rapid cycling allows approximately 1–3 seconds for annealing (3). If this annealing time (at or below Tm-5°C) is reduced 10-fold in extreme PCR, a similar ignition efficiency can be expected if primer concentration is increased 10-fold. As available annealing times become increasingly shorter, primer concentrations should be increased to approximately the same fold. Typical rapid PCR protocols use 500 nM of primers per primer. If the annealing time in extreme PCR is reduced by 3–40-fold, the primer concentration required to obtain the same priming efficiency is 1,500–20,000 nM of primers per primer. This equates to 3,000–40,000 nM of total primers, higher than any primer concentration in Table 1. This indicates that one reason for poor efficiency in earlier attempts with cycles of <20 seconds is poor annealing efficiency, inferior to insufficient primer concentrations. In extreme PCR, primer concentrations are increased to 1.5–20 µM each to obtain excellent annealing efficiency, despite annealing times of 0.05–0.3 seconds. For consistently shorter annealing times, consider consistently higher primer concentrations, using increased primer concentrations to offset reduced annealing times to obtain the same annealing efficiency. Note that most commercially available instruments require a holding time of at least 1 second. While a few instruments allow a holding time of "0" seconds, commercially available instruments do not allow holding times of even a fraction of a second. For some illustrative examples of extreme PCR, holding times in increments of 0.1 or 0.01 seconds may be necessary.

[0084] Another way to increase the annealing rate and shorten the annealing time without compromising efficiency is to increase the Mg content. ++ Concentration. It is known in the art to increase annealing rate by increasing ionic strength, and divalent cations are particularly effective for increasing hybridization rate, including primer annealing.

[0085] Illustratively, the rate of approach to the target annealing temperature should be as fast as possible. For example, at 200-800℃ / s ( Figure 2a and 6aThe annealing temperature can be reached in 0.05–0.2 seconds. Rapid cooling also minimizes rehybridization of full-length products. Regarding the formation of double-stranded amplification products during cooling, PCR efficiency decreases because the primers cannot anneal to the double-stranded products. Although this is rare in the early stages of PCR, more and more double strands form during cooling as product concentration increases. Continuous monitoring with SYBR® Green I suggests that this product reannealing may be a major cause of PCR stationarity (44).

[0086] Polymerase extension also takes time, and short extension times can limit PCR efficiency. It is known that longer products during PCR require longer extension times, and a final extension of a few minutes is often added at the end of the PCR cycle, presumably to complete the extension of all products. A common approach for long products is to extend the extension time. This is further extended by using a lower extension temperature, as is seen in some cases of two-step cycles where primer annealing and polymerase extension are performed at the same temperature.

[0087] For optimal PCR efficiency, essentially complete extension of the primer template is required for each cycle. The extension rate of most polymerases reaches a maximum with increasing temperature. For Taq polymerase, the maximum is approximately 100 nucleotides / s at 75–80 °C, decreasing by approximately four-fold for every 10 °C decrease in temperature (50). For the 536 bp β-globin product, 76 °C was found to be optimal for rapid cyclic PCR (4). Faster polymerases have recently been introduced, commercially claimed to reduce overall PCR time, suggesting their ability to eliminate or shorten the extension retention time of longer products.

[0088] As an alternative or supplement to faster polymerase extension rates, increasing polymerase concentration has been found to reduce the extension time required. Considering the standard Taq polymerase concentration for PCR using 500 nM primers (0.04 U / µl) or 1.5 nM (49), if each primer is ligated to the template, only enough polymerase extends 0.15% of the template at a time, requiring repeated recycling of the polymerase to new primers and templates to extend it completely. By increasing the polymerase concentration, more available primers and templates are extended simultaneously, reducing the time required to extend all the template, presumably not through a faster extension rate, but through extending a larger proportion of the primers and templates within any given time.

[0089] Generally, for small PCR products (<100 bp), the required polymerization time appears to be directly proportional to the enzyme's polymerization rate (which itself varies with temperature) and the polymerase concentration. The required time is also inversely proportional to the length of the template to be extended (product length minus primer length). Extreme PCR with <20-second cycles can produce high yields of specific products by increasing polymerase activity 20-300 times higher than the standard 0.04 U / µl activity in PCR. That is, an activity of 0.8–12 U / µl (1–16 µM KlenTaq) enables two-step extreme PCR with combined annealing / extension times of 0.1–1.0 seconds. The highest polymerase activity previously used was 0.5 U / µl (Table 1). For the two-step PCR used as an illustrative example of extreme PCR, a combined annealing / extension step at 70–75 °C is advantageous for a faster polymerization rate. Furthermore, because two-step PCR simplifies temperature cycling, it is typically used in illustrative examples of extreme cycling (<20-second cycles), and both rapid annealing and rapid extension must occur during the combined annealing / extension step. Therefore, both elevated primer and polymerase concentrations are used in illustrative examples to produce robust PCR under extreme two-temperature cycling. Illustratively, primer concentrations of 1.5–20 µM each and polymerase concentrations of 0.4–12 U / µl of any standard polymerase (0.5–16 µM KlenTaq) are necessary for a combined annealing / extension time of 0.05–5.0 seconds at 50–75 °C, as illustrated in the examples below. Because there is only one PCR cycle for both annealing and extension, extreme PCR conditions require amplification of both processes, as illustrated by increasing the concentrations of both primers and polymerase.

[0090] Extreme three-temperature cycles were also envisioned, where the annealing and extension steps were kept separate at different temperatures. In this case, the time allocated to the annealing and extension steps can be individually controlled and adjusted to suit specific needs. For example, if only the annealing time is short (0.05–0.2 seconds) and the extension time remains relatively long (1, 2, 5, 10, or 15 seconds are used as illustrations), then only the primer concentration needs to be increased for effective PCR. Alternatively, if the extension time is short (< 1 sec within 70–80 °C) but the annealing time is long, then only the polymerase concentration is considered to need to be increased to obtain effective PCR. It should be understood that effective PCR has an illustrative efficiency of at least 70%, more illustratively at least 80%, and even at least 90%.

[0091] For products longer than 100 bp, efficient extension using extreme PCR may require a combination of high polymerase concentration and extended extension time. If polymerase is in excess, the minimum time should be illustratively defined as the extension length in bases (defined as product length minus primer length) divided by the polymerase extension rate in bases per second. However, as mentioned earlier, polymerase is typically saturated only at the start of PCR until the template concentration increases to a level greater than the polymerase concentration. One way to reduce cycle time is to use two-temperature PCR close to the polymerase's maximum temperature (typically 70-80°C). Real-time PCR can be used, and quantification cycles (Cq) can be monitored to determine the required extension time experimentally. For example, if the polymerase concentration is in excess, at 75°C and a polymerase extension rate of 100 bases per second, a 200 bp product can be expected to take approximately 2 seconds. Similarly, using the same polymerase, a 400 bp product can be expected to take approximately 4 seconds, provided its concentration is greater than the template being extended. If the polymerase is not excessive, adding more polymerase allows more template to extend at the same time, reducing the extension time required in proportion to the polymerase concentration.

[0092] The practicality of any DNA analysis method depends on how quickly it can be performed, how much information it can yield, and how difficult it is to perform. Compared to conventional cloning techniques, PCR is fast and simple. Rapid cycling and extreme PCR are dedicated to continuously reducing the time required. Real-time PCR increases the information content by acquiring data with each cycle. Stroke analysis can be performed during or after PCR to continuously monitor DNA hybridization as the temperature rises.

[0093] Returning to the equilibrium and kinetics paradigm of PCR ( Figures 15a-15b Extreme PCR with products <100 bp is an example of good application of kinetic models. Temperature is always changing, and the rates of denaturation, annealing, and extension are temperature-dependent; therefore, a proper evaluation of PCR can be obtained by integrating the compositional reaction rates across temperature. For products greater than 100 bp, longer extension times may be necessary, and both kinetic and equilibrium models are appropriate.

[0094] When reaction conditions were set according to at least one embodiment described herein, PCR was found to proceed at extremely rapid rates; for example, in some embodiments where amplification was completed in less than one minute, the cycle time was less than two seconds. Illustratively, various combinations of increased polymerase and primer concentrations were used for this extreme PCR. While not bound by any particular theory, it is believed that excessive primer concentrations generally allow for complete primer annealing, thereby improving PCR efficiency. Furthermore, without being bound by any particular theory, it is believed that increasing polymerase concentration improves PCR efficiency by allowing for more complete extension. Increased polymerase concentrations favor binding to annealed primers and also facilitate recombination if the polymerase detaches before complete extension. The examples below demonstrate that extreme PCR is successful, even when starting with complex eukaryotic genomic DNA.

[0095] Although KlenTaq was used in the following examples, it is believed that any thermostable polymerase with similar activity could be used in a similar manner in extreme PCR, taking into account polymerase extension rates. Examples include Herculase, Kapa2G FAST, KODPhusion, and natural or cloned thermophilic bacteria. Thermus aquaticus Polymerase, Platinum Taq, GoTaq, and Fast Start are commercially available polymerase preparations that, when used at the high concentrations provided herein (with illustrative adjustments for differences in enzyme activity rates), enable extreme PCR.

[0096] Because no recent commercially available PCR instrument allows for a 2-second cycle time, System 4 was set up to test a proof-of-concept for extreme PCR. However, it should be understood that System 4 is illustrative, and other systems capable of rapid thermal cycling are also within the scope of this disclosure. Figure 1a As shown, a hot water bath 10 at 95.5°C (the boiling point of Salt Lake City, UT, where this example was conducted) and a cold water bath 14 at 30–60°C were used to change the temperature of the 1–5 µl sample contained in sample container 20. The illustrative water baths 10 and 14 are 4.5-quart stainless steel tanks (Lab Safety Supply, #41634), but 500 ml glass beakers were used in some examples and heated on hot plates 12 and 16 while being magnetically stirred (Fisher Scientific Isotemp Digital Hotplates (#11-300-49SHP)). However, it should be understood that other embodiments can be used to heat and cool the sample. Figure 1aIn the illustrated embodiment, sample container 20 is a composite glass / plastic reaction tube (BioFire Diagnostics #1720, 0.8 mm ID, 1.0 mm OD). However, in other instances, a composite stainless steel / plastic reaction tube made of stainless steel tubing (Small Parts, 0.042” ID / 0.075” OD, 0.035” ID / 0.042” OD, or 0.0265” ID / 0.035” OD) fitted with a plastic cap to a BioFire tube is used as sample container 20. While other sample containers are within the scope of this invention, it is preferable that the sample container has a large surface area / volume ratio and a fast heat transfer rate. In some embodiments, the open end of the metal tube is sealed by heating to a reddish-white color with flame gas and clamping with clamps. For real-time PCR, optically transparent tubes or tubes with optically transparent portions are required. By briefly centrifuging, the samples are centrifuged to the bottom of each tube.

[0097] Sample containers 20 are clamped by tube clamps 22 connected to stepper motor shafts 26 via arms 21. Tube clamps 22 are machined from black Delrin plastic to hold 2-5 sample containers 20. Figure 1a Only one sample container 20 is visible, but a row of such sample containers 20 may exist, thus keeping the reaction solution within a radius of 6.5–7.5 cm. Although in Figure 1a Although not visible in the image, the thermocouple (Omega Type T Precision Fine Wire Thermocouple #5SRTC-TT-T-40-36, 36” leads, 0.003” diameter, with Teflon insulator) can be used to measure temperature. (See reference...) Figure 1d The figure shows similar parts that represent similar components. Figure 1bSimilar clamps and arms exist, with clamp 222 designed to hold two sample containers, one position of which is occupied by a thermocouple 228. It should be understood that any embodiment described herein can use any number of sample containers 20 or 220, with or without a thermocouple, as shown in Figure 1d. Thermocouple amplification and linearization were performed using an Analog Devices AD595 chip (not shown). The thermocouple voltage was first calculated from the AD595 output as T-type voltage = (AD595 output / 247.3) – 11 µV. The thermocouple voltage was then converted to temperature using the National Institute of Standards and Technology (NIST) coefficient for the voltage / temperature dependence of the T-type thermocouple. The analog signal was digitized (PCIe-6363 acquisition board) and processed using LabVIEW software (version 2010, National Instruments) mounted on CPU 40 and observed on user interface 42. The stepper motor is illustratively started with a strong start at 87-92℃ and 60-75℃, or can be maintained in each water bath for a period of time under computer control. Typically, 30-50 cycles are performed.

[0098] Stepper motor 24 (Applied Motion Products, #HT23-401, 3V, 3A) is placed between water baths 10 and 14, allowing all sample containers 20 in the clamps 22 to tumble between water baths 10 and 14, ensuring that the portions of each sample container 20 containing the sample are fully immersed in the water. Stepper motor 24 is illustratively driven by a 4SX-411 nuDrive (National Instruments, not shown) and controlled by a PCI-7344 motion controller mounted on CPU 40 and NI-Motion software (version 8.2, National Instruments). Stepper motor 24 rotates between water baths 10 and 14 in approximately 0.1 seconds. Figure 2a The figure shows the sample temperature trace (—) alongside the trace for the sample container 20 position (-----) when the stepper motor is started at 90°C and 50°C. As can be seen in Figure 2, there is some overshoot to temperatures below 50°C, presumably caused by the time required to move the sample container 20 out of the water bath 14. Therefore, as mentioned above, it may be necessary to start the stepper motor 24 at a higher temperature. In the examples below, the given temperature refers to the achieved sample temperature, not the start-up temperature. Figure 2aThe calculated maximum heating rate was 385 °C / s, and the maximum cooling rate was 333 °C / s. Illustratively, extreme PCR can be performed at a slow-release rate of at least 200 °C / s. In other embodiments, the slow-release rate can be 300 °C / s or greater.

[0099] In some instances, System 4 is also configured for real-time monitoring. For example... Figure 1a As shown, for real-time monitoring, the fiber optic head 50 of the optical unit 25 is mounted above the sample container 20, such that when the sample container 20 is moved from the hot water bath 10 to the cold water bath by the stepper motor 24, the sample container 20 passes through the fiber optic head 50, whether it remains in the monitoring position or not. In this illustrative embodiment, the fiber optic head is provided in the air above the water bath. The thermal cycling device 4 is controlled by the CPU 40 and observed through the user interface 42.

[0100] Figure 1b Display similar to Figure 1a The implementation scheme includes hot plates 212 and 216 for controlling the temperatures of the hot water bath 210 and the cold water bath 214. A stepper motor 224 is provided for moving the sample container 220 and the thermocouple 228 via a moving arm 221 and a clamp 222, illustratively made of aluminum. Figure 1d (As shown in the image). However, in this embodiment, the end 250 of the fiber optic cable 252 is held in the water bath 214 by the positioning unit 254. The fiber optic cable 252 enters the water bath 214 through the port 248 and provides signals to the optical unit 225. The thermal circulation device 204 can be controlled by the CPU 240 and observed on the user interface 242.

[0101] Light from the Ocean Optics LLS-455 LED Light Source 256 was guided by fiber optic cable 252 (OceanOptics P600-2-UV-VIS, 600 µm core diameter) to a Hamamatsu optical unit 258 with a 440 + / - 20 nm excitation interference filter, a beam-splitting 458 nm dichroic filter, and a 490 + / - 5 nm emission filter (all from Semrock, not shown). Surface fluorescence illumination of the capillary was achieved using another fiber optic cable (not shown), which, when placed in a cooler water bath, was positioned approximately 1–2 mm away from and parallel to one sample capillary. Emission detection was performed using a Hamamatsu PMT 62.

[0102] Figure 1cAn illustrative system 304 for three-temperature PCR is shown. A 95.5°C hot water bath 310, a 30-60°C cold water bath 314, and a 70-80°C medium-temperature water bath 313 are used to change the temperature of 1-5 µl of sample contained in sample container 320, heated on three hot plates 312, 316, and 318 while magnetically stirred. Sample container 320 is clamped by a clamp 322 connected to a stepper motor 324 via an arm 321. A thermocouple 328 is also clamped by the clamp 322. Arm 321 can be lifted when the stepper motor 324 rotates. A fiber optic head 350 in the medium-temperature water bath 313 is provided illustratively, but it should be understood that it can be exposed to air and... Figure 1a Similarly, due to the setup of this illustrative embodiment, it is impossible to place the three water baths 310, 313, and 314 equidistant from each other. Therefore, the maximum space is placed between the hot water bath 310 and the cold water bath 314, as cooling of the sample between these baths is required while the sample moves between the other water baths to be heated. However, it should be understood that this configuration is merely illustrative, and other configurations are within the spirit of this disclosure. Because two stepper motors are used simultaneously (one to lift the capillary out of the water, and one to transfer the sample between the water baths), the angular motion of each can be minimized to reduce the time spent moving between the baths. In a two-water-bath system, the stepper motor required to transfer the sample between the baths requires an angular motion greater than 270 degrees. However, in a three-water-bath system, the stepper motor lifting the sample requires less than 45 degrees of lateral movement, while the stepper motor moving the sample between the water baths only needs to move 90 degrees or less. The water baths can also be configured in a circular fan shape (disc wedge) to further limit the required angular motion. Minimizing angular motion reduces the time spent moving between the water baths. Expected movement time of less than 100 milliseconds, or even less than 50 milliseconds. Other components of this system (304) are similar. Figure 1a The system shown in -b is 4, 204, in Figure 1c Not displayed.

[0103] Example 1

[0104] Unless otherwise specified, PCR is performed in a 5 µl reaction volume containing 50 mM Tris (pH 8.3, 25°C), 3 mM MgCl2, 200 µM of each dNTP (dATP, dCTP, dGTP, dTTP), 500 µg / ml unacetylated bovine serum albumin (Sigma), 2% (v / v) glycerol (Sigma), 50 ng purified human genomic DNA, and 1X LC-Green. ® Plus (BioFire Diagnostics). Primer and polymerase concentrations vary depending on the specific experimental protocol. Klentaq1 TMDNA polymerases were obtained from AB Peptides, St. Louis, MO or Wayne Barnes, Washington University (St. Louis). KlenTaq has a molecular weight of 62.1 kD and an extinction coefficient of 69,130 ​​M at 280 nm. -1 cm -1 The sequence was calculated (US Patent No. 5,436,149). Mass spectrometry confirmed a dominant molecular weight of 62 kD, and denaturing polyacrylamide gel electrophoresis showed a purity greater than 80% by integrating the major band. Concentrations were calculated using absorbance and purity, indicating an 80 µM stock solution in 10% glycerol. The final polymerase concentration was typically 0.25–16 µM. 1 µM KlenTaq equals 0.75 U / µl, defined as 10 nmol of product synthesized from activated salmon sperm DNA over 30 minutes at 72°C. Primers were synthesized at the University of Utah Core Facility, desalted, and their concentrations determined by A 260 Determination. The final concentration of each primer is usually between 2.5 and 20 µM.

[0105] Using primers CCCATTCAACGTCTACATCGAGTC (SEQ ID NO:1) and TCCTTCTCTTGCCAGGCAT (SEQ ID NO:2), human genomic DNA was amplified. KCNE1 The 45 bp fragment was enclosed in primers for variant rs#1805128 (c.253G>A) and the amplified sequence was: CCCATTCAACGTCTACATCGAGTCC(G / A)ATGCCTGGCAAGAGAAGGA (SEQ ID NO:3).

[0106] Figure 3a Display usage Figure 1a The apparatus shown is used to generate a melting curve of PCR products via extreme PCR, employing 0.64 µM KlenTaq and 10 µM primers, and cycling between 91 °C and 50 °C. Figure 2b As shown, the process lasted for 35 cycles, with a total amplification time of 28 seconds. Each cycle took 0.8 seconds. Figure 3a The melting curve of the same amplicon generated by rapid cycling in LightCycler is also shown, using 0.064 µM KlenTaq and 0.5 µM primers, with 35 cycles between 90 °C and 50 °C, for a total amplification time of 12 minutes. Figure 2c Each loop takes 10.3 seconds. Note that because... Figure 2b and 2c Different time scales, so Figure 2b The entire extreme PCR protocol was completed in fewer than two cycles than its rapid cycling counterpart. Both reactions produced amplicons with similar Tm values ​​and strong bands on gel electrophoresis. Figure 3b The negative control showed no amplification by either helical analysis or gel electrophoresis. In this illustrative example, when analyzed by gel electrophoresis, the extreme PCR conditions showed a higher yield than the rapid cycling PCR conditions. Figure 3b The 0.5°C difference in the melting curve Tm is attributed to the varying amounts of glycerol in each reaction, which is determined by the glycerol content in the polymerase storage buffer (the final glycerol concentration in PCR is 1.3% under extreme conditions and 0.1% under rapid conditions). Figure 3b The amplicon sizes were also confirmed to be similar and as predicted. Furthermore, despite high concentrations of polymerase and primers, the reaction appeared specific, with no signs of nonspecific products. However, high-resolution melting analysis was unable to distinguish the three genotypes. The stoichiometric percentage of polymerase to total primer concentration was 3% for extreme PCR and 6.4% for rapid cycling PCR.

[0107] 45 bp was synthesized using 1 µM polymerase, 10 µM primers, and 1.3% glycerol. KCNE1 Real-time monitoring of the reaction. Using... Figure 1a The device monitors the sample in the air between two water baths per cycle. The air temperature in the sealed chamber is maintained at 70°C, and the sample is probed for 0.2 seconds per cycle. As measured by the temperature reference capillary, the sample is cyclicated between 60 and 90°C. Figure 3c As shown. Because of the additional time used for positioning and measurement, the cycle time increased from 0.8 seconds to 1.12 seconds. Therefore, 50 cycles were completed in 56 seconds. Amplification was evident after approximately 30 cycles, or after approximately 34 seconds, based on the increase in fluorescence. Figure 3c The temperature was kept close to 60°C; however, the sample was measured in air, which limited the polymerase extension rate.

[0108] like Figure 3c As observed, the reaction has a quantification cycle (Cq) of approximately 25 cycles, but appears to reach a plateau only after at least 50 cycles. Furthermore, since the reaction stops after 64 cycles, the number of amplicones may continue to increase until a plateau is reached quite late. Without being bound by theory, it is thought that increasing primer concentration could allow for improved yield and delayed plateau, illustrated by 20 cycles after Cq, and more illustratively, 25 cycles or more after Cq.

[0109] Example 2

[0110] In this embodiment, a 58 bp fragment (rs#2834167) of the interleukin-10β receptor containing the A>G variant enclosed in parentheses was amplified using primers CTACAGTGGGAGTCACCTGC (SEQ ID NO:4) and GGTACTGAGCTGTGAAAGTCAGGTT (SEQ ID NO:5) to generate the following amplicon: CTACAGTGGGAGTCACCTGCTTTTGCC(A / G)AAGGGAACCTGACTTTCACAGCTCAGTACC (SEQ ID NO:6). Using Figure 1a The instrument shown was used for extreme PCR as described in Example 1. 1 µM polymerase, 10 µM primers, and 1.3% glycerol (polymerase to total primer percentage = 5%) were used. To increase the polymerase extension temperature to 70-80°C, at which temperature the polymerase exhibits a higher extension rate, different localization schemes were employed. After reaching the annealing temperature, the sample was transferred to a hot water bath until the extension temperature was reached, rather than being immediately localized in the air for monitoring. The sample was then localized in the air just above the hot water bath to produce… Figure 4a and 4b The temperature cycling was shown, allowing for faster polymerase extension at the optimal temperature between 70 and 77°C. Using a 0.97-second cycle, 39 cycles were completed in 38 seconds, amplifying three different genotypes separately via extreme PCR. Following extreme PCR, high-resolution melting curves for each genotype were obtained on an HR-1 instrument modified to accept LC24 capillaries. Figure 4c As expected, all three genotypes were amplified and identified.

[0111] Example 3

[0112] The reaction mixture in Example 1 is the same for both extreme PCR and fast cycling PCR, the difference being the amount of polymerase and primers, and as follows: Figure 3aThe observed small differences in glycerol concentration clearly caused the Tm shift. In this example and all future examples, the glycerol concentration was maintained at 2% by supplementing it if necessary. For extreme PCR, 1 µM polymerase and 10 µM primers were used, while for rapid cyclic PCR, 0.064 µM polymerase and 0.5 µM primers were used. As mentioned above, a faster annealing time is considered to provide improved primer specificity. With this improved specificity, higher primer concentrations can be used, which is considered to favor primer binding and allow for a reduction in annealing time. Similarly, an increased polymerase concentration favors binding to annealed primers and also favors rebinding to incomplete amplicon if the polymerase detaches before complete extension. Additionally, due to the higher polymerase concentration, a larger proportion of the primer template can be extended at once, even in the later stages of PCR, reducing the number of templates that a single polymerase must extend and reducing the overall extension time.

[0113] Figure 5a The results of extreme PCR cycles with different polymerase and primer concentrations are summarized. In this example, a 49 bp fragment of the interleukin-10β receptor was amplified using primers GGGAGTCACCTGCTTTTGCC (SEQ ID NO:7) and TACTGAGCTGTGAAAGTCAGGTTCC (SEQ ID NO:8) and 3 mM MgCl2, producing: GGGAGTCACCTGCTTTTGCCAAAGGGAACCTGACTTTCACAGCTCAGTA (SEQ ID NO:9). For each extreme PCR reaction, the following steps were performed: Figure 1b The device shown lacks real-time monitoring. The temperature is cyclically changed between 90°C and 63°C for 35 cycles, with a total reaction time of just under 26 seconds (0.73 seconds per cycle). Figure 5b As shown. The reaction conditions are as described in Example 1, except for the amounts of polymerase and primers. Figure 5a As shown. Figure 5a The vertical axis in the graph is quantified as the peak of the negative derivative plot of the melting curve, which was obtained on the HR-1 instrument without normalization. At 0.5 µM polymerase, almost no amplification was observed at any primer concentration level. However, at 1.0 µM polymerase, and at primer concentrations of 5 µM and above, recognizable levels of amplicon amplification were observed. The amount of amplicon increased with increasing polymerase level, up to approximately 4 µM. At 8 µM polymerase, the amount of amplicon reached a plateau or decreased, depending on the primer concentration, with a significant decrease at lower primer concentrations at 16 µM. It appears that for 49 bp products under these extreme temperature cycling conditions, the polymerase has a favorable concentration range between approximately 1 and 8 µM, more particularly between 2 and 8 µM, depending on the primer concentration.

[0114] Similarly, almost no amplification was observed with a primer concentration of 2.5 µM. However, amplification was successful with 5 µM primers, where KlenTaq concentrations ranged from 2–8 µM, and amplification improved with increasing concentration. Excellent amplification was achieved with primer concentrations of approximately 10–20 µM. Figure 5c The melting curves for different primer concentrations are shown at 4 µM KlenTaq. Figure 5d The size of the product was confirmed to vary with polymerase concentration, while the primer concentration was maintained at 10 µM. Despite high concentrations of polymerase and primers, no nonspecific amplification was observed.

[0115] Without being bound by theory, it seems that the ratio between the amount of enzyme and the amount of primers is important for extreme PCR cycles, provided that both are above a threshold amount. Note that the amounts mentioned above are based on the individual primers provided. Assuming that the polymerase binds to each of the doublet primers, the total primer concentration is likely the most important. For KlenTaq, a suitable ratio is 0.03–0.4 (3–40% enzyme / total primer concentration), and for extreme PCR, the illustrative minimum KlenTaq concentration is about 0.5 µM, more illustratively about 1.0 µM. As for asymmetric PCR, primers can be provided in equimolar amounts, or one can be provided in excess. The optimal polymerase:primer percentage can also depend on the temperature cycling conditions and product size. For example, standard (slow) temperature cycling often uses a much lower polymerase:primer percentage, typically 1.5 nM (0.04 U / µl) polymerase (49) and 1,000 nM total primer concentration for a 0.15% percentage, which is less than 1 / 10 of the percentages found to be effective for extreme PCR.

[0116] Example 4

[0117] The same PCR target as in Example 3 was amplified using 8 µM polymerase and 20 µM primers in a 19-gauge steel hypodermic needle to improve heat transfer and cycling speed. The polymerase / total primer percentage was 20%. Amplification was performed in... Figure 1b The experiment was conducted in an instrument and completed within 16 seconds using 35 cycles, each lasting 0.46 seconds, between 91°C and 59-63°C. Figure 6a The maximum heating rate during cycling was 407 °C / s, and the maximum cooling rate was 815 °C / s, indicating that PCR can occur at a slow-change rate greater than 400 °C / s without hold. Analysis of the products on a 4% NuSieve 3:1 agarose gel showed strong specific bands of the correct size. Figure 6b The template-free control showed no product at 49 bp, but did show a prominent primer band similar to that of the positive sample.

[0118] Example 5

[0119] In the absence of real-time components, primers CCTGTGCTTTCTGTATCCTCAGAGTGGCATTCT (SEQ ID NO:10) and CGTCTGCTGGAGTGTGCCCAATGCTATA (SEQ ID NO:11) were used. Figure 1b The instrument was used to amplify a 102 bp fragment of the NQO1 gene. Polymerase concentrations were varied between 0.25 and 4 µM, while primer concentrations varied between 0.5 and 8 µM. Primers were designed for annealing at higher temperatures (less than 70 s) so that the extension during the combined annealing / extension phase was within the optimal temperature range for the polymerase. Higher polymerization rates at these temperatures were expected to amplify longer products. The cooler water bath was maintained at 72 °C, and the end of the annealing / extension phase was initiated by time (1 second) rather than temperature. For 30 cycles, a cycle time of 1.93 seconds was used, with cycles between 72 and 90 °C requiring 58 seconds. Figure 7a ).like Figure 7a As observed, the sample temperature dropped by approximately 3°C below the annealing / stretching temperature, while simultaneously being transferred to the hot water bath via air. Figure 7b The display is achieved through quantization. Figure 5a The amount of product amplified by the melting curves is shown. Melting curve analysis revealed only a single product at Tm 84°C. Very little product was observed with 0.25 µM polymerase or 1 µM primers. Some amplification occurred with 2 µM primers, with optimal amplification at 2–4 µM polymerase and 8 µM primers. At 2–4 µM primer concentrations, yield decreased with increasing polymerase concentration, although this was not observed at 8 µM primer concentration. Although the thermal cycling and target length differed from Example 3, optimal amplification occurred at a polymerase / total primer concentration of 3.1–50%.

[0120] Example 6

[0121] Use with real-time monitoring Figure 1b The instrument uses extreme PCR amplification. BBS2The 135 bp and 337 bp fragments of the gene were amplified. To investigate the effect of product length on extreme PCR and the control of potential confusion with different primers, primers with a common 5' overhang were used to amplify the fragments from genomic DNA. For the 135 bp fragment, the primers were ACACACACACACACACACACACACACACACACACACAAAAATTCAGTGGCATTAAATACG (SEQ ID NO:12) and GAGAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAAACCAGAGCTAAAGGGAAG (SEQ ID NO:13). For the 337 bp fragment, the primers were ACACACACACACACACACACACACACACACACACACACAAAAAGCTGGTGTCTGCTATAGAACTGATT (SEQ ID NO:14) and GAGAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAAAGTTGCCAGAGCTAAAGGGAAGG (SEQ ID NO:15). Following standard PCR amplification of genomic DNA, primers and dNTPs were degraded using ExoSAP-IT (Affymetrix, CA), followed by purification of the PCR products using the QuickStep™ 2 PCR Purification Kit (catalog number 33617, Edge BioSystems, Gaithersburg, MD). The PCR products were diluted approximately 1 million-fold and adjusted to an equal concentration by adding Cq obtained from standard real-time PCR to obtain 25 cycles of Cq (approximately 10,000 copies / 10 µl reaction).

[0122] Extreme PCR was performed on 1,000 copies of amplified template in a total volume of 5 µl using two µM universal primers: ACACACACACACACACACACACACACACACACACACAAAAA (SEQ ID NO:16) and GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAA (SEQ ID NO:17), along with 2 µM polymerase and 2% glycerol. The 135 bp BBS2 fragment produced a 226 bp product requiring an extension of 176 or 185 bases (depending on the primers), while the 337 bp BBS2 fragment produced a 428 bp PCR product requiring an extension of 378 or 387 bases. Specific amplification was verified on an agarose gel by melting analysis. Figure 8aExtreme PCR temperature profiles for the 226 bp product are shown, including a 1-second combined annealing / extension at 75°C and denaturation at 87°C. A 2-second annealing / extension phase at the same temperature was also performed (trace not shown). Figure 8c The real-time PCR results of these amplifications are shown, demonstrating a change to a higher Cq with approximately 5 cycles of extension at 1 second compared to a 2-second extension, presumably reflecting a decrease in efficiency as extension time decreases. Figure 8 shows extreme PCR temperature profiles for the 428 bp product, illustrating combined annealing / extension at 75°C for 4 seconds and denaturation at 87°C. Annealing / extension for 5 seconds at the same temperature was also performed (trace not shown). Figure 8d The real-time PCR results of these amplifications are shown, demonstrating a shift to higher Cq with approximately two cycles of extension at 4 seconds compared to a 5-second extension, presumably reflecting a decrease in efficiency as extension time decreases.

[0123] Example 7

[0124] Using diluted human genomic DNA, 2 µM KlenTaq and 8 µM primers were used for NQO1, and 8 µM KlenTaq and 20 µM primers were used for KNCE1. For the 102 bp fragment of NQO1 from Example 5 and the 45 bp fragment of KCNE1 from Example 1, the following methods were employed. Figure 1b The quantitative performance of PCR was evaluated using real-time instruments. The dynamic range was measured in at least four decimal units, such as... Figure 9a and 9b As observed, the amplification efficiency calculated from the standard curve was 95.8% for NQO1 and 91.7% for KCNE1. The template-free control reaction did not amplify after 50 cycles, and for higher concentrations, the amplification curve shape and intensity for single-copy replication (average copy number / reaction of 1.5 copies) were similar (Figures 9A and 9C). At an average copy number of 0.15 copies / reaction, 2 out of 17 reactions were positive (combining both NQO1 and KCNE1 assays), with an expected value of 0.13 copies / reaction calculated using a binomial expansion.

[0125] Example 8

[0126] Extension time required for different product lengths in real-time PCR ( Figure 10a -c). To control for potential confusion from different primers, the following universal high Tm (77℃) primers with a synthesis template of 100-500 bp were used:

[0127] ACTCGCACGAACTCACCGCACTCC (SEQ ID NO:18) and GCTCTCACTCGCACTCTCACGCACA (SEQ ID NO:19).

[0128] The synthetic template sequences are:

[0129] 100 bp template:

[0130] ACTCGCACGAACTCACCGCACTCCGGATGGATTGTGAAGAGGCCCAAGATACTGGTCATATTATCCTTTGATCTAGCTCTCACTCGCACTCTCACGCACA (SEQ ID NO:20).

[0131] 200 bp template:

[0132] ACTCGCACGAACTCACCGCACTCCTCAATGCTGACAAATCGAAAGAATAGGAATAGCGTAATTACTAGAGGACTCCAATATAGTATATTACCCTGGTGACCGCCTGTACTGTAGGAACACTACCGCGGTTATATTGACAGCTTAGCAATCTACCCTGTTGGGATCTGTTTAAGTGGCTCTCACTCGCACTCTCACGCACA (SEQ ID NO:21).

[0133] 300 bp template:

[0134] ACTCGCACGAACTCACCGCACTCCCCTTCGAATATAAAGTACGACATTACTAGCAATGACAGTTCCAGGATTTAAGAAAGTAGTGTTCCACATCAATGCATATCCAGTGAAAGCATAACGTCAAAAAAAGCCTGGCACCGTTCGCGATCTGGACTTACTTAGATTTGTTGTAGTCAAGCCGGCTATCAGCGATTTATCCCGGAAACACATACTAGTGAGTTATTTGTATGTTACCTAGAATAGCTGTCACGAATCACTAATACATTCACCCACCAGCTCTCACTCGCACTCTCACGCACA (SEQ ID NO:22).

[0135] 400 bp template:

[0136] ACTCGCACGAACTCACCGCACTCCTGAATACAAGACGACAGTCCTGATTATATTTTCATTTAATTACGCCAATTTAATTATGATGAATATTAACGGAATTAAATATGTATTGATAAGTACTAAGTAATGGTTTACCCACGGCGATCTATATGCAAGGGAAACATTAACAAATTTAAACATCTGATGTGGACAAAACTTGTAATGTGGTATAGTTAAAAATATAGGTTTCAGGGACACGTAAGTATCTATCTTGAATGTTTAAGTAGGTCCTGTCTACCATTCTGAAATTTAGAAAATCGCGTTCATCGGGCTGTCGGCTACACCTCAGAAAACCATTTCGTGTTGCACAGGAGGAACTTTCGAGGGTTCGTATGAGCTCTCACTCGCACTCTCACGCACA (SEQ ID NO:23).

[0137] 500 bp template:

[0138] ACTCGCACGAACTCACCGCACTCCACCGCTTGACGACGTAGGGTATTTGGTATCTGAATCTACTCATTTACCTACATACTGAAGATTTTGCGATCGTCTAATATATTGGACTAATGCCCGATTTCTGATCAATTACTCTAGGCGATACTTCATCGCTGGCCTTATTTGGATTTTGCTCAAGTGCTAAACTCTCTGCGCGTCAATACTAGTCTGACATCAGTCAAGACCTGCTATCTGAAAACTACTAGAG AGATATACCTAACAACTTTAGTGGATAAATCAGGTCTGGAGATTGTCATATAATGCCACTAGGGTCAGAAGGCTGTGTCAAAGTTAGTGGTTAGTAGGTCTCCGCTCTGCGGTACTATTCTTATATTCTCTTACTATGCATCAAACAAAATAGAATGCATAGACAAACCGCCTGCCAAGTTTACAAGATAACTTGCGTATAGGTTTATAAGGGTTCTTCTGTATCGCTCTCACTCGCACTCTCACGCACA (SEQ ID NO:24).

[0139] The optimal concentrations of primers and polymerase for intermediate-length 300-bp products were first determined using a 4-second combined annealing / extension cycle at 4.9 seconds per cycle. Figure 10a The same primer (4 µM) and polymerase (2 µM) concentrations were then used for all product lengths, and the minimum extension time was determined. Figure 11a -e). Depending on the product length, increasing the extension time leads to a decrease in the fractional quantification cycle (Cq) until no further change is observed, reflecting the minimum extension time required for effective PCR. For example, Figure 10b The amplification curves for a 500 bp product using KAPA2G™ FAST polymerase (Kapa Biosystems) are shown. The minimum extension time using KAPA2G FAST polymerase is 3 s, compared to 7 s using KlenTaq1 (a deletion mutant of Taq polymerase, AB Peptides). Longer products require longer extension times when polymerase properties remain constant. Figure 10cFor KlenTaq1 polymerase, approximately 1 second is required for each 60 bp length, while for KAPA2G FAST, approximately 1 second is required for each 158 bp length. Note that these two polymerases were chosen because they are commercially available at sufficient concentrations, whereas most other polymerases are not commercially available at such high concentrations. It is important to understand that the extension time depends directly on and is linearly related to the length to be extended, and is negatively correlated with polymerase concentration and polymerase rate. A proportionality constant (k2) can be defined to link these three parameters:

[0140] Required extension time = k2 * (extension length) / ([polymerase] * (polymerase speed))

[0141] Example 9

[0142] High Mg ++ Concentration can also reduce extreme PCR time. The 60 bp fragment of AKAP10 was amplified using the following primers: GCTTGGAAGATTGCTAAAATGATAGTCAGTG (SEQ ID NO:25) and TTGATCATACTGAGCCTGCTGCATAA (SEQ ID NO:26) to generate the amplicon GCTTGGAAGATTGCTAAAATGATAGTCAGTGAC(A / G)TTATGCAGCAGGCTCAGTATGATCAA (SEQ ID NO:27).

[0143] Using 2–7 mM MgCl2, each reaction was performed in a 1 µl volume, with 35 cycles based on time control (0.07 s in a 94°C water bath, 0.1–0.4 s in a 60°C water bath). The sample volume was 1 µl, containing 5 ng of human genomic DNA, 20 µM primers, and 8 µM polymerase. Using a 0.42 s / cycle protocol, when MgCl2 was 2–3 mM, the melting curve (…) was observed. Figure 12a ) or gel ( Figure 12b No product was observed at 4 mM MgCl2. Very little product was observed at 4 mM, but a large amount of product was observed after amplification with 5-7 mM MgCl2. At 5 mM MgCl2, the melting curve at a cycle time of 0.32 seconds showed... Figure 13a ) or gel ( Figure 13bNo product was observed at 0.42 sec, 0.52 sec, and 0.62 sec, but abundant product was observed at cycle times of 0.42 sec, 0.52 sec, and 0.62 sec, indicating that a specific, high-yield 60 bp product can be obtained in PCR performed at 15 sec (35 cycles within 14.7 sec). Therefore, illustrative Mg++ concentrations are at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, or higher, and it should be understood that these illustrative Mg++ concentrations can be used with any of the embodiments described herein.

[0144] Example 10

[0145] When used at slower cycling rates, the high concentrations of primers and polymerases used for extreme PCR can have detrimental effects. Non-specific products were obtained on instruments based on rapid cycling or modules at 1 / 32 or 1 / 106 times slower speeds, respectively. Figure 14a -b shows the results of amplification of the AKAP10 60 bp product used in Example 9, under the following conditions, using 20 µM primers, 8 µM KlenTaq and 10 ng human genomic DNA for 40 cycles: (1) extreme PCR at 94°C for 0.5 s and at 60°C for 0.2 s, for a total time of about 17 seconds; (2) rapid cycling PCR (Roche LightCycler) at 94°C for 10 s for initial denaturation, followed by cycling at 85°C for 0 s and at 60°C for 0 s, for a total time of about 9 minutes; and (3) Legacy (module) temperature cycling (BioRad CFX96) at 94°C for 10 s for initial denaturation, followed by cycling at 85°C for 0 s and at 60°C for 5 s, for a total time of about 30 minutes. As can be seen, even though the rapid cycling of LightCycler produces a considerable amount of nonspecific amplification, extreme cycling conditions produce a single melting peak and minimal nonspecific amplification on the gel.

[0146] It was also noted that the yield was increased in extreme PCR, which resulted from high primer and polymerase concentrations. Compared to rapid cycling PCR, extreme PCR produced more than 30-fold amounts of product, compared using quantitative PCR (data not shown).

[0147] use Figure 1aAll Examples 1–10 may be performed using one or more of the devices described in – 1d or with minor variations in these constructions. However, it should be understood that the methods and reactions described herein can occur in a variety of instruments. The water baths and tubes used in these embodiments allow for sufficiently rapid temperature changes to study the effects of increased concentrations of primers and polymerases. However, other embodiments may be more commercially suitable. Microfluidic systems with low volume and high surface area / volume ratios may be well suited for extreme PCR. Such systems allow for the rapid temperature changes required for the high concentrations of primers and polymerases used in extreme PCR. Microfluidic systems include microfluidic systems (35, 53) that integrate miniaturized channels for repeated sample transport through denaturation, annealing, and extension temperature zones. Some of these systems have been shown to be effective for PCR of lower complexity targets, with cycle times as fast as 3 seconds. More complex targets are expected to be amplified in this system if the polymerase is provided at a concentration of at least 0.5 µM and the primers at a concentration of at least 2 µM each. Static PCR chips and PCR droplet systems (54) can also benefit from increased primer and probe concentrations because the volume can be as small as 1 nl or less and low enough to allow for extremely fast cycling. It should be understood that the exact instrument is not important to this invention, as long as the instrument temperature cycling is fast enough to take advantage of the increased primer and polymerase concentrations without suffering the loss of specificity associated with higher primer concentrations at lower cycling rates.

[0148] Although all the above examples use PCR, it should be understood that PCR is only illustrative, and the expected increased primer and enzyme concentrations combined with shorter amplification times are intended for nucleic acid amplification methods other than PCR. Illustrative enzyme activities that can be increased include polymerization (DNA polymerase, RNA polymerase, or reverse transcriptase), ligation, unwinding (helicase) or exonuclease activity (5'-3' or 3'-5'), strand substitution and / or cleavage, endonuclease activity, and RNA digestion of DNA / RNA hybrids (RNAseH). Amplification reactions include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-mediated amplification (including transcription-based amplification systems, auto-maintained sequence replication, and nucleic acid sequence-based amplification), strand substitution amplification, whole-genome amplification, multiple substitution amplification, antisense RNA amplification, loop-mediated amplification, linear-linked amplification, rolling circle amplification, branching amplification, isothermal oligonucleotide amplification, helicase chain reaction (LCR), and sequential invasive signal amplification.

[0149] Generally, amplification time changes inversely with the same factor when enzyme activity changes. For reactions involving primers, amplification time changes inversely with the same factor when primer concentration changes. When amplification requires both primers and enzymes, both enzyme and primer concentrations should be varied to maximize reaction rate. If primer annealing occurs at a specific amplification cycle segment (e.g., a specific temperature during 3°C PCR), the expected time required for satisfactory primer annealing at that segment is negatively correlated with primer concentration. Similarly, if enzyme activity is required at a specific amplification cycle segment (e.g., a specific temperature during 3°C PCR), the expected time required for satisfactory enzyme activity at that segment is negatively correlated with a range of enzyme concentrations. Changing primer or enzyme concentrations can be used to alter the time required for individual segments, or if both occur under the same conditions (e.g., in 2°C PCR or during isothermal reaction processes), changes in both concentrations may be necessary to prevent one reaction from limiting the rate. Increased Mg++ concentrations can also be used in combination with increased enzyme and primer concentrations to further accelerate the amplification process. A higher Mg++ concentration simultaneously increases the rate of primer annealing and reduces the time required for many enzyme reactions used in nucleic acid amplification.

[0150] Higher concentrations of Mg++, enzymes, and primers are particularly useful when accompanied by shorter amplification times or segments. When higher concentrations are used without shortening the time, nonspecific amplification products may occur in some cases because the "rigor" of the reaction is reduced. Reducing the amplification time or segment time introduces higher rigidity, which seems to offset the loss of rigidity caused by the increased reactant concentration. Conversely, if these lower concentrations are offset by longer amplification times or segments, reagent costs can be minimized by reducing reactant concentrations.

[0151] Increasing polymerase concentration can reduce the time required for long-range PCR, taking a target of 5-50 kb as an example. Typically, a 10-30 minute extension period is used to amplify large targets because the target is so long that the required time is needed for: 1) the polymerase to complete the extension of a single target, and 2) the enzyme to be recycled for the polymerization of other primer templates. At the start of PCR, when there are more enzyme molecules available than primer template molecules, this recycling of polymerase is not necessary. However, even before the exponential phase is complete, the number of polymerase molecules often becomes limiting, and enzyme recycling is essential. By increasing the polymerase concentration, the required extension period can be reduced to less than 5 minutes, and possibly less than 2 minutes, while maintaining the high yield resulting from the high primer concentration. Although the actual enzyme rate is not increased, the need for fewer recyclings affects the minimum time required, which is roughly linear with the enzyme concentration.

[0152] Cyclic sequencing time can also be reduced by increasing primer and polymerase concentrations. Typically, in standard cycle sequencing, the primer concentration is 0.16 µM, and the co-annealing / extension period is 10 minutes at 50–60°C. Increasing primer and polymerase concentrations by up to 10-fold can reduce the annealing / extension time by approximately 10-fold. In both long PCR and cycle sequencing, the expected time is inversely proportional to either polymerase or primer concentration, whichever is the limiting factor.

[0153] PCR with ligation adapters that are used as primers in the preparation of mass-parallel sequencing can be completed in much less time than current methods that combine extreme temperature cycling with higher concentrations of primers, polymerases, and / or Mg++.

[0154] In all the applications described above, it is expected that the specificity of the reaction will be maintained through shorter amplification times. While those skilled in the art will expect that high primer and polymerase concentrations will cause difficulties from nonspecific amplification, minimizing the overall cycle time and / or the time for each segment results in high PCR specificity and efficiency.

[0155]

[0156]

[0157]

[0158] The specific conditions for extreme PCR are shown in Table 2. All data are provided except for simultaneous optimization experiments for the three polymerases and primer concentrations of the target. Table 3 details the quantitative relationships between the variables. The inverse proportionality relating the required annealing time to primer concentration is approximately a constant (k1), defined by the equation (required annealing time) = k1 / [primer]. Using various typical values ​​of these variables under legacy (standard) PCR, rapid cycling PCR, and extreme PCR conditions, a range of largely overlapping inverse proportionality constants was generated (legacy 0.75–30, rapid cycling 0.2–10, extreme 1–20). Due to this inverse proportionality, the required annealing time beyond what has been performed can be used to predict the required primer concentration for the desired time. For example, using a constant of 5 (s * µM), a primer concentration of 500 µM can be calculated for an annealing time of 0.01 s. Conversely, if a primer concentration of 0.01 µM is required, the required annealing time should be 500 seconds. Although these conditions fall outside the boundaries of legacy and extreme PCR, they predict the relationship between primer concentrations and annealing times that can be used for successful PCR. A reasonable boundary for k1 between legacy, rapid cycling, and extreme PCR is 0.5–20 (s x µM), more preferably 1–10 (s x µM), and most preferably 3–6 (s x µM).

[0159] Similar calculations can be performed to correlate the required extension time with polymerase concentration, polymerase rate, and the length of the product to be amplified. However, due to the many other variables (polymerase, Mg++, buffer) affecting PCR over time between legacy, rapid cycling, and extreme PCR performed in different laboratories, it is best to pay attention to the tightly controlled conditions of extreme PCR provided in this article to establish an inverse proportionality between the variables. This allows for a quantitative expression of the relationship between polymerase concentration, polymerase rate, product length, and required extension time under extreme PCR conditions. The equation is defined as (required extension time) = k2(product length) / ([polymerase]*(polymerase rate)). k2, determined experimentally, is defined as the value of polymerase concentration, polymerase rate, product length, and required extension time under constant temperature and Mg++ conditions. ++The proportionality constants of the above equations are determined under conditions of polymerase type, buffer, additives, and dsDNA dye concentration. For a 2D optimized 3-extreme PCR target with [polymerase] and [primers], the [polymerase] at the edge of successful amplification can be identified between primer concentrations and correlated with the other three variables. As shown in Table 3, the value of k2 varies less than 2-fold for these three different targets, suggesting that k2 is a constant and can be used to predict one variable if the other variables are known. The required extension time is proportional to the extension length (product length minus primer length) and inversely proportional to the polymerase rate and polymerase concentration. k2 has units of (1 / µM) and an optimal value of 0.5 (1 / µM) in the extreme PCR conditions used in this paper, within the range of 0.3–0.7 (1 / µM). Similar values ​​for k2 can be derived for other reaction conditions with different polymerase types, Mg++ concentrations, or different buffers or dyes.

[0160] Extreme PCR can be performed in any type of container, as long as the sample temperature can be changed rapidly. Microdroplets of aqueous reactants suspended in oil streams or thin 2D rice paper, in addition to standard tubes and capillary tubes, provide good thermal contact. Continuous flow PCR, which involves sample streams passing through spatial segments at different temperatures (or as droplets, separated by bubbles, or otherwise prevented from mixing), is a good method for achieving the required speed and temperature control for extreme PCR.

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[0218] Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the appended claims. sequence list <110> University of Utah Research Foundation Farrar, Jared S Wittwer, Carl T <120> Extreme PCR <130> 2185.0023WO <150> 61 / 651,161 <151> 2012-05-24 <150> 61 / 811,145 <151> 2013-04-12 <160> 27 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> DNA <213> Homo sapiens <400> 1 cccattcaac gtctacatcg agtc 24 <210> 2 <211> 19 <212> DNA <213> Homo sapiens <400> 2 tccttctctt gccaggcat 19 <210> 3 <211> 45 <212> DNA <213> Homo sapiens <220> <221> mutation <222> (23)..(23) <223> G or A residues <400> 3 cccattcaac gtctacatcg agtccgatgc ctggcaagag aagga 45 <210> 4 <211> 20 <212> DNA <213> Homo sapiens <400> 4 ctacagtggg agtcacctgc 20 <210> 5 <211> 25 <212> DNA <213> Homo sapiens <400> 5 ggtactgagc tgtgaaagtc aggtt 25 <210> 6 <211> 58 <212> DNA <213> Homo sapiens <220> <221> mutation <222> (28)..(28) <223> A or G residues <400> 6 ctacagtggg agtcacctgc ttttgccaaa gggaacctga ctttcacagc tcagtacc 58 <210> 7 <211> 20 <212> DNA <213> Homo sapiens <400> 7 gggagtcacc tgcttttgcc 20 <210> 8 <211> 25 <212> DNA <213> Homo sapiens <400> 8 tactgagctg tgaaagtcag gttcc 25 <210> 9 <211> 49 <212> DNA <213> Homo sapiens <400> 9 gggagtcacc tgcttttgcc aaagggaacc tgactttcac agctcagta 49 <210> 10 <211> 34 <212> DNA <213> Homo sapiens <400> 10 ctctgtgctt tctgtatcct cagagtggca ttct 34 <210> 11 <211> 28 <212> DNA <213> Homo sapiens <400> 11 cgtctgctgg agtgtgccca atgctata 28 <210> 12 <211> 60 <212> DNA <213> Homo sapiens <220> <221> Other features <222> (1)..(41) <223> 5' Standard Synthesis Area <400> 12 acacacacac acacacacac acacacacac acacacaaaa attcagtggc attaaatacg 60 <210> 13 <211> 67 <212> DNA <213> Homo sapiens <220> <221> Other features <222> (1)..(50) <223> 5' Standard Synthesis Area <400> 13 gagagagaga gagagagaga gagagagaga gagagagaga gagagaaaaa ccagagctaa 60 agggaag 67 <210> 14 <211> 66 <212> DNA <213> Homo sapiens <220> <221> Other features <222> (1)..(41) <223> 5' Standard Synthesis Area <400> 14 acacacac acacacac acacacac acacacaaaa agctggtgtc tgctatagaa 60 ctgatt 66 <210> 15 <211> 72 <212> DNA <213> Homo sapiens <220> <221> Other features <222> (1)..(50) <223> 5' Standard Synthesis Area <400> 15 gagagagaga gagagagaga gagagagaga gagagagaga gagagaaaaa gttgccagag 60 ctaaagggaa gg 72 <210> 16 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Synthetic universal primers <400> 16 acacacac acacacac acacacac acacacaaaa a 41 <210> 17 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Synthetic universal primers <400> 17 gagagagaga gagagagaga gagagagaga gagagagaga gagagaaaaa 50 <210> 18 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic universal primers <400> 18 actcgcacga actcaccgca ctcc 24 <210> 19 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic universal primers <400> 19 actcgcacga actcaccgca ctcc 24 <210> 20 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthesized template <400> 20 actcgcacga actcaccgca ctccggatgg attgtgaaga ggcccaagat actggtcata 60 ttatcctttg atctagctct cactcgcact ctcacgcaca 100 <210> twenty one <211> 200 <212> DNA <213> Artificial sequence <220> <223> Synthesized template <400> 21 actcgcacga actcaccgca ctcctcaatg ctgacaaatc gaaagaatag gaatagcgta 60 attactagag gactccaata tagtatatta ccctggtgac cgcctgtact gtaggaacac 120 taccgcggtt atattgacag cttagcaatc taccctgttg ggatctgttt aagtggctct 180 cactcgcact ctcacgcaca 200 <210> 22 <211> 300 <212> DNA <213> Artificial sequence <220> <223> Synthetic template <400> 22 actcgcacga actcaccgca ctccccttcg aatataaagt acgacattac tagcaatgac 60 agttccagga tttaagaaag tagtgttcca catcaatgca tatccagtga aagcataacg 120 tcaaaaaaag cctggcaccg ttcgcgatct ggacttactt agatttgttg tagtcaagcc 180 ggctatcagc gatttatccc ggaaacacat actagtgagt tatttgtatg ttacctagaa 240 tagctgtcac gaatcactaa tacattcacc caccagctct cactcgcact ctcacgcaca 300 <210> 23 <211> 400 <212> DNA <213> Artificial sequence <220> <223> Synthetic template <400> 23 actcgcacga actcaccgca ctcctgaata caagacgaca gtcctgatta tattttcatt 60 taattacgcc aatttaatta tgatgaatat taacggaatt aaatatgtat tgataagtac 120 taagtaatgg tttacccacg gcgatctata tgcaagggaa acattaacaa atttaaacat 180 ctgatgtgga caaaacttgt aatgtggtat agttaaaaat ataggtttca gggacacgta 240 agtatctatc ttgaatgttt aagtaggtcc tgtctaccat tctgaaattt agaaaatcgc 300 gttcatcggg ctgtcggcta cacctcagaa aaccatttcg tgttgcacag gaggaacttt 360 cgagggttcg tatgagctct cactcgcact ctcacgcaca 400 <210> 24 <211> 500 <212> DNA <213> Artificial sequence <220> <223> Synthetic template <400> 24 actcgcacga actcaccgca ctccaccgct tgacgacgta gggtatttgg tatctgaatc 60 tactcattta cctacatact gaagattttg cgatcgtcta atatattgga ctaatgcccg 120 atttctgatc aattactcta ggcgatactt catcgctggc cttatttgga ttttgctcaa 180 gtgctaaact ctctgcgcgt caatactagt ctgacatcag tcaagacctg ctatctgaaa 240 actactagag agatatacct aacaacttta gtggataaat caggtctgga gattgtcata 300 taatgccact agggtcagaa ggctgtgtca aagttagtgg ttagtaggtc tccgctctgc 360 ggtactattc ttatattctc ttactatgca tcaaacaaaa tagaatgcat agacaaaccg 420 cctgccaagt ttacaagata acttgcgtat aggtttataa gggttcttct gtatcgctct 480 cactcgcact ctcacgcaca 500 <210> 25 <211> 31 <212> DNA <213> Homo sapiens <400> 25 gcttggaaga ttgctaaaat gatagtcagt g 31 <210> 26 <211> 26 <212> DNA <213> Homo sapiens <400> 26 ttgatcatac tgagcctgct gcataa 26 <210> 27 <211> 60 <212> DNA <213> Homo sapiens <220> <221> Mutation <222> (34)..(34) <223> A or G residue <400> 27 gcttggaaga ttgctaaaat gatagtcagt gacattatgc agcaggctca gtatgatcaa 60

Claims

1. A PCR system, the PCR system comprising means for performing PCR, comprising: Sample containers for holding PCR samples, wherein the sample containers contain Target nucleic acid, dNTP, Polymerase provided at a concentration of at least 8 µM, and Configure primer pairs for amplifying the target nucleic acid, wherein each primer is provided at a concentration of at least 20 µM; A heat transfer device used to change the temperature of a sample in a sample container; and A temperature controller is used to control the heat transfer device to repeatedly heat and cool the sample at a slow rate of at least 400°C / s.

2. The PCR system according to claim 1, wherein The heat transfer device includes multiple water baths, and The temperature controller is a motor connected to an arm that moves the sample container between the water baths.

3. The PCR system according to claim 1, wherein... The heat transfer device includes multiple temperature zones. The sample container defines the routes for each temperature zone, and The temperature controller directs the sample flow to various temperature zones.

4. The PCR system according to claim 1, wherein The heat transfer device contains hot gas.

5. The PCR system according to claim 1, wherein... The heat transfer device includes Joule heating.

6. The PCR system according to claim 1, wherein the heat transfer device is a heating block.

7. The PCR system of claim 1, wherein the sample container further comprises a dye, and the device further comprises an optical unit configured for detecting dye emission.

8. The PCR system according to claim 7, wherein the optical unit detects dye emission in real time.

9. The PCR system of claim 1, wherein the temperature controller is triggered by the sample temperature.

10. The PCR system of claim 1, wherein the temperature controller is programmed to hold the sample at a certain temperature for a specific time.

11. The PCR system of claim 10, wherein the duration of the specific time is less than 1 second.

12. The PCR system according to claim 1, wherein the device is controlled by a CPU.

13. The PCR system of claim 1, wherein the transfer between temperature zones is achieved in less than 100 milliseconds.

14. A PCR system comprising means for performing PCR, the PCR system including: A sample container for holding PCR samples, wherein the sample container contains eukaryotic genomic DNA, a thermostable polymerase, and primers for amplifying a target nucleic acid sequence of the eukaryotic genomic DNA, wherein the ratio of polymerase to primers is 0.03 to 0.4 polymerase:total primer concentration, wherein the polymerase concentration is at least 8 µM, and each primer is provided at a concentration of at least 20 µM. A heat transfer device is used to change the temperature of the sample in the sample container, and A temperature controller is used to control the heat transfer device to repeatedly heat and cool the sample at a slow rate of at least 400°C / s.

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