Patent Description:
PCR is conceptually divided into <NUM> reactions, each usually assumed to occur over time at each of three temperatures. Such an "equilibrium paradigm" of PCR is easy to understand in terms of three reactions (denaturation, annealing, and extension) occurring at <NUM> temperatures over <NUM> time periods each cycle. However, this equilibrium paradigm does not fit well with physical reality. Instantaneous temperature changes do not occur; it takes time to change the sample temperature. Furthermore, individual reaction rates vary with temperature, and once primer annealing occurs, polymerase extension immediately follows. More accurate, particularly for rapid PCR, is a kinetic paradigm where reaction rates and temperature are always changing. Holding the temperature constant during PCR is not necessary as long as the products denature and the primers anneal. Under the kinetic paradigm of PCR, product denaturation, primer annealing, and polymerase extension may temporally overlap and their rates continuously vary with temperature. Under the equilibrium paradigm, a cycle is defined by <NUM> temperatures each held for a time period, whereas the kinetic paradigm requires transition rates and target temperatures. Illustrative time/temperature profiles for the equilibrium and kinetic paradigms are shown in <FIG>. However, it is understood that these temperature profiles are illustrative only and that in some implementations of PCR, the annealing and extension steps are combined so that only <NUM> temperatures are needed.

Paradigms are not right or wrong, but they vary in their usefulness. The equilibrium paradigm is simple to understand and lends itself well to the engineering mindset and instrument manufacture. The kinetic paradigm is more relevant to biochemistry, rapid cycle PCR, and melting curve analysis.

When PCR was first popularized in the late <NUM>, the process was slow. A typical protocol was <NUM> minute for denaturation at <NUM>, <NUM> minutes for annealing at <NUM>, and <NUM> minutes for extension at <NUM>. When the time for transition between temperatures was included, <NUM> minute cycles were typical, resulting in completion of <NUM> cycles in <NUM> hours. Twenty-five percent of the cycling time was spent in temperature transitions. As cycling speeds increased, the proportion of time spent in temperature transitions also increased and the kinetic paradigm became more and more relevant. During rapid cycle PCR, the temperature is usually changing. For rapid cycle PCR of short products (<<NUM> bps), <NUM>% of the time may be spent in temperature transition and no holding times are necessary. For rapid cycle PCR of longer products, a temperature hold at an optimal extension temperature may be included.

In isolation, the term "rapid PCR" is both relative and vague. A <NUM> hour PCR is rapid compared to <NUM> hours, but slow compared to <NUM> minutes. Furthermore, PCR protocols can be made shorter if one starts with higher template concentrations or uses fewer cycles. A more specific measure is the time required for each cycle. Thus, "rapid cycle PCR" (or "rapid cycling") was defined in <NUM> as <NUM> cycles completed in <NUM>-<NUM> minutes (<NUM>), resulting in cycles of <NUM>-<NUM> seconds each. This actual time of each cycle is longer than the sum of the times often programmed for denaturation, annealing and extension, as time is needed to ramp the temperatures between each of these stages. Initial work in the early <NUM> established the feasibility of rapid cycling using capillary tubes and hot air for temperature control. Over the years, systems have become faster, and the kinetic requirements of denaturation, annealing, and extension have become clearer.

In one early rapid system, a heating element and fan from a hair dryer, a thermocouple, and PCR samples in capillary tubes were enclosed in a chamber (<NUM>). The fan created a rapid flow of heated air past the thermocouple and capillaries. By matching the thermal response of the thermocouple to the sample, the temperature of the thermocouple closely tracked the temperature of the samples, even during temperature changes. Although air has a low thermal conductivity, rapidly moving air against the large surface area exposed by the capillaries was adequate to cycle the sample between denaturation, annealing, and extension temperatures. Electronic controllers monitored the temperature, adjusted the power to the heating element, and provided the required timing and number of cycles. For cooling, the controller activated a solenoid that opened a portal to outside air, introducing cooling air to the otherwise closed chamber.

Temperatures could be rapidly changed using the capillary/air system. Using a low thermal mass chamber, circulating air, and samples in glass capillaries, PCR products ><NUM> bp were visualized on ethidium bromide stained gels after only <NUM> minutes of PCR (<NUM> cycles of <NUM> seconds each) (<NUM>). Product yield was affected by the extension time and the concentration of polymerase. With <NUM> second cycle times (about <NUM> seconds between <NUM> and <NUM> for extension), the band intensity increased as the polymerase concentration was increased from <NUM> to <NUM> Units per <NUM>µl reaction. It is noted that polymerase Unit definitions can be confusing. For native Taq polymerase, <NUM> U/<NUM>µl is about <NUM> under typical rapid cycling conditions (<NUM>).

Rapid protocols use momentary or "<NUM>" second holds at the denaturation and annealing temperatures. That is, the temperature-time profiles show temperature spikes for denaturation and annealing, without holding the top and bottom temperatures. Denaturation and annealing can occur very quickly.

Rapid and accurate control of temperature allowed analytical study of the required temperatures and times for PCR. For an illustrative <NUM> bp fragment of human genomic DNA (β-globin), denaturation temperatures between <NUM> and <NUM> were equally effective, as were denaturation times from <<NUM> second to <NUM> seconds. However, it was found that denaturation times longer than <NUM> seconds actually decreased product yield. Specific products in good yield were obtained with annealing temperatures of <NUM>-<NUM>, as long as the time for primer annealing was limited. That is, best specificity was obtained by rapid cooling from denaturation to annealing and an annealing time of <<NUM> second. Yield was best at extension temperatures of <NUM>-<NUM>, and increased with extension time up to about <NUM> seconds.

Conclusions from this early work were: <NUM>) denaturation of PCR products is very rapid with no need to hold the denaturation temperature, <NUM>) annealing of primers can occur very quickly and annealing temperature holds may not be necessary, and <NUM>) the required extension time depends on PCR product length and polymerase concentration. Also, rapid cycle PCR is not only faster, but better in terms of specificity and yield (<NUM>, <NUM>) as long as the temperature was controlled precisely. PCR speed is not limited by the available biochemistry, but by instrumentation that does not control the sample temperature closely or rapidly.

However, most current laboratory PCR instruments perform poorly with momentary denaturation and annealing times, and many don't even allow programming of "<NUM>" second holding periods. Time delays from thermal transfer through the walls of conical tubes, low surface area-to-volume ratios, and heating of large samples force most instruments to rely on extended times at denaturation and annealing to assure that the sample reaches the desired temperatures. With these time delays, the exact temperature vs time course becomes indefinite. The result is limited reproducibility within and high variability between commercial products (<NUM>). Many instruments show marked temperature variance during temperature transitions (<NUM>, <NUM>). Undershoot and/or overshoot of temperature is a chronic problem that is seldom solved by attempted software prediction that depends on sample volume. Such difficulties are compounded by thermal properties of the instrument that may change with age.

Over time, conventional heat block instruments have become faster, with incremental improvements in "thin wall" tubes, more conductive heat distribution between samples, low thermal mass blocks and other "fast" modifications. Nevertheless, it is unusual for these systems to cycle rapidly enough to complete a cycle in less than <NUM> seconds. A few heat block systems can achieve <<NUM> second cycles, usually restricted to <NUM>-temperature cycling between a limited range of temperatures. By flattening the sample container, rapid cycling can be achieved by resistive heating and air cooling (<NUM>), or by moving the sample in a flexible tube between heating zones kept at a constant temperature (<CIT>).

Commercial versions of the air/capillary system for PCR have been available since <NUM> (<NUM>) and for real-time PCR since <NUM> (<NUM>, <NUM>). Rapid cycling capabilities of other instruments are often compared against the air/capillary standard that first demonstrated <NUM>-<NUM> second cycles. Oddly enough, there has been a trend to run the capillary/air systems slower over the years, perhaps reflecting discomfort with "<NUM>" second denaturation and annealing times by many users. Also, heat-activated enzymes require long activation periods, often doubling run times even when "fast" activation enzymes are used. Another compromise away from rapid cycling is the use of plastic capillaries. These capillaries are not thermally matched to the instrument, so <NUM> second holds at denaturation and annealing are often required to reach the target temperatures (<NUM>).

Some progress in further decreasing the cycle times for PCR has occurred in microsystems, where small volumes are naturally processed (<NUM>, <NUM>). However, even with high surface area-to-volume sample chambers, cycles may be long if the heating element has a high thermal mass and is external to the chamber (<NUM>). With thin film resistive heaters and temperature sensors close to the samples, <NUM>-<NUM> minute amplification can be achieved (<NUM>, <NUM>).

While cooling of low thermal mass systems is usually by passive thermal diffusion and/or by forced air, several interesting heating methods have been developed. Infrared radiation can be used for heating (<NUM>) with calibrated infrared pyrometry for temperature monitoring (<NUM>). Alternatively, thin metal films on glass capillaries can serve as both a resistive heating element and a temperature sensor for rapid cycling (<NUM>). Finally, direct Joule heating and temperature monitoring of the PCR solution by electrolytic resistance is possible and has been implemented in capillaries (<NUM>). All of the above methods transfer heat to and from fixed samples.

Instead of heat transfer to and from stationary samples, the samples can be physically moved to different temperature baths, or through channels with fixed temperature zones. Microfluidic methods have become popular, with the PCR fluid passing within channels through different segments kept at denaturation, annealing, and extension temperatures. Continuous flow PCR has been demonstrated within serpentine channels that pass back and forth through <NUM> temperature zones (<NUM>) and within loops of increasing or decreasing radius that pass through <NUM> temperature sectors (<NUM>). A variant with a serpentine layout uses stationary thermal gradients instead of isothermal zones, to more closely fit the kinetic paradigm of PCR (<NUM>). To limit the length of the microchannel necessary for PCR, some systems shuttle samples back and forth between temperature zones by bi-directional pressure-driven flow (<NUM>), pneumatics (<NUM>), or electrokinetic forces (<NUM>). Instead of linear shuttling of samples, a single circular channel can be used with sample movement driven as a magnetic ferrofluid (<NUM>) or by convection (<NUM>). One potential advantage of microsystem PCR, including continuous flow methods, is cycling speed.

Although some microsystems still require ><NUM> second cycles, many operate in the <NUM>-<NUM> second cycle range of rapid cycle PCR (<NUM>, <NUM>). Minimum cycle times ranging from <NUM>-<NUM> seconds have been reported for infrared heating (<NUM>, <NUM>). Metal coated capillaries have achieved <NUM> second PCR cycles (<NUM>), while direct electrolytic heating has amplified with <NUM> second cycles (<NUM>). Minimum cycle times reported for closed loop convective PCR range from <NUM>-<NUM> seconds (<NUM>, <NUM>). Several groups have focused on reducing PCR cycle times to <<NUM> seconds, faster than the original definition of rapid cycle PCR that was first demonstrated in <NUM>. Thin film resistive heating of stationary samples has reduced cycle times down to <NUM> seconds for <NUM>µl samples (<NUM>) and <NUM> seconds for <NUM> nl samples (<NUM>). Continuous flow systems have achieved <NUM>-<NUM> second cycles with thermal gradient PCR (<NUM>) and sample shuttling (<NUM>), while a ferrofluid loop claims successful PCR with <NUM> second cycles (<NUM>). Continuous flow systems through glass and plastic substrates have achieved cycle times of <NUM> seconds (<NUM>) and <NUM> seconds (<NUM>) for various size PCR products. Alternating hot and cool water conduction through an aluminum substrate amplified <NUM>µl droplets under oil with <NUM> second cycles (<NUM>). Similarly, water conduction through a porous copper block amplified <NUM>µl samples with <NUM> second cycles (<NUM>). A continuous flow device of <NUM>µl reaction plugs augmented by vapor pressure achieved <NUM> second cycles (<NUM>). Additionally, there are reports that claim to amplify an <NUM> bp fragment of the Stx bacteriophage of E. coli in capillaries with <NUM> second cycles by immersion of the capillaries in gallium sandwiched between Peltier elements (<NUM>). Alternatively, PCR amplification in capillaries cycled by pressurized hot and cool gases obtained <NUM> second cycles (<NUM>).

Table <NUM> summarizes work to minimize PCR cycle times to less than the <NUM> second cycles that originally defined "Rapid PCR". Over the past <NUM> years, new prototype instruments have been developed that incrementally improve cycling speed. However, practical PCR performance (efficiency and yield) is often poor. As a general rule, as cycles become increasingly shorter, claims for successful PCR correlate with lower complexity targets (bacteria, phage, multicopy plasmids, or even PCR products) that are used at higher starting concentrations (see, e.g., <CIT>, wherein <NUM> ng of amplicon was used as the starting sample). Indeed, none of the studies listed in Table <NUM> with <<NUM> second cycles used complex eukaryotic DNA such as human DNA. The starting copy number of template molecules is often very high (e.g., <NUM>,<NUM>,<NUM> copies of lambda phage/µl), so that little amplification is needed before success is claimed. Furthermore, the lack of no template controls in many studies raises questions regarding the validity of positive results, especially in an environment with high template concentrations. One instrument-oriented report focuses extensively on the design and modeling of the thermal cycling device, with a final brief PCR demonstration using a high concentration of a low complexity target. Heating and cooling rates (up to <NUM>/s) have been reported based on modeling and measurements without PCR samples present (<NUM>).

<CIT> describes very rapid PCR procedures using thermostable DNA polymerases.

One way to decrease cycle time is to introduce variations to the PCR protocol to ease the temperature cycling requirements. Longer primers with higher Tms allow higher annealing temperatures. By limiting the product length and its Tm, denaturation temperatures can be lowered to just above the product Tm. In combination, higher annealing and lower denaturation temperatures decrease the temperature range required for successful amplification. Reducing <NUM>-step cycling (denaturation, annealing, and extension) to <NUM>-steps (denaturation and a combined annealing/extension step) also simplifies the temperature cycling requirements. Both decreased temperature range and <NUM>-step cycling are typical for the studies in Table <NUM> with cycle times <<NUM> seconds. Two-step cycling can, however, compromise polymerase extension rates if the combined annealing/extension step is performed at temperatures lower than the <NUM> to <NUM> temperature optimum where the polymerase is most active. Polymerase extension rates are log-linear with temperature until about <NUM>-<NUM>, with a reported maximum of <NUM>-<NUM> bp/s (<NUM>).

Even with protocol variations, amplification efficiency and yield are often poor when cycle times are <<NUM> seconds when compared to control reactions (<NUM>, <NUM>). These efforts towards faster PCR appear dominated by engineering with little focus on the biochemistry. As cycle times decrease from <NUM> seconds towards <NUM> seconds, PCR yield decreases and finally disappears, reflecting a lack of robustness even with simple targets at high copy number.

The instrumentation in various references disclosed in Table <NUM> may be suitable for extremely fast PCR, if reaction conditions are compatible. As disclosed herein, a focus on increased concentrations of primers, polymerase, and Mg++ allows for "extreme PCR" (PCR with <<NUM> second cycles (<NUM> cycles in <<NUM>)), while retaining reaction robustness and yield.

The invention provides a method for amplifying a target nucleic acid sequence in a biological sample during amplification as defined in the appended claims.

Additional features of the present disclosure will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.

As used herein, the terms "a," "an," and "the" are defined to mean one or more and include the plural unless the context is inappropriate. The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of <NUM>%. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

The word "or" as used herein means any one member of a particular list and also includes any combination of members of that list.

By "sample" is meant an animal; a tissue or organ from an animal; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; a solution containing one or more molecules derived from a cell, cellular material, or viral material (e.g. a polypeptide or nucleic acid); or a solution containing a naturally or non-naturally occurring nucleic acid, which is assayed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells, cell components, or nucleic acids.

The phrase "nucleic acid" as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the disclosure can also include nucleotide analogs (e.g., BrdU, dUTP, <NUM>-deaza-dGTP), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

By "probe," "primer," or "oligonucleotide" is meant a single-stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target"). The stability of the resulting hybrid depends upon the length, GC content, nearest neighbor stacking energy, and the extent of the base-pairing that occurs. The extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art. Probes, primers, and oligonucleotides may be detectably-labeled, either radioactively, fluorescently, or non-radioactively, by methods well-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) may be used to detect dsDNA. It is understood that a "primer" is specifically configured to be extended by a polymerase, whereas a "probe" or "oligonucleotide" may or may not be so configured.

By "specifically hybridizes" is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid (for example, a sample nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.

By "high stringency conditions" is meant conditions that allow hybridization comparable with that resulting from the use of a DNA probe of at least <NUM> nucleotides in length, in a buffer containing <NUM> NaHPO4, pH <NUM>, <NUM>% SDS, <NUM> EDTA, and <NUM>% BSA (Fraction V), at a temperature of <NUM>, or a buffer containing <NUM>% formamide, <NUM>. 8X SSC, <NUM> Tris-Cl, pH <NUM>, 1X Denhardt's solution, <NUM>% dextran sulfate, and <NUM>% SDS, at a temperature of <NUM>. Other conditions for high stringency hybridization, such as for PCR, northern, Southern, or in situ hybridization, DNA sequencing, etc., are well known by those skilled in the art of molecular biology (<NUM>).

Methods are provided for PCR using <<NUM> second, <<NUM> second, <<NUM> second, <<NUM> second, and <<NUM> second cycle times. With these cycle times, a <NUM> cycle PCR is completed in <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM> seconds, and <<NUM> seconds, respectively. As PCR speeds become increasingly faster, the primer and polymerase concentrations are increased, thereby retaining PCR efficiency and yield.

Compromising any of the <NUM> component reactions of PCR (primer annealing, polymerase extension, and template denaturation) can limit the efficiency and yield of PCR. For example, if primers anneal to only <NUM>% of the template, the PCR efficiency cannot be greater than <NUM>%, even if <NUM>% of the templates are denatured and <NUM>% of the primed templates are extended to full length products. Similarly, if extension is only <NUM>% efficient, the maximum possible PCR efficiency is only <NUM>%. In order for the PCR product concentration to double each cycle, all the components must reach <NUM>% completion. Denaturation, annealing and extension will be considered sequentially in the following paragraphs.

Inadequate denaturation is a common reason for PCR failure, in slow (><NUM> second cycles), rapid (<NUM>-<NUM> second cycles), and extreme (<<NUM> second cycles) PCR temperature cycling. The goal is complete denaturation each cycle, providing quantitative template availability for primer annealing. Initial denaturation of template before PCR, particularly genomic DNA, usually requires more severe conditions than denaturation of the amplification product during PCR. The original optimization of rapid cycle PCR (<NUM>) was performed after boiling the template, a good way to assure initial denaturation of genomic DNA. Incomplete initial denaturation can occur with high Tm targets, particularly those with flanking regions of high stability (<NUM>). This can compromise quantitative PCR, illustratively for genomic insertions or deletions, particularly if minor temperature differences during denaturation affect PCR efficiency (<NUM>-<NUM>). If prior boiling or restriction digestion (<NUM>) is not desired, and higher denaturation temperatures compromise the polymerase, adjuvants that lower product Tm can be used to help with denaturation.

Although <NUM> is often used as a default target temperature for denaturation, it is seldom optimal. PCR products melt over a <NUM> range, depending primarily on GC content and length (<NUM>). Low denaturation target temperatures have both a speed and specificity advantage when the PCR product melts low enough that a lower denaturation temperature can be used. The lower the denaturation temperature, the faster the sample can reach the denaturation temperature, and the faster PCR can be performed. Added specificity arises from eliminating all potential products with higher denaturation temperatures, as these potential products will remain double-stranded and will not be available for primer annealing. To amplify high Tm products, the target temperature may need to be increased above <NUM>. However, most current heat stable polymerases start to denature above <NUM> and the PCR solution may boil between <NUM> and <NUM>, depending on the altitude, so there is not much room to increase the temperature. Lowering the monovalent salt and Mg++ concentration lowers product Tm. Similarly, incorporating dUTP and/or <NUM>-deaza-dGTP also lowers product Tm, but may decrease polymerase extension rates. Most proprietary PCR "enhancers" are simple organics that lower product Tm, enabling denaturation (and amplification) of high Tm products. Most popular among these are DMSO, betaine, glycerol, ethylene glycol, and formamide. In addition to lowering Tm, some of these additives also raise the boiling point of the PCR mixture (particularly useful at high altitudes). As the concentration of enhancer increases, product Tms decrease, but polymerase inhibition may increase.

Denaturation, however, need not be rate limiting even under extreme cycling conditions, because DNA unwinding is first order and very fast (<NUM>-<NUM> msec), even when the temperature is only slightly above the product Tm. Denaturation occurs so rapidly at <NUM>-<NUM> above the Tm of the amplification product that it is difficult to measure, but complete denaturation of the amplicon probably occurs in less than <NUM> second. If the product melts in multiple domains, the target denaturation temperature should be <NUM>-<NUM> above the highest melting domain. As long as the sample reaches this temperature, denaturation is very fast, even for long products. Using capillaries and water baths (<NUM>), complete denaturation of PCR products over <NUM> kB occured in less than one second (<NUM>). Product Tms and melting domains are illustratively determined experimentally with DNA dyes and high resolution melting (<NUM>). Although Tm estimates can be obtained by software predictions (<NUM>), their accuracy is limited. Furthermore, observed Tms strongly depend on local reaction conditions, such as salt concentrations and the presence of any dyes and adjuvants. Thus, observed Tms are usually better matched to the reaction conditions.

Without any effect on efficiency, the approach rate to denaturation can be as fast as possible, for example <NUM>-<NUM>/s, as shown in <FIG> and <FIG>. At these rates, only about <NUM>-<NUM> seconds are required to reach denaturation temperatures. However, a slower rate as the target temperature is approached decreases the risk of surpassing the target temperature and avoids possible polymerase inactivation or boiling of the solution. One illustrative method to achieve a slower approach temperature is to submerge the sample in a hot bath that exceeds the target temperature by <NUM>-<NUM>. The temperature difference between the target and bath temperatures determines the exponential approach curve that automatically slows as the difference decreases. By continuously monitoring the temperature, the next phase (cooling toward annealing) is triggered when the denaturation target is achieved. In summary, complete product denaturation in PCR requires <<NUM> at temperatures <NUM>-<NUM> above the highest melting domain temperature of the product and the denaturation temperature can be approached as rapidly as possible, illustratively at <NUM>-<NUM>/second. Since denaturation is first order, its rate depends only on the product concentration, and the efficiency (or percentage of the product that is denatured) is independent of the product concentration.

Incomplete and/or misdirected primer annealing can result in poor PCR. Low efficiency results if not all template sites are primed. Furthermore, if priming occurs at undesired sites, alternative products may be produced. The goal is essentially complete primer annealing to only the desired sites each cycle, providing quantitative primed template for polymerase extension.

Rapid PCR protocols with <NUM>-<NUM> second cycles suggest an annealing time of <<NUM> second at <NUM> below the Tm of <NUM> primers (<NUM>). Primer concentrations for instruments attempting <<NUM> second cycles range from <NUM>-<NUM>,<NUM> each (Table <NUM>). These concentrations are similar to those used in conventional PCR (><NUM> second cycles), where long annealing times are used. Lowering the primer concentration is often used to improve specificity, and increasing the primer concentration is seldom considered due to concerns regarding nonspecific amplification. However, with rapid cycling, improved specificity has been attributed to shorter annealing times (<NUM>). If this trend is continued, one would expect that very short annealing times of extreme PCR should tolerate high primer concentrations. To promote annealing, an annealing temperature <NUM> below the primer Tm is recommended for <NUM>-<NUM> second cycles. Tms are best measured experimentally by melting analysis using saturating DNA dyes and oligonucleotides under the same buffer conditions used for amplification. The primer is combined with its complementary target with a <NUM>'-extension as a dangling end, to best approximate the stability of a primer annealed to its template, and melting analysis is performed.

In contrast to denaturation, annealing efficiency depends on the primer concentration. Primer annealing can become limiting at very fast cycle speeds. Primer annealing is a second order reaction dependent on both primer and target concentrations. However, during most of PCR, the primer concentration is much higher than the target concentration and annealing is effectively pseudo-first order and dependent only on the primer concentration. In this case, the fraction of product that is primed (the annealing efficiency) depends only on the primer concentration, not the product concentration, so that higher primer concentrations should allow for shorter annealing times. Furthermore, without being bound to theory, it is believed that the relationship is linear. As the annealing time becomes shorter and shorter, increased primer concentrations become necessary to maintain the efficiency and yield of PCR. For example, rapid cycling allows about <NUM>-<NUM> seconds for annealing at temperatures <NUM> below primer Tm (<NUM>). If this annealing time (at or below Tm-<NUM>) is reduced <NUM>-fold in extreme PCR, a similar priming efficiency would be expected if the primer concentration were increased <NUM>-fold. As the available annealing time becomes increasingly shorter, the primer concentration should be made increasingly higher by approximately the same multiple. Typical rapid PCR protocols use <NUM> each primer. If the annealing time in extreme PCR is reduced <NUM> to <NUM>-fold, the primer concentrations required to obtain the same priming efficiency are <NUM>,<NUM> - <NUM>,<NUM> each primer. This is equivalent to <NUM>,<NUM> - <NUM>,<NUM> total primers, higher than any primer concentration in Table <NUM>. This suggests that one reason for poor efficiency in prior attempts at <<NUM> second cycling is poor annealing efficiency secondary to inadequate primer concentrations. In extreme PCR, the primer concentrations are increased to <NUM>-<NUM> each to obtain excellent annealing efficiency despite annealing times of <NUM> - <NUM> seconds. Ever greater primer concentrations can be contemplated for ever shorter annealing times, using increased primer concentrations to offset decreased annealing times to obtain the same annealing efficiency. It is noted that most commercial instruments require a hold time of at least <NUM> second, while a few instruments allow a hold time of "<NUM>" seconds, but no commercial instrument allows a hold time of a fractional second. For some illustrative examples of extreme PCR, hold times in increments of <NUM> or <NUM> seconds may be desirable.

Another way to increase the annealing rate and shorten annealing times without compromising efficiency is to increase the Mg++ concentration. Annealing rates are known in the art to increase with increasing ionic strength, and divalent cations are particularly effective for increasing rates of hybridization, including primer annealing.

Illustratively, the approach rate to the annealing target temperature may be as fast as possible. For example, at <NUM>-<NUM>/s (<FIG> and <FIG>), annealing temperatures can be reached in <NUM>-<NUM> seconds. Rapid cooling also minimizes full length product rehybridization. To the extent that duplex amplification product forms during cooling, PCR efficiency is reduced because primers cannot anneal to the duplex product. Although this is rare early in PCR, as the product concentration increases, more and more duplex forms during cooling. Continuous monitoring with SYBR® Green I suggests that such product reannealing can be a major cause of the PCR plateau (<NUM>).

Polymerase extension also requires time and can limit PCR efficiency when extension times are short. Longer products are known to require longer extension times during PCR and a final extension of several minutes is often appended at the end of PCR, presumably to complete extension of all products. The usual approach for long products is to lengthen the time for extension. Using lower extension temperatures further increases required times, as in some cases of <NUM>-step cycling where primer annealing and polymerase extension are performed at the same temperature.

Essentially complete extension of the primed template each cycle is required for optimal PCR efficiency. Most polymerase extension rates increase with temperature, up to a certain maximum. For Taq polymerase, the maximum is about <NUM> nucleotides/s at <NUM>-<NUM> and it decreases about <NUM>-fold for each <NUM> that the temperature is reduced (<NUM>). For a <NUM> bp beta-globin product, <NUM> was found optimal in rapid cycle PCR (<NUM>). Faster polymerases have recently been introduced with commercial claims that they can reduce overall PCR times, suggesting that they may be able to eliminate or shorten extension holding times for longer products.

As an alternative or complement to faster polymerase extension rates, it has been found that increasing the concentration of polymerase reduces the required extension time. Given a standard Taq polymerase concentration in PCR (<NUM> U/µl) or <NUM> (<NUM>) with <NUM> of each primer, if each primer is attached to a template, there is only enough polymerase to extend <NUM>% of the templates at a time, requiring recycling of the polymerase over and over again to new primed templates in order to extend them all. By increasing the concentration of polymerase, more of the available primed templates are extended simultaneously, decreasing the time required to extend all the templates, presumably not by faster extension rates, but by extending a greater proportion of the primed templates at any given time.

To a first approximation, for small PCR products (<<NUM> bp), the required polymerization time appears to be directly proportional to the polymerization rate of the enzyme (itself a function of temperature) and the polymerase concentration. The required time is also inversely proportional to the length of template to be extended (product length minus the primer length). By increasing the polymerase activity <NUM>-<NUM> fold over the standard activity of <NUM> U/µl in the PCR, extreme PCR with <<NUM> second cycles can result in high yields of specific products. That is, activities of <NUM> - <NUM> U/µl (<NUM>-<NUM> of KlenTaq) enable two-step extreme PCR with combined annealing/extension times of <NUM>-<NUM> second. The highest polymerase activity used previously was <NUM> U/µl (Table <NUM>). For two-step PCR that is used in illustrative examples of extreme PCR, a combined annealing/extension step at <NUM>-<NUM> is advantageous for faster polymerization rates. Furthermore, because it simplifies temperature cycling, two-step PCR is typically used in illustrative examples of extreme cycling (<<NUM> second cycles) and both rapid annealing and rapid extension must occur during the combined annealing/extension step. Therefore, both increased primer concentrations and increased polymerase concentrations are used in illustrative examples, resulting in robust PCR under extreme two-temperature cycling. Illustratively, primer concentrations of <NUM>-<NUM> each and polymerase concentrations of <NUM> - <NUM> U/µl of any standard polymerase (<NUM>-<NUM> of KlenTaq) are necessary with combined annealing/extension times of <NUM> - <NUM> seconds at <NUM>-<NUM>, as illustrated in the Examples to follow. Because there is only one PCR cycling segment for both annealing and extension, extreme PCR conditions require enhancement of both processes, illustratively by increasing the concentrations of both the primers and the polymerase.

Extreme three-temperature cycling is also envisioned, where the annealing and extension steps are kept separate at different temperatures. In this case, the time allotted to annealing and extension steps can be individually controlled and tailored to specific needs. For example, if only the annealing time is short (<NUM> - <NUM> seconds) and the extension time is kept comparatively long (illustratively for <NUM>, <NUM>, <NUM>, <NUM> or <NUM> seconds), only the primer concentrations need to be increased for efficient PCR. Alternatively, if the extension time is short (< <NUM> sec within <NUM>-<NUM>), but the annealing time is long, it is believed that only the polymerase concentration needs to be increased to obtain efficient PCR. It is understood that efficient PCR has an illustrative efficiency of at least <NUM>%, more illustratively of at least <NUM>%, and even at least <NUM>%.

For products longer than <NUM> bp, efficient extension using extreme PCR may need a combination of high polymerase concentration and increased extension time. If the polymerase is in excess, the minimum time illustratively should be the extension length (defined as the product length minus the primer length) in bases divided by the polymerase extension rate in bases/second. However, as previously noted, the polymerase is usually only saturating in the beginning of PCR, before the concentration of template increases to greater than the concentration of polymerase. One way to decrease cycle time is to use two-temperature PCR near the temperature maximum of the polymerase, typically <NUM>-<NUM>. The required extension time can be determined experimentally using real-time PCR and monitoring the quantification cycle or Cq. For example, at a polymerase extension rate of <NUM> bases/second at <NUM>, a <NUM> bp product would be expected to require about <NUM> seconds if the concentration of polymerase is in excess. Similarly, a <NUM> bp product would be expected to require about <NUM> seconds using this same polymerase as long as its concentration is greater than the template being extended. If the polymerase is not in excess, adding more polymerase allows more templates to be extended at the same time, decreasing the required extension time in proportion to the concentration of polymerase.

The utility of any DNA analysis method depends on how fast it can be performed, how much information is obtained, and how difficult it is to do. Compared to conventional cloning techniques, PCR is fast and simple. Rapid cycle and extreme PCR focus on continued reduction of the time required. Real-time PCR increases the information content by acquiring data each cycle. Melting analysis can be performed during or after PCR to monitor DNA hybridization continuously as the temperature is increased.

Returning to the equilibrium and kinetic paradigms of PCR (<FIG>), extreme PCR of products <<NUM> bps exemplifies a good application of the kinetic model. Temperatures are always changing and rates of denaturation, annealing, and extension depend on temperature, so an adequate assessment of PCR can only be obtained by integrating the rates of the component reactions across temperature. For products greater than <NUM> bp, longer extension times may be necessary, and components of both the kinetic and equilibrium models are appropriate.

When the reaction conditions are configured according to at least one embodiment herein, it has been found that PCR can be performed at very fast rates, illustratively with some embodiments in less than one minute for complete amplification, with cycle times of less than two seconds. Illustratively, various combinations of increased polymerase and primer concentrations are used for this extreme PCR. Without being bound to any particular theory, it is believed that an excess concentration of primers will allow for generally complete primer annealing, thereby increasing PCR efficiency. Also without being bound to any particular theory, it is believed that an increase in polymerase concentration improves PCR efficiency by allowing more complete extension. Increased polymerase concentration favors binding to the annealed primer, and also favors rebinding if a polymerase falls off prior to complete extension. The examples below show that extreme PCR has been successful, even when starting with complex eukaryotic genomic DNA.

Although KlenTaq was used in the Examples to follow, it is believed that any thermostable polymerase of similar activity will perform in a similar manner in extreme PCR, with allowances for polymerase extension rates. For example, Herculase, Kapa2G FAST, KOD Phusion, natural or cloned Thermus aquaticus polymerase, Platinum Taq, GoTaq and Fast Start are commercial preparation of polymerases that should enable extreme PCR when used at the increased concentrations presented here, illustratively adjusted for differences in enzyme activity rates.

Because no current commercial PCR instrument allows for two second cycle times, a system <NUM> was set up to test proof of concept for extreme PCR. However, it is understood that the system <NUM> is illustrative and other systems that can thermocycle rapidly are within the scope of this disclosure. As shown in <FIG>, a hot water bath <NUM> of <NUM> (the temperature of boiling water in Salt Lake City, UT, the location where the present examples were performed), and a cool water bath <NUM> of <NUM>-<NUM> are used to change the temperature of <NUM>-<NUM>µl samples contained in a sample container <NUM>. The illustrative water baths <NUM>, <NUM> are <NUM> quart stainless steel dressing jars (Lab Safety Supply, #<NUM>), although <NUM> glass beakers were used in some examples, and are heated on electric hotplates <NUM>, <NUM> with magnetic stirring (Fisher Scientific Isotemp Digital Hotplates (#<NUM>-<NUM>-49SHP). However, it is understood that other examples may be used to heat and cool the samples. In the example shown in <FIG>, the sample container <NUM> is a composite glass/plastic reaction tube (BioFire Diagnostics #<NUM>, <NUM> ID and <NUM> OD). However, in other examples, hypodermic needles (Becton Dickenson #<NUM>, <NUM>" ID, <NUM>" OD) and composite stainless steel/plastic reaction tubes constructed from stainless steel tubing (Small Parts, <NUM>" ID/<NUM>" OD, <NUM>" ID/<NUM>" OD, or <NUM>" ID/<NUM>" OD) and fit into the plastic tops of the BioFire tubes were used as the sample container <NUM>. While other sample containers are within the scope of this disclosure, it is desirable that the sample containers have a large surface area to volume ratio and have a fast heat transfer rate. For certain examples, the open end of the metal tubing was sealed by heating to a red-white color using a gas flame and compressing in a vise. For real-time PCR, tubes that are optically clear or have an optically clear portion are desirable. Samples were spun down to the bottom of each tube by brief centrifugation.

The sample container <NUM> is held by a tube holder <NUM> attached to a stepper motor shaft <NUM> by arm <NUM>. The tube holder <NUM> was machined from black Delrin plastic to hold <NUM>-<NUM> sample containers <NUM> (only one sample container <NUM> is visible in <FIG>, but a row of such sample containers <NUM> may be present) so that the reaction solutions were held at a radius of <NUM> - <NUM>. While not visible in <FIG>, a thermocouple (Omega type T precision fine wire thermocouple #5SRTC-TT-T-<NUM>-<NUM>, <NUM>" lead, <NUM>' diameter with Teflon insulation) may be used to measure temperature. With reference to <FIG>, which shows a similar tube holder and arm of <FIG> with like numbers representing similar components, a tube holder <NUM> designed to hold two sample containers is present, with one location in tube holder <NUM> occupied by a thermocouple <NUM>. It is understood that any number of sample containers <NUM> or <NUM> may be used in any of the examples described herein, with or without a thermocouple, as shown in <FIG>. Thermocouple amplification and linearization is performed with an Analog Devices AD595 chip (not shown). The thermocouple voltage was first calculated from the AD595 output as Type T voltage = (AD595 output/<NUM>) - <NUM>µV. Then the thermocouple voltage was converted to temperature using National Institute of Standards and Technology coefficients for the voltage/temperature correlation of Type T thermocouples. The analog signal was digitized (PCIe-<NUM> acquisition board) and processed by LabView software (version <NUM>, National Instruments) installed on CPU <NUM> and viewed on user interface <NUM>. Stepper motion illustratively is triggered dynamically at <NUM>-<NUM> and <NUM>-<NUM> or may be held in each water bath for a computer-controlled period of time. Thirty to fifty cycles are typically performed.

The stepper motor <NUM> (Applied Motion Products, #HT23-<NUM>, 3V,3A) is positioned between the water baths <NUM> and <NUM> so that all sample containers <NUM> in the tube holder <NUM> could flip between each water bath <NUM> and <NUM>, so that the portion of each sample container <NUM> containing samples are completely submerged. The stepper motor <NUM> is powered illustratively by a 4SX-<NUM> nuDrive (National Instruments, not shown) and controlled with a PCI-<NUM> motion controller and NI-Motion Software (version <NUM>, National Instruments) installed on CPU <NUM>. Stepper motor <NUM> rotates between water baths <NUM> and <NUM> in about <NUM> second. <FIG> shows a sample temperature trace (-) juxtaposed over a trace of the position of the sample container <NUM> (-----), for a run where stepper motion was triggered at <NUM> and <NUM>. As can be seen in <FIG>, there is some overshoot to a temperature lower than <NUM>, presumably due do the time required to move the sample container <NUM> out of water bath <NUM>. Thus, as discussed above, it may be desirable to trigger stepper motor <NUM> at a somewhat higher temperature. In the examples below, the temperatures given are for the sample temperature reached, not the trigger temperature. The maximum heating rate calculated from <FIG> is <NUM>/s and maximum cooling rate <NUM>/s. Illustratively, extreme PCR may be performed with ramp rates of at least <NUM>/s. In other examples, the ramp rate may be <NUM>/s or greater.

In some examples, system <NUM> is also configured for real-time monitoring. As shown in <FIG>, for real time monitoring, a fiber optics tip <NUM> of optics block <NUM> is mounted above sample container <NUM>, such that when sample container <NUM> is being moved from hot water bath <NUM> to the cold water bath by stepper motor <NUM>, sample container <NUM> passes by the fiber optics tip <NUM>, with or without a hold in this monitoring position. In this illustrative example, fiber optics tip is provided in air above the water baths. Thermocycling device <NUM> may be controlled by CPU <NUM> and viewed on user interface <NUM>.

<FIG> shows an example similar to <FIG>. Hot plates <NUM> and <NUM> are provided for controlling temperature of hot water bath <NUM> and cold water bath <NUM>. A stepper motor <NUM> is provided for moving sample container <NUM> and thermocouple <NUM> (shown in <FIG>), by moving arm <NUM> and tube holder <NUM>, which is illustratively made of aluminum. However, in this example, the tip <NUM> of the fiber optics cable <NUM> is held in water bath <NUM> by positioning block <NUM>. Fiber optics cable <NUM> enters water bath <NUM> through port <NUM> and provides signal to optics block <NUM>. Thermocycling device <NUM> may be controlled by CPU <NUM> and viewed on user interface <NUM>.

Light from an Ocean Optics LLS-<NUM> LED Light Source <NUM> was guided by fiber optics cable <NUM> (Ocean Optics P600-<NUM>-UV-VIS, <NUM> fiber core diameter) into a Hamamatsu Optics Block <NUM> with a <NUM> +/-<NUM> excitation interference filter, a beamsplitting <NUM> dichroic and a <NUM> +/- <NUM> emission filter (all from Semrock, not shown). Epifluorescent illumination of the capillary was achieved with another fiber optic cable (not shown) placed approximately <NUM>-<NUM> distant from and in-line with the one sample capillary when positioned in the cooler water bath. Emission detection was with a Hamamatsu PMT <NUM>.

<FIG> shows an illustrative system <NUM> for three-temperature PCR. A hot water bath <NUM> of <NUM>, a cool water bath <NUM> of <NUM>-<NUM>, and a medium water bath <NUM> of <NUM>-<NUM> are used to change the temperature of <NUM>-<NUM>µl samples contained in a sample container <NUM>, and are heated on three electric hotplates <NUM>, <NUM>, and <NUM> with magnetic stirring. The sample container <NUM> is held by a tube holder <NUM> attached to a stepper motor <NUM> by arm <NUM>. Thermocouple <NUM> is also held by tube holder <NUM>. Arm <NUM> may be raised as stepper motor <NUM> rotates. A fiber optics tip <NUM> is illustratively provided in medium water bath <NUM>, although it is understood that it may be placed in air, as with <FIG>. Due to the set-up of this illustrative example, it was not possible to place the three water baths, <NUM>, <NUM>, and <NUM> equidistant from one another. Accordingly, the largest space was placed between hot water bath <NUM> and cool water bath <NUM>, as cooling of the sample between these baths is desirable, whereas the sample moves between the other water baths to be heated. However, it is understood that this configuration is illustrative only.

Because two stepper motors are used simultaneously (one to raise the capillary out of the water and one to transfer between water baths) the angular motion of each can be minimized to decrease the time of movement between baths. In the <NUM> water bath system, the required angular motion of the stepper to transfer the sample between baths is greater than <NUM> degrees. However, in the <NUM> water bath system, the stepper motor that raises the samples needs to traverse less than <NUM> degrees while the stepper moving the samples between water baths needs to move only <NUM> degrees or less. The water baths can also be configured as sectors of a circle (pie-shaped wedges) to further limit the angular movement required. Minimizing the angular movement decreases the transfer time between water baths. Transfer times less than <NUM> msec or even less than <NUM> msec are envisioned. Other components of this system <NUM> are similar to the systems <NUM>, <NUM> shown in <FIG> and are not shown in <FIG>.

Unless otherwise indicated, PCR was performed in <NUM>µl reaction volumes containing <NUM> Tris (pH <NUM>, at <NUM>), <NUM> MgCl<NUM>, <NUM> each dNTP (dATP, dCTP, dGTP, dTTP), <NUM>µg/ml non-acetylated bovine serum albumin (Sigma), <NUM>% (v/v) glycerol (Sigma), <NUM> ng of purified human genomic DNA, and 1X LCGreen® Plus (BioFire Diagnostics). The concentration of the primers and the polymerase varied according to the specific experimental protocols. Klentaq1™ DNA polymerase was obtained from either AB Peptides, St. Louis, MO, or from Wayne Barnes at Washington University (St. The molecular weight of KlenTaq is <NUM> kD with an extinction coefficient at <NUM> of <NUM>,<NUM>-<NUM>cm-<NUM>, as calculated from the sequence (<CIT>). Mass spectrometry confirmed a predominate molecular weight of <NUM> kD, and denaturing polyacrylamide gels showed that the major band was greater than <NUM>% pure by integration. Using the absorbance and purity to calculate the concentration indicated an <NUM> stock in <NUM>% glycerol. Final polymerase concentrations were typically <NUM>-<NUM>. One µM KlenTaq is the equivalent of <NUM> U/µl, with a unit defined as <NUM> nmol of product synthesized in <NUM> at <NUM> with activated salmon sperm DNA. Primers were synthesized by the University of Utah core facility, desalted, and concentrations determined by A<NUM>. The final concentrations of each primer typically varied from <NUM>-<NUM>.

A <NUM> bp fragment of KCNE1 was amplified from human genomic DNA using primers CCCATTCAACGTCTACATCGAGTC (SEQ ID NO:<NUM>) and TCCTTCTCTTGCCAGGCAT (SEQ ID NO:<NUM>). The primers bracketed the variant rs#<NUM> (c. <NUM>>A) and amplified the sequence: CCCATTCAACGTCTACATCGAGTCC(G/A)ATGCCTGGCAAGAGAAGGA (SEQ ID NO:<NUM>).

<FIG> shows a melting curve of the PCR product generated by extreme PCR using the device shown in <FIG>, where <NUM> KlenTaq and <NUM> of each primer were used, and cycled between <NUM> and <NUM>, as shown in <FIG>, for <NUM> cycles and a total amplification time of <NUM> seconds. Each cycle required <NUM> seconds. Also shown in <FIG> is a melting curve of the same amplicon generated by rapid cycling in the LightCycler, where <NUM> KlenTaq and <NUM> of each primer were used, and cycling was between <NUM> and <NUM> for <NUM> cycles and a total amplification time of <NUM> minutes (<FIG>). Each cycle required <NUM> seconds. Note that because of the different time scales in <FIG> and <FIG>, the entire extreme PCR protocol of <FIG> is completed in less than <NUM> cycles of its rapid cycle counterpart. Both reactions produced amplicons having similar Tms and strong bands on gel electrophoresis (<FIG>), whereas neither negative control showed amplification by either melting analysis or gel electrophoresis. In this illustrative example, extreme PCR conditions showed greater yield than rapid cycle PCR conditions when analyzed on gels (<FIG>). The <NUM> difference in Tm on the melting curves is believed to be due to the different amounts of glycerol in each reaction, arising from the glycerol content in the polymerase storage buffer (final concentration of glycerol in the PCR was <NUM>% under extreme conditions and <NUM>% under rapid conditions). <FIG> also confirms that the size of the amplicons were similar and as predicted. In addition, despite the high concentrations of polymerase and primers, the reaction appears specific with no indication of nonspecific products. However, high resolution melting analysis was unable to distinguish the <NUM> genotypes. The stoichiometric percentage of polymerase to total primer concentration was <NUM>% for extreme PCR and <NUM>% for rapid cycle PCR.

Real-time monitoring of the <NUM> bp KCNE1 reaction was performed using <NUM> polymerase, <NUM> of each primer, and <NUM>% glycerol. The sample was monitored each cycle in air between the <NUM> water baths using the device of <FIG>. The enclosed chamber air temperature was held at <NUM> and the sample was interrogated for <NUM> seconds each cycle. As measured by the temperature reference capillary, samples were cycled between <NUM> and <NUM>, as shown in <FIG>. The cycle time increased from <NUM> seconds to <NUM> seconds because of the added time for positioning and measuring. Thus, fifty cycles were completed in <NUM> seconds. Amplification was apparent from an increase in fluorescence at about <NUM> cycles or after about <NUM> seconds (<FIG>). The temperature remained near <NUM> while the sample was in air for measurement, limiting the extension rate of the polymerase.

As seen in <FIG>, this reaction has a quantification cycle (Cq) of about <NUM> cycles, but it does not seem to plateau until at least <NUM> cycles. Also, because the reaction was stopped after <NUM> cycles, it is possible that the quantity of amplicon may continue to increase and not plateau until significantly later. Without being bound to theory, it is believed that the increase in primer concentration allows for improved yield and delayed plateau, illustratively <NUM> cycles after Cq, and more illustratively <NUM> cycles or more after Cq.

In this example, a <NUM> bp fragment bracketing an A>G variant (rs#<NUM>) in the interleukin <NUM> beta receptor was amplified with primers CTACAGTGGGAGTCACCTGC (SEQ ID NO:<NUM>) and GGTACTGAGCTGTGAAAGTCAGGTT (SEQ ID NO:<NUM>) to generate the following amplicon: CTACAGTGGGAGTCACCTGCTTTTGCC(A/G)AAGGGAACCTGACTTTCACAGC TCAGTACC (SEQ ID NO:<NUM>). Extreme PCR was performed as described in Example <NUM> using the instrument shown in <FIG>. One µM polymerase, <NUM> each primer and <NUM>% glycerol were used (polymerase to total primer percentage = <NUM>%). In order to increase the temperature for polymerase extension to <NUM>-<NUM>, where the polymerase has higher extension rates, a different positioning protocol was used. After reaching the annealing temperature, instead of immediately positioning in air for monitoring, the sample was transferred to the hot water bath until the extension temperature was reached. Then the sample was positioned in air just above the hot water bath, producing the temperature cycles shown in <FIG>, and enabling faster polymerase extension at optimal temperatures between <NUM> and <NUM>. The <NUM> different genotypes were each amplified by extreme PCR using <NUM> second cycles, completing <NUM> cycles in <NUM> seconds. After extreme PCR, high resolution melting curves were obtained for each genotype on an HR-<NUM> instrument modified to accept LC24 capillaries. <FIG> reveals that all three genotypes were amplified and distinguished, as expected.

The reaction mixtures in Example <NUM> were the same for both the extreme PCR and rapid cycle PCR, except for the amounts of polymerase and primers, and a minor difference in glycerol concentration that apparently caused the shift in Tm seen in <FIG>. In this and all future examples, the glycerol concentration was held at <NUM>% by equalizing its concentration as necessary. For extreme PCR, <NUM> polymerase and <NUM> of each primer were used, while for rapid cycle PCR, <NUM> polymerase and <NUM> of each primer were used. As discussed above, it is believed that faster annealing times provide for improved primer specificity. With this improved specificity, increased concentrations of primers may be used, which is believed to favor primer binding and allow reduced annealing times. Similarly, increased polymerase concentrations favor binding to the annealed primer, and also favor rebinding to the incomplete amplicon if a polymerase falls off prior to complete extension. In addition, because of the higher polymerase concentration, a greater proportion of the primed templates can be extended at once even late in PCR, reducing the number of templates that a single polymerase must extend and reducing the overall extension time.

<FIG> summarizes the results of extreme PCR cycling with various polymerase and primer concentrations. In this example, a <NUM> bp fragment of the interleukin <NUM> beta receptor was amplified with primers GGGAGTCACCTGCTTTTGCC (SEQ ID NO:<NUM>) and TACTGAGCTGTGAAAGTCAGGTTCC (SEQ ID NO:<NUM>) and <NUM> MgCl<NUM>, to generate: GGGAGTCACCTGCTTTTGCCAAAGGGAACCTGACTTTCACAGCTCAGTA (SEQ ID NO:<NUM>). For each extreme PCR reaction, the device shown in <FIG> was used without real time monitoring. The temperature was cycled between <NUM> and <NUM> for <NUM> cycles, for a total reaction time of just under <NUM> seconds (<NUM> second cycles) as shown in <FIG>. Reaction conditions were as discussed in Example <NUM>, except that the amounts of polymerase and primers were varied, as shown in <FIG>. The vertical axis in <FIG> is quantified as the peak of the negative derivative plot of the melting curve, obtained without normalization on the HR-<NUM> instrument. At <NUM> polymerase, virtually no amplification was seen at any level of primer concentration. However, at <NUM> polymerase, discernible levels of amplification were seen at primer concentrations of <NUM> and above. As the polymerase levels increase, so do the amount of amplicon, up to levels of about <NUM>. At <NUM> polymerase, the amount of amplicon plateaued or dropped off, depending on the primer concentration, with a significant drop off at <NUM> at lower primer concentrations. It appears that under these extreme temperature cycling conditions for a <NUM> bp product, the polymerase has a favored concentration range between about <NUM> and <NUM>, and more specifically between <NUM> and <NUM>, depending on the primer concentration.

Similarly, little amplification was seen with primer concentrations of <NUM>. However, amplification was successful at <NUM> primer, with KlenTaq concentrations of <NUM>-<NUM>, and amplification continued to improve with increasing concentrations. Excellent amplification was achieved with primer concentrations of about <NUM>-<NUM> primer. <FIG> shows melting curves for various primer concentrations at <NUM> KlenTaq, while <FIG> verifies the size of the product as the polymerase concentration varies while the primer concentration is held at <NUM>. Despite the high concentrations of polymerase and primers, no nonspecific amplification is seen.

Without being bound to theory, it appears that the ratio between the amount of enzyme and amount of primer is important for extreme PCR cycling, provided that both are above a threshold amount. It is noted that the above amounts are provided based on each primer. Given that the polymerase binds to each of the duplexed primers, the total primer concentration may be the most important. For KlenTaq, suitable ratios are <NUM>-<NUM> (<NUM>-<NUM>% enzyme to total primer concentration), with an illustrative minimum KlenTaq concentration of about <NUM>, and more illustratively about <NUM>, for extreme PCR. The primers may be provided in equimolar amounts, or one may be provided in excess, as for asymmetric PCR. The optimal polymerase:primer percentage may also depend on the temperature cycling conditions and the product size. For example, standard (slow) temperature cycling often uses a much lower polymerase to primer percentage, typically <NUM> (<NUM> U/µl) polymerase (<NUM>) and <NUM>,<NUM> total primer concentration, for a percentage of <NUM>%, over <NUM> times lower than the percentages found effective for extreme PCR.

The same PCR target as in Example <NUM> was amplified with <NUM> polymerase and <NUM> each primer in a <NUM> gauge steel hypodermic needle, to increase thermal transfer and cycling speeds. The polymerase to total primer percentage was <NUM>%. Amplification was performed on the instrument of <FIG> and was completed in <NUM> seconds using <NUM> cycles of <NUM> seconds each (<FIG>), cycling between <NUM> and <NUM>-<NUM>. The maximum heating rate during cycling was <NUM>/s and the maximum cooling rate was <NUM>/s, demonstrating that PCR can occur with ramp rates of greater than <NUM>/s with no holds. Analysis of the products on a <NUM>% NuSieve <NUM>:<NUM> agarose gel revealed strong specific bands of the correct size (<FIG>). The no template control showed no product at <NUM> bp, but did show a prominent primer band similar to the positive samples.

A <NUM> bp fragment of the NQO1 gene was amplified using primers CTCTGTGCTTTCTGTATCCTCAGAGTGGCATTCT (SEQ ID NO:<NUM>) and CGTCTGCTGGAGTGTGCCCAATGCTATA (SEQ ID NO:<NUM>) and the instrument of <FIG> without the real-time components. The polymerase concentration was varied between <NUM> and <NUM>, while each primer concentration was varied between <NUM> and <NUM>. The primers were designed to anneal at higher temperatures (low <NUM>) so that extension at a combined annealing/extension phase would be at a more optimal temperature for the polymerase. Greater polymerization rates at these temperatures were expected to enable amplification of longer products. The cooler water bath was controlled at <NUM> and the end of the annealing/extension phase triggered by time (<NUM> second), rather than temperature. Cycling between <NUM> and <NUM> for <NUM> cycles required <NUM> seconds using <NUM> second cycles (<FIG>). As seen in <FIG>, the sample temperature drops about <NUM> below the annealing/extension temperature while it travels through the air to the hot water bath. <FIG> shows the amount of product amplified by quantifying the melting curves as in <FIG>. Melting curve analysis showed only a single product of Tm <NUM>. Very little product was observed at <NUM> polymerase or at <NUM> each primer. Some amplification occurs at <NUM> each primer, with the best amplification at <NUM>-<NUM> polymerase and <NUM> each primer. At primer concentrations of <NUM>-<NUM>, yield decreases as the polymerase concentration increases, although this was not seen at <NUM> primer concentration. Although the thermal cycling and target length are different from Example <NUM>, the best amplification occurs at polymerase to total primer concentrations of <NUM> to <NUM>%.

Extreme PCR was used to amplify <NUM> bp and <NUM> bp fragments of the BBS2 gene using the instrument shown in <FIG> with real time monitoring. In order to study the effect of product length on extreme PCR and control for possible confounding effects of different primers, the fragments were first amplified from genomic DNA using primers with common <NUM>'-end extensions. For the <NUM> bp fragment the primers were ACACACACACACACACACACACACACACACACACACAAAAATTCAGTGGCAT TAAATACG (SEQ ID NO:<NUM>) and GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAAA CCAGAGCTAAAGGGAAG (SEQ ID NO:<NUM>). For the <NUM> bp fragment the primers were ACACACACACACACACACACACACACACACACACACAAAAAGCTGGTGTCTG CTATAGAACTGATT (SEQ ID NO:<NUM>) and GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAAA GTTGCCAGAGCTAAAGGGAAGG (SEQ ID NO:<NUM>). After standard PCR amplification from genomic DNA, primers and dNTPs were degraded by ExoSAP-IT (Affymetrix, CA), followed by PCR product purification using the QuickStep™ <NUM> PCR Purification Kit (Catalog # <NUM>, Edge BioSystems, Gaithersburg, MD). PCR products were diluted approximately <NUM> million-fold and adjusted to equal concentrations by equalizing the Cq obtained by standard real-time PCR to obtain a Cq of <NUM> cycles (approximately <NUM>,<NUM> copies/<NUM>µl reaction).

Extreme PCR was performed on <NUM>,<NUM> copies of the amplified templates in a total volume of <NUM>µl using the common primers ACACACACACACACACACACACACACACACACACACAAAAA(SEQ ID NO:<NUM>) and GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAAA (SEQ ID NO:<NUM>) each at <NUM> with <NUM> polymerase and <NUM>% glycerol. The <NUM> bp BBS2 fragment resulted in a <NUM> bp product requiring extension of <NUM> or <NUM> bases (depending on the primer), while the <NUM> bp BBS2 fragment resulted in a <NUM> bp PCR product requiring extension of <NUM> or <NUM> bases. Specific amplification was verified on agarose gels and by melting analysis. The extreme PCR temperature profile used for the <NUM> bp product is shown in <FIG>, which included a <NUM> second combined annealing/extension at <NUM> and denaturation at <NUM>. Also performed was a <NUM> second annealing/extension phase at the same temperature (trace not shown). Real time PCR results for these amplifications are shown in <FIG>, revealing about a <NUM> cycle shift to higher Cq with the <NUM> second extension as compared to the <NUM> second extension, presumably reflecting a decrease in efficiency as the extension time is decreased. The extreme PCR temperature profile used for the <NUM> bp product is shown in <FIG>, showing a <NUM> second combined annealing/extension at <NUM> and denaturation at <NUM>. Also performed was a <NUM> second annealing/extension phase at the same temperature (trace not shown). Real time PCR results for these amplifications are shown in <FIG>, revealing about a <NUM> cycle shift to higher Cq with the <NUM> second extension as compared to the <NUM> second extension, presumably reflecting a decrease in efficiency as the extension time is decreased.

Quantitative performance of PCR was assessed using the real-time instrument of <FIG> for the <NUM> bp fragment of NQO1 of Example <NUM> and the <NUM> bp fragment of KCNE1 of Example <NUM> using a dilution series of human genomic DNA, using <NUM> KlenTaq and <NUM> each primer for NQO1 and <NUM> KlenTaq and <NUM> each primer for KNCE1. With a dynamic range of at least <NUM> decades, as seen in <FIG>, the amplification efficiencies calculated from the standard curves were <NUM>% for NQO1 and <NUM>% for KCNE1. Control reactions without template did not amplify after <NUM> cycles and single copy replicates (mean copy number of <NUM> copies per reaction) were similar in amplification curve shape and intensity to higher concentrations (<FIG> and <FIG>). At a mean copy number of <NUM> copies/reaction, <NUM> reactions were positive out of <NUM> (combining both NQO1 and KCNE1 trials), with a calculated expectation of <NUM> copies/reaction by binomial expansion.

The extension time required for different product lengths using real-time PCR (<FIG>). To control for the possible confounding effects of different primers, synthetic templates of <NUM>-<NUM> bp using the following common high Tm (<NUM>) primers: ACTCGCACGAACTCACCGCACTCC (SEQ ID NO: <NUM>) and GCTCTCACTCGCACTCTCACGCACA (SEQ ID NO: <NUM>).

Optimal concentrations of primers and polymerase were first determined for the intermediate length <NUM>-bp product using a <NUM> second combined annealing/extension segment with <NUM> seconds per cycles (<FIG>). Identical primer (<NUM>) and polymerase (<NUM>) concentrations were then used for all product lengths and minimum extension times were determined (<FIG>). Depending on the product length, increased extension times resulted in decreased fractional quantification cycles (Cq) until no further change was observed, reflecting the minimum extension time required for efficient PCR. For example, amplification curves using the KAPA2G™ FAST polymerase (Kapa Biosystems) for the <NUM> bp product are shown in <FIG>. The minimum extension time using KAPA2G FAST polymerase was <NUM>, compared to <NUM> using KlenTaq1 (a deletion mutant of Taq polymerase, AB Peptides). When the identity of the polymerase is kept constant, longer products required longer extension times (<FIG>). For KlenTaq1 polymerase, about <NUM> second is required for each <NUM> bps, while for KAPA2G FAST, <NUM> second is required for each <NUM> bp. It is noted that these two polymerases were chosen because they are commercially available at sufficient concentrations, while most other polymerases are not commercially available at such high concentrations. It is understood that the required time for extension depends directly and linearly with the length to be extended, and inversely with the concentration of polymerase and the polymerase speed. A proportionality constant (k2) can be defined that relates these <NUM> parameters:<MAT>.

Extreme PCR times can also be reduced with high Mg++ concentrations. A <NUM> bp fragment of AKAP10 was amplified with primers: GCTTGGAAGATTGCTAAAATGATAGTCAGTG (SEQ ID NO:<NUM>) and TTGATCATACTGAGCCTGCTGCATAA (SEQ ID NO:<NUM>), to generate the amplicon
<IMG>.

Each reaction was in a <NUM>µl volume with time based control (<NUM> seconds in a <NUM> water bath, <NUM>-<NUM> seconds in a <NUM> water bath) for <NUM> cycles using <NUM>-<NUM> MgCl<NUM>. The sample volume was <NUM>µl, with <NUM> ng human genomic DNA, <NUM> primers, and <NUM> polymerase. Using a <NUM> second per cycle protocol, when the MgCl<NUM> was <NUM>-<NUM>, no product was observed on melting curves (<FIG>) or gels (<FIG>). Minimal product was present at <NUM>, but a large amount of product was observed after amplification with <NUM>-<NUM> MgCl<NUM>. At <NUM> MgCl<NUM>, no products were observed on melting curves (<FIG>) or gels (<FIG>) with cycle times of <NUM> seconds, but large amounts of product were present at cycle times of <NUM> seconds, <NUM> seconds, and <NUM> seconds, demonstrating that specific, high yield <NUM> bp products can be obtained in PCR performed in under <NUM> seconds (<NUM> cycles in <NUM> seconds). Thus, illustrative Mg++ concentrations are at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or more, and it is understood that these illustrative Mg++ concentrations may be used with any of the embodiments described herein.

The high concentrations of primer and polymerase used in extreme PCR can have detrimental effects when used at slower cycling speeds. Non-specific products were obtained on rapid cycle or block based instruments that are <NUM>- or <NUM>-fold slower, respectively. <FIG> shows the results comparing amplification of the AKAP10 <NUM> bp product used in Example <NUM>, wherein amplification was performed using <NUM> of each primer, <NUM> KlenTaq and <NUM> ng human genomic DNA for <NUM> cycles using: (<NUM>) extreme PCR with set times of <NUM> at <NUM>° and <NUM> seconds at <NUM>°giving a total time of approximately <NUM> seconds, (<NUM>) Rapid cycle PCR (Roche LightCycler) using set times of <NUM> at <NUM>°for an initial denaturation, followed by cycles of <NUM>° for <NUM> seconds, and <NUM>° for <NUM> seconds, giving a total time of approximately <NUM> minutes, and (<NUM>) Legacy (block) temperature cycling (BioRad CFX96) with a <NUM> initial denaturation at <NUM>°, following by temperature cycling for <NUM> at <NUM>°and <NUM> at <NUM>° with a total time of approximately <NUM> minutes. As can be seen, even the rapid cycling of the LightCycler resulted in quite a bit of non-specific amplification, while the extreme cycling conditions resulted in a single melting peak and minimal non-specific amplification on the gel.

It also noted that the yield is enhanced in extreme PCR, resulting from high primer and polymerase concentrations. Extreme PCR produced over <NUM>-fold the amount of product compared to rapid cycle PCR, using quantitative PCR for comparison (data not shown).

Examples <NUM> - <NUM> were all performed using one or more of the devices described in <FIG>, or minor variations on those configurations. However, it is understood that the methods and reactions described herein may take place in a variety of instruments. The water baths and tubes used in these examples allow for sufficiently rapid temperature change to study the effects of elevated concentrations of primers and polymerase. However, other exampless may be more suitable commercially. Microfluidics systems, with low volume and high surface area to volume ratios, may be well suited to extreme PCR. Such systems allow for rapid temperature changes required by the high concentrations of primers and polymerase that are used in extreme PCR. Microfluidics systems include micro-flow systems (<NUM>, <NUM>) that incorporate miniaturized channels that repeatedly carry the samples through denaturation, annealing, and extension temperature zones. Some of these systems have already demonstrated effective PCR with cycle times as fast as <NUM> seconds for lower complexity targets. It is expected that more complex targets may be amplified in such systems if the polymerase is provided at a concentration of at least <NUM> and primers are each provided at a concentration of at least <NUM>. Stationary PCR chips and PCR droplet systems (<NUM>) may also benefit from increased primer and probe concentrations, as the volumes may be as small as <NUM> nl or smaller and may be low enough to permit very fast cycling. It is understood that the exact instrumentation is unimportant to the present invention, provided that the instrumentation temperature cycles fast enough to take advantage of increased primer and polymerase concentrations without suffering from the loss of specificity associated with higher primer concentrations at slower cycle speeds.

While the above examples all employ PCR, it is understood that PCR is illustrative only, and increased primer and enzyme concentrations combined with shorter amplification times are envisioned for nucleic acid amplification methods other than PCR. Illustrative enzymatic activities whose magnitude may be increased include polymerization (DNA polymerase, RNA polymerase or reverse transcriptase), ligation, helical unwinding (helicase), or exonuclease acitivity (<NUM>' to <NUM>' or <NUM>' to <NUM>'), strand displacement and/or cleavage, endonuclease activity, and RNA digestion of a DNA/RNA hybrid (RNAse H). Amplification reactions include without limitation the polymerase chain reaction, the ligase chain reaction, transcription medicated amplification (including transcription-based amplification system, self-sustained sequence replication, and nucleic acid sequence-based amplification), strand displacement amplification, whole genome amplification, multiple displacement amplification, antisense RNA amplification, loop-mediated amplification, linear-linked amplification, rolling circle amplification, ramification amplification, isothermal oligonucleotide amplification, helicase chain reaction, and serial invasive signal amplification.

In general, as the enzyme activity is varied, the amplification time varies inversely by the same factor. For reactions that include primers, as the primer concentration is varied, the amplification time varies inversely by the same factor. When both primers and enzymes are required for amplification, both enzyme and primer concentrations should be varied in order to maximize the reaction speed. If primer annealing occurs in a unique segment of the amplification cycle (for example, a unique temperature during <NUM>-temperature PCR), then the time required for satisfactory completion of primer annealing in that segment is expected to be inversely related to the primer concentration. Similarly, if the enzyme activity is required in a unique segment of the amplification cycle (for example, a unique temperature during <NUM>-temperature PCR), then the time required for satisfactory completion of the enzymatic process in that segment is expected to be inversely related to the enzyme concentration within a certain range. Varying the primer or enzyme concentrations can be used to change the required times of their individual segments, or if both occur under the same conditions (such as in <NUM>-temperature PCR or during an isothermal reaction process), it is expected that a change in both concentrations may be necessary to prevent one reaction from limiting the reaction speed. Increased Mg++ concentration can also be used in combination with increased enzyme and primer concentrations to further speed amplification processes. Higher Mg++ concentrations both increase the speed of primer annealing and reduce the time for many enzymatic reactions used in nucleic acid amplification.

Higher concentrations of Mg++, enzymes, and primers are particularly useful when they are accompanied by shorter amplification times or segments. When higher concentrations are used without shortening times, non-specific amplification products may occur in some cases, as the "stringency" of the reaction has been reduced. Reducing the amplification time or segment time(s) introduces a higher stringency that appears to counterbalance the loss of stringency from increased reactant concentrations. Conversely, reagent costs can be minimized by reducing the concentration of the reactants if these lower concentrations are counterbalanced by increased amplification times or segment times.

Increasing polymerase concentrations can reduce the time necessary for long-range PCR, illustratively where the target is <NUM>-<NUM> kb. Typically, <NUM> to <NUM> extension periods are used to amplify large targets because the target is so long that such times are needed: <NUM>) for the polymerase to complete extension of a single target, and <NUM>) for enzyme recycling to polymerize additional primed templates. This recycling of polymerase is not needed at the beginning of PCR, when the available enzyme outnumbers the primed template molecules. However, even before the exponential phase is finished, the number of polymerase molecules often becomes limiting and enzyme recycling is necessary. By increasing the concentration of the polymerase, the required extension period can be reduced to less than <NUM> minutes and possibly less than <NUM> minutes, while maintaining increased yield due to the high primer concentration. Although the actual enzyme speed is not increased, less recycling is necessary, affecting the minimum time required, approximately in a linear fashion with the enzyme concentration.

Cycle sequencing times can also be reduced by increasing primer and polymerase concentrations. Typically, in standard cycle sequencing primer concentrations are <NUM> and the combined annealing/extension period is <NUM> at <NUM>-<NUM> degrees C. By increasing the primer and polymerase concentrations by <NUM>-fold, the time required for annealing/extension can be reduced approximately <NUM>-fold. In both long PCR and cycle sequencing, the expected time required is inversely proportional to the polymerase or primer concentration, whichever is limiting.

PCR of fragments with ligated linkers that are used as primers in preparation for massively parallel sequencing can be completed in much less time than currently performed by combining extreme temperature cycling with higher concentrations of primers, polymerase, and/or Mg++.

In all of the above applications, it is expected that the specificity of the reaction is maintained by shorter amplification times. Although high primer and polymerase concentrations are expected by those well versed in the art to cause difficulty from non-specific amplification, minimizing the overall cycle time and/or individual segment times results in high specificity and efficiency of the PCR.

Specific conditions for extreme PCR are shown in Table <NUM>. All data are presented except for the simultaneous optimization experiments for polymerase and primer concentrations for <NUM> of the targets. In Table <NUM>, the quantitative relationships between variables are detailed. The inverse proportionality that relates the required annealing time to the primer concentration is approximately constant (k1) and defined by the equation (Required annealing time) = k1/[primer]. Using a range of typical values for these variables under conditions of legacy (standard) PCR, rapid cycle PCR, and extreme PCR produces ranges for the inverse proportionality constant that largely overlap (legacy <NUM> - <NUM>, rapid cycle <NUM> - <NUM>, and extreme <NUM> - <NUM>). Because of this constant inverse proportionality, desired annealing times outside of those currently performed can be used to predict the required primer concentrations for the desired time. For example, using a constant of <NUM> (s * µM), for an annealing time of <NUM>, a primer concentration of <NUM> can be calculated. Conversely, if a primer concentration of <NUM> were desired, the required annealing time would be <NUM> seconds. Although these conditions are outside the bounds of both legacy and extreme PCR, they predict a relationship between primer concentrations and annealing times that are useful for PCR success. Reasonable bounds for k1 across legacy, rapid cycle and extreme PCR are <NUM> - <NUM> (s x µM), more preferred <NUM> - <NUM> (s x µM) and most preferred <NUM> - <NUM> (s x µM).

Similar calculations can be performed to relate desired extension times to polymerase concentration, polymerase speed, and the length of the product to be amplified. However, because of many additional variables that affect PCR between legacy, rapid cycle and extreme PCR (polymerase, Mg++, buffers), performed in different laboratories over time, it may be best to look at the well-controlled conditions of extreme PCR presented here to establish an inverse proportionality between variables. This allows a quantitative expression between polymerase concentration, polymerase speed, product length, and the required extension time under extreme PCR conditions. The defining equation is (Required Extension Time) = k2(product length)/([polymerase]*(polymerase speed)). The experimentally determined k2 is defined as the proportionality constant in the above equation under conditions of constant temperature, Mg++, type of polymerase, buffers, additives, and concentration of dsDNA dye. For the <NUM> extreme PCR targets with two dimensional optimization of [polymerase] and [primer], the [polymerase] at the edge of successful amplification can be discerned across primer concentrations and related to the other <NUM> variables. As shown in Table <NUM>, the values of k2 for these <NUM> different targets vary by less than a factor of <NUM>, from which it is inferred that k2 is a constant and can be used to predict one variable if the others are known. The required extension time is proportional to the extension length (product length minus the primer length) and inversely proportional to the polymerase speed and concentration of polymerase. k2 has units of (<NUM>/µM) and an optimal value for the extreme PCR conditions used here of <NUM> (<NUM>/µM) with a range of <NUM> - <NUM> (<NUM>/µM). Similar values for k2 could be derived for other reaction conditions that vary in polymerase type, Mg++ concentration or different buffer or dye conditions.

Claim 1:
A method for amplifying a target nucleic acid sequence in a biological sample during amplification comprising the steps of:
adding a thermostable polymerase and primers configured for amplification of the target nucleic acid sequence to the biological sample, wherein the ratio of polymerase to total primer concentration is <NUM>-<NUM>: <NUM>, and the polymerase concentration is at least <NUM>; and
amplifying the target nucleic acid sequence by polymerase chain reaction by thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature through a plurality of amplification cycles using an extreme temperature cycling profile wherein each cycle is completed in less than <NUM> seconds.