DESCRIPTION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT Application No. PCT/US2017/018748, filed Feb. 21, 2017, entitled “DEVICES AND METHODS FOR RAPID PCR”, which claims the benefit of and priority to U.S. Provisional Application Nos. 62/298,311, filed Feb. 22, 2016, U.S. Prov. App. Ser. No. 62/330,701, filed May 2, 2016, and U.S. Prov. App. Ser. No. 62/409,829, filed Oct. 18, 2016. All the aforementioned applications are incorporated by reference herein in their entirety.

 

GOVERNMENT INTEREST

This invention was made with government support under W911QY-13-D-0080 awarded by the U.S. Department of Defense. The government has certain rights in the invention.

BACKGROUND1. Technical Field

Embodiments of the present disclosure relate generally to methods and devices for amplifying nucleic acids.

 

2. Background

In the United States, Canada, and Western Europe infectious disease accounts for approximately 7% of human mortality, while in developing regions infectious disease accounts for over 40% of human mortality. Infectious diseases lead to a variety of clinical manifestations. Among common overt manifestations are fever, pneumonia, meningitis, diarrhea, and diarrhea containing blood. While the physical manifestations suggest some pathogens and eliminate others as the etiological agent, a variety of potential causative agents remain, and clear diagnosis often requires a variety of assays be performed. Traditional microbiology techniques for diagnosing pathogens can take days or weeks, often delaying a proper course of treatment.

 

In recent years, the polymerase chain reaction (PCR) has become a method of choice for rapid diagnosis of infectious agents. PCR can be a rapid, sensitive, and specific tool to diagnose infectious disease. A challenge to using PCR as a primary means of diagnosis is the variety of possible causative organisms or viruses and the low levels of organism or virus present in some pathological specimens. It is often impractical to run large panels of PCR assays, one for each possible causative organism or viruses, most of which are expected to be negative. The problem may be exacerbated when pathogen nucleic acid is at low concentration and requires a large volume of sample to gather adequate reaction templates. In some cases there is inadequate sample to assay for all possible etiological agents. A solution is to run “multiplex PCR” wherein the sample is concurrently assayed for multiple targets in a single reaction. While multiplex PCR has proven to be valuable in some systems, shortcomings exist concerning robustness of high level multiplex reactions and difficulties for clear analysis of multiple products. To solve these problems, the assay may be subsequently divided into multiple secondary PCRs. Nesting secondary reactions within the primary product increases robustness. Closed systems such as the FilmArray® (BioFire Diagnostics, LLC, Salt Lake City, Utah) reduce handling, thereby diminishing contamination risk.

 

PCR may be conceptually divided into 3 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 3 temperatures over 3 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 3 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 FIGS. 5a-5b . 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 2 temperatures are needed.

 

When PCR was first popularized in the late 1980s, the process was slow. A typical protocol was one minute for denaturation at 94° C., two minutes for annealing at 55° C., and three minutes for extension at 72° C. When the time for transition between temperatures was included, 8 minute cycles were typical, resulting in completion of 30 cycles in four 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 (<100 bps), 100% 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 one-hour PCR is rapid compared to four hours, but slow compared to 15 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 1994 as 30 cycles completed in 10-30 minutes, resulting in cycles of 20-60 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 1990s 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.

 

Rapid protocols use momentary or “0” 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.

Conclusions from this early work were: 1) denaturation of PCR products is very rapid with no need to hold the denaturation temperature, 2) annealing of primers can occur very quickly, particularly with higher primer concentrations, and annealing temperature holds may not be necessary, and 3) 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 as long as the temperature was controlled precisely.

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 3-step cycling (denaturation, annealing, and extension) to 2-steps (denaturation and a combined annealing/extension step) also simplifies the temperature cycling requirements. Two-step cycling can, however, compromise polymerase extension rates if the combined annealing/extension step is performed at temperatures lower than the 70 to 80° C. temperature optimum where the polymerase is most active, particularly with fast ramp rates. Polymerase extension rates are log-linear with temperature until about 70-80° C., with a reported maximum of 60-120 bp/s.

Even with protocol variations, amplification efficiency and yield are often poor when cycle times are <20 seconds when compared to control reactions. These efforts towards faster PCR appear dominated by engineering with little focus on biochemistry. As cycle times decrease from 20 seconds towards 2 seconds, PCR yield decreases and finally disappears, reflecting a lack of robustness even with simple targets at high copy number.

 

Recently, a system has been reported using thin walled capillaries and water baths to thermocycle or using induction heating (US 2015/0118715; WO 2015/069743, herein incorporated in their entireties by reference) at speeds of less than 10 seconds per cycle, and in some embodiments less than one second per cycle. Adjustments in chemistry for this “extreme PCR”, wherein polymerase and primer concentration are increased, permit the polymerase chain reaction to proceed at such fast rates.

In one example of extreme PCR, the polymerase is provided at a concentration of at least 0.5 μM and primers are each provided at a concentration of at least 2 μM, and in some examples the primer concentration is 2.5 μM or more. By non-limiting example, annealing time may be defined by annealing time=k1/[primer], wherein k1 is a constant and [primer] is the concentration of each primer, and time at the elongation temperature may be defined by elongation time=k2(extension length)/([polymerase]*(polymerase speed)), wherein k2 is a proportionality constant, [polymerase] is the concentration of the polymerase, and polymerase speed is a rate of polymerase incorporation of bases in nucleotides. In another example of extreme PCR, the polymerase to primer ratio is illustratively (about 0.03 to about 0.4 polymerase):(total primer concentration), and the polymerase concentration is at least 0.5 μM. It is noted that polymerase Unit definitions can be confusing. For native Taq polymerase, 0.4 U/10 μl is about 1.5 nM under typical rapid cycling conditions.

 

While improvements in chemistry are reported in WO 2013/177429, the device requires large water baths, and it is ideally placed inside a water-resistant cabinet. Rapid temperature cycling having cycle times of 10 seconds or less using the chemistry of WO 2013/177429 in commercial instrumentation would be desired. It would also be desirable to perform such rapid temperature cycling in a closed container.

The present invention addresses various issues relating to fast PCR, including contamination risks by providing devices, kits, and methods for fast PCR, illustratively in a closed container.

 

BRIEF SUMMARY

Described herein are devices (instruments and systems) and methods for rapid amplification of nucleic acids in a flexible sample container. In an illustrative embodiment, a flexible sample container may include a first-stage chamber fluidly connected to a second-stage reaction zone, the second-stage reaction zone comprising a plurality of second-stage reaction wells. Conventionally, thermocycling devices for nucleic acid amplification include a heater that raises and lowers the temperature of a sample to accomplish a number of cycling of annealing, elongation, and denaturation. In contrast, the devices described herein may include a temperature control element that includes a first temperature zone and a second temperature zone. In one example, the temperatures of the first temperature zone and the second temperature zone may be held constant, wherein, illustratively, one zone may be held at an elongation temperature and the other zone may be held at a denaturation temperature. Alternatively, the first temperature zone and the second temperature zone may be thermally cycled in a limited range (e.g., a 5-20° C. range). The temperature control unit and various portions of a flexible sample container may be aligned to accomplish temperature cycling for nucleic acid amplification. Other components of the device described in detail herein may work cooperatively with the temperature control unit to accomplish thermal cycling. Because the temperatures of the first temperature zone and the second temperature zone are held constant or are cycled in a narrow range, the temperature changes for nucleic acid amplification can be accomplished more quickly.

In one embodiment, a method of thermal cycling is described. The method includes

(a) providing a sample container comprising a first-stage chamber fluidly connected to a second-stage reaction zone, the second-stage reaction zone comprising a plurality of second-stage reaction wells,

(b) introducing a sample into the sample container, and

(c) inserting the sample container into an instrument, the instrument comprising a temperature control element. The method further includes

(d) aligning the temperature control element and the first-stage chamber to effect thermal cycling of the sample in the first-stage chamber,

(e) after effecting thermal cycling of the sample in the first-stage chamber, moving at least a fraction of a product derived from the sample from the first-stage chamber into the plurality of second-stage reaction wells in the second-stage reaction zone, and

(f) aligning the temperature control element and the second-stage reaction zone to effect thermal cycling of the fraction of the sample in the second-stage reaction zone.

In one aspect, the temperature control element may include one or more heater or cooler devices such as, but not limited to, Peltier devices, resistance heaters, induction heaters, electromagnetic heaters, thin film heaters, printed element heaters, positive temperature coefficient heaters, and combinations thereof. In one aspect, the temperature control element comprises a first temperature zone and a second temperature zone, wherein the first temperature zone is hotter than the second temperature zone.

In one aspect, aligning the temperature control element and the second-stage reaction zone in step (f) includes repeatedly translating the temperature control element relative to the second-stage reaction zone. In another aspect, aligning the temperature control element and the second-stage reaction zone in step

(f) includes repeatedly translating the second-stage reaction zone relative to the temperature control element.

In one aspect, the instrument further includes a wiper element, and step (d) further includes aligning the wiper element with the temperature control element and the first-stage chamber such that rotational movement of the wiper element moves a first portion of the sample from thermal control of the first temperature zone to thermal control of the second temperature zone, while simultaneously moving a second portion of the sample from thermal control of the second temperature zone to thermal control of the first temperature zone.

In one aspect, the sample container includes a sample preparation zone fluidly connected to the first-stage chamber, and, prior to step (d), the method further includes: introducing the sample into the sample preparation zone, contacting the sample preparation zone with a lysis apparatus to produce a lysate, recovering nucleic acids from the lysate, and moving the recovered nucleic acids into the first-stage chamber. In another aspect, recovering nucleic acids from the sample further includes: contacting the lysate with a plurality of magnetic beads, deploying a magnet to separate the magnetic beads from the lysate, washing the magnetic beads, recapturing the magnetic beads with the magnet, contacting the magnetic beads with an elution buffer to release the nucleic acids from the magnetic beads, and recapturing the magnetic beads with the magnet and separating the eluted nucleic acids from the magnetic beads.

In one aspect, the step (f) of the method includes aligning the second nucleic acid amplification zone with the first temperature zone and then the second temperature zone of the temperature control element to thermocycle the sample in the second nucleic acid amplification zone.

In one aspect, the steps of the method may be are completed in 20 minutes or less, 15 minutes or less, or, preferably, 10 minutes or less. In another aspect, each thermal cycle of the first and second nucleic acid amplification zones is completed in 8 seconds or less, 6 seconds or less, or, preferably, 4 seconds or less.

In another embodiment, an instrument for thermocycling a sample provided in a flexible sample container is described. The instrument includes a first heater adjacent to a first portion of the flexible sample container for adjusting a first portion of the sample to a first temperature, a second heater adjacent to a second portion of the flexible sample container for adjusting a second portion of the sample to a second temperature, the second temperature different from the first temperature, and a wiper element that moves the first portion of the sample to the second portion of the flexible sample container while moving the second portion of the sample to the first portion of the flexible sample container such that portions of the sample are under control of each of the heaters simultaneously. In one aspect, the wiper element includes a blade that divides the sample into at least two discrete sections comprising at least a first section and a second section, such that the first portion is contained in the first section and the second portion is contained in the second section. In one aspect, the wiper element repeatedly moves portions of the sample to opposite portions of the sample container to thermocycle the sample.

In an embodiment, another instrument for thermocycling a sample is described. The instrument includes a receptacle for positioning a flexible sample container having at least a first reaction chamber in the instrument, and a heater assembly that includes a first heater element and a second heater element, and a translator mechanically coupled to at least one of the receptacle, the flexible sample container, or the heater assembly to laterally align the first reaction chamber relative to the first and second heater elements of the heater assembly such that the first reaction chamber is under temperature control of at least one of the first or the second heater elements. Wherein the instrument is configured to repeatedly align the first reaction chamber with the first heater element and then the second heater element for thermocycling a fluid sample in the at least one reaction chamber.

In yet another embodiment, a thermocycling system is described. The thermocycling system includes a receptacle for receiving a flexible sample container therein, the flexible sample container having a first-stage chamber including a sample to be thermocycled therein, a heater element that includes at least a first temperature zone and a second temperature zone positioned on a first side of the flexible sample container, and a wiper element positioned on a second side of the sample container, the wiper element being configured for contacting the first-stage chamber to divide the sample into at least a first portion and a second portion. Wherein one or more of the receptacle, the heater element, the wiper element, or the flexible sample container are movable such that movement aligns the first-stage chamber relative to the wiper element and the first and second temperature zones of the heater element, and wherein the wiper element is configured to rotate adjacent to the first-stage chamber to move the first portion of the sample to the second portion while moving the second portion of the sample to the first portion such that the first and second portion are under temperature control of the first and second temperature zones of the heater element.

In yet another embodiment, another instrument for thermocycling a sample is described. The instrument includes a receptacle for positioning a flexible sample container in the instrument. In one embodiment, the flexible sample container includes a first-stage chamber and a second-stage reaction chamber having an array of second-stage reaction wells. The instrument further includes a heater element comprising at least a first temperature zone and a second temperature zone, wherein one or more of the receptacle, the sample container, or the heater element are movable such that movement aligns the first-stage chamber and the second-stage reaction chamber relative to the heater element, and wherein the receptacle and the heater element are configured to allow the heater element to heat first the first-stage chamber and second the second-stage reaction chamber to effect thermal cycling and nucleic acid amplification in the first-stage chamber and then the second-stage reaction chamber.

In one aspect, the instrument further includes a wiper element having at least one blade configured to contact the first-stage chamber and divide the first-stage chamber into at least a first volume and a second volume. Wherein the heater element is aligned beneath the first-stage chamber such that the first volume is positioned over the first temperature zone and a second volume is positioned over the second temperature zone, and wherein the wiper element is configured to rotate to move the first volume to the second temperature zone while moving the second volume to the first temperature zone such that first and second volumes are under control of each of the temperature zones.

In yet another embodiment, yet another instrument for thermocycling a sample is described. The instrument includes a receptacle for positioning a flexible sample container in the instrument. In one aspect, the flexible sample container includes at least one reaction chamber. The instrument further includes a heater assembly that includes a first heater element, a second heater element, and a third heater element. Wherein the first and third heater elements are held at a temperature higher than the second heater element, and wherein the instrument is configured to align the at least one reaction chamber with the first heater element, the second heater element, and the third heater element for thermocycling a fluid sample in the at least one reaction chamber. In one aspect, the first and third heater elements are set at a temperature in a range of about 90° C.-110° C. and the second heater element is set to a temperature of about 55° C.-65° C.

In still yet another embodiment, a polymerase chain reaction method using the instrument described in the previous paragraph is included. The method includes (a) providing the sample container comprising the at least one reaction chamber, (b) introducing a sample into the reaction chamber, wherein the sample includes a target nucleic acid, at least one primer for amplifying the target nucleic acid, and a thermostable DNA polymerase, (c) inserting the sample container into the instrument, (d) aligning the first heater element with the reaction chamber, then aligning the second heater element with the reaction chamber, and then aligning third heater element with the reaction chamber. Wherein the first and third heater elements are set at a denaturation temperature and the second heater is set at an annealing temperature, and wherein step (d) comprises one cycle of denaturation, annealing, and elongation/denaturation. The method further includes repeating step (d) for a selected number of cycles (at least once) to accomplish nucleic acid amplification.

In still yet another embodiment, yet another instrument for amplifying nucleic acids is described. The nucleic acids are provided in a sample container wherein the nucleic acids are configured in an array. The instrument includes an opening for receiving the sample container, a plurality of heaters, each heater provided at a different temperature, and a mover for moving the heaters sequentially to a position adjacent to the opening, such that only the heater in the position controls temperature of the nucleic acids in the sample container. In one aspect, the sample container further includes components for amplifying nucleic acids, and the plurality of heaters includes at least a first heater at an annealing temperature and a second heater at a denaturation temperature. In another aspect, the plurality of heaters further includes a third heater at an elongation temperature.

In still yet another embodiment, yet another method for performing PCR is described. The method includes (a) providing a PCR mixture in a sample vessel the PCR mixture having a volume, (b) thermocycling for a first number of cycles, each of the first number of cycles having a first cycle time, (c) reducing the volume of the PCR mixture in the sample container to a second volume, the second volume being smaller than the first volume, and (d) thermocycling for a second number of cycles, each of the second number of cycles having a second cycle time, the second cycle time being shorter than the first cycle time. In one aspect, the method further includes (e) reducing the volume of the PCR mixture in the sample container to a third volume, the third volume being smaller than the second volume, and (f) thermocycling for a third number of cycles, each of the second number of cycles having a third cycle time, the third cycle time being shorter than the second cycle time.

In still yet another embodiment, yet another method of amplifying nucleic acids in a sample is described. The method includes introducing a fluid sample into a sample compartment of a container, the fluid sample containing a target nucleic acid and reagents for amplifying the target nucleic acid, introducing the container into a heating apparatus, the heating apparatus including a first heater, a second heater, and a mover (e.g., a wiper or a squisher) for moving the fluid sample within the sample compartment, the first heater being set to a first temperature and the second heater being set to a second temperature, the first temperature being greater than the second temperature, a first part of the sample compartment being disposed proximal to the first heater so that the first heater exhibits thermal control on the first part of the sample compartment and a second part of the sample compartment being disposed proximal to the second heater so that the second heater exhibits thermal control on the second part of the sample compartment, and selectively moving at least a portion of the fluid sample between the first part of the sample compartment and the second part of the sample compartment, such that portions of the sample are under control of each of the heaters simultaneously.

In still yet another embodiment, yet another method of amplifying nucleic acids in a sample is described. The method includes (a) introducing a fluid sample into a first compartment of a container, the fluid sample comprising a target nucleic acid and reagents for amplifying the target nucleic acid, wherein the first compartment is under control of a first heater that is set at a temperature that is below an annealing temperature (a low annealing temperature), (b) raising the temperature of the first heater to the annealing temperature, (c) moving the fluid sample into a second compartment of a container, wherein the second compartment is under control of a second heater that is set at a temperature that is above an elongation temperature (a high elongation temperature), and lowering the temperature of the first heater to the low annealing temperature subsequent to moving the fluid sample into the second compartment, (d) lowering the temperature of the second heater to the elongation temperature, (e) raising the temperature of the second heater to at least a denaturation temperature, and (f) repeating steps (a) through (e). In one aspect, when step (a) is repeated, the temperature of the second heater is moved to the high elongation temperature. In another aspect, step (e) includes raising the temperature of the second heater to a temperature above the denaturation temperature, and step (a) is repeated as soon as the fluid sample reaches the denaturation temperature.

In still yet another embodiment, yet another instrument is provided for thermocycling a sample provided in a flexible sample container having at least a first-stage chamber. In one embodiment, the instrument includes a receptacle for positioning the flexible sample container in the instrument and a heater element that includes at least a first temperature zone and a second temperature zone. Wherein one or more of the receptacle or the heater element are movable such that movement positions the first-stage chamber relative to the first temperature zone and the second temperature zone of the heater element, and wherein the receptacle and the heater element are configured to allow the first temperature zone and the second temperature zone to control the temperature of the first-stage chamber to effect thermal cycling and nucleic acid amplification therein.

In one aspect, the heater element may be positionable beneath the first-stage chamber such that a first portion of the sample may be positioned over the first temperature zone and a second portion of the sample may be positioned over the second temperature zone. The instrument further includes a mixing component having a wiper with at least one blade that may be configured to contact the first-stage chamber and to rotate to move the first portion of the sample to the second temperature zone while moving the second portion of the sample to the first temperature zone such that first and second portions of the sample may be under control of each of the temperature zones to effect thermal cycling and nucleic acid amplification of the contents of the first-stage chamber.

In another aspect, the flexible sample container may include a sample preparation zone where, for example, cells in a sample may be lysed and nucleic acids may be recovered for amplification in the first-stage chamber. In another aspect, the flexible sample container may include a second nucleic acid amplification zone downstream from the first-stage chamber. The second nucleic acid amplification zone may be configured to receive a portion of a diluted amplification product from the first-stage chamber and further amplify the diluted amplification product in an array of wells with specific primer sets selected for assaying the contents of the sample.

In still yet another embodiment, yet another instrument for amplifying nucleic acids in a sample is described. The instrument includes an opening for receiving a flexible sample container, the flexible sample container comprising at least one reaction zone, and a plurality of heaters, wherein each of the heaters is configured to be set at a different temperature, and wherein the heaters are positioned on a substantially planar mount such that each heater can be sequentially aligned with the at least one reaction zone to heat or cool a sample therein. In one aspect, the substantially planar mount comprises a circular mount that is configured to be rotated adjacent to the flexible sample container.

The instruments and methods described herein may include or be configured for automated sample preparation, first-stage PCR, second-stage PCR, and automated analysis of the second-stage PCR product in the flexible sample container. For instance, one or more of the receptacle, the heater element, or the mixing component may be positionable relative to the flexible sample container for heated and chilled sample preparation, first-stage PCR, and second stage PCR.

In still yet another embodiment a flexible sample container is described. The flexible sample container includes a reaction chamber having an array of reaction wells, wherein each of the wells of the array is fluidly connected to a selectively openable and selectively sealable fill channel and fill hole. In one aspect, the fill hole is fluidly connected to a well-filling channel that flows adjacent to and over a well of the array, wherein the well filling channel is formed by making a cutout in a layer adjacent to the well of the array and another cutout in a second layer. In another aspect, the fill hole is fluidly connected to a well filling channel that flows into a well of the array, wherein the well-filling channel is formed by making a cutout in a layer that fluidly connects the fill hole to the well of the array.

Additional features and advantages of the embodiments of the invention will be set forth in the description which follows or may be learned by the practice of such embodiments. The features and advantages of such embodiments may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features will become more fully apparent from the following description and appended claims, or may be learned by the practice of such embodiments as set forth hereinafter.

 

What is claimed is:1. A method for performing PCR, comprising:

(a) providing a PCR mixture in a sample vessel, the sample vessel having a volume and the PCR mixture having a volume;

(b) thermocycling the volume of the PCR mixture in the volume of the sample vessel for a first number of cycles, each of the first number of cycles having a first cycle time;

(c) reducing the volume of the sample vessel to a second volume of the sample vessel thereby reducing the volume of the PCR mixture to a second volume of the PCR mixture, the second volume of the sample vessel being smaller than the volume of the sample vessel and the second volume of the PCR mixture being smaller than the volume of the PCR mixture,

wherein the sample vessel is compressible, and the reducing step is performed by compressing the sample vessel to the second volume of the sample vessel to expel a portion of the sample from the sample vessel; and

(d) thermocycling the second volume of the PCR mixture for a second number of cycles in the second volume of the sample vessel, each of the second number of cycles having a second cycle time, the second cycle time being shorter than the first cycle time.

2. The method of claim 1, further comprising:

(e) reducing the second volume of the PCR mixture in the sample vessel to a third volume, the third volume of the PCR mixture being smaller than the second volume of the PCR mixture; and

(f) thermocycling the third volume of the PCR mixture for a third number of cycles in the sample vessel, each of the third number of cycles having a third cycle time, the third cycle time being shorter than the second cycle time.

3. The method of claim 1, wherein the sample vessel is in contact with a first temperature zone and a second temperature zone, and one or more of the thermocycling steps includes moving the sample between the first and second temperature zones.

4. The method of claim 3, further comprising moving a first portion of the PCR mixture to a second portion of the sample vessel while moving a second portion of the PCR mixture to a first portion of the sample vessel such that the first and second portions of the PCR mixture are under control of the first and second temperature zones simultaneously, wherein the first temperature zone is hotter than the second temperature zone.

5. The method of claim 1, wherein the sample vessel is configured to contact a first temperature zone and a second temperature zone, and the thermocycling step includes alternatingly contacting the sample vessel with the first temperature zone and then the second temperature zone.

6. The method of claim 1, wherein the sample vessel is heated by a heater that thermocycles between two or more temperatures, the heater cycling at the first cycle time in step (b) and at the second cycle time at step (d).

7. The method of 1, wherein the first number of cycles is 2 to 10 cycles.

8. The method of claim 1, wherein the second volume of the PCR mixture in the second volume of the sample vessel is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% smaller than the volume of the PCR mixture in the volume of the sample vessel.

9. The method of claim 1, wherein (i) the second volume of the PCR mixture in the second volume of the sample vessel is 50% smaller than the volume of the PCR mixture in the volume of the sample vessel and (ii) the second cycle time is 25% to 50% shorter than the first cycle time.

10. The method of claim 1, wherein the reducing step comprises reducing the volume of the PCR mixture in the sample vessel 2 to 5 times and reducing the cycle time after each volume reduction.

11. The method of claim 1, wherein the sample vessel is part of a closed sample container having at least one additional fluidly connected sample vessel therein.

12. A method for performing PCR, comprising:

(a) providing a PCR mixture in a compressible sample vessel, the sample vessel having a volume and the PCR mixture having a volume;

(b) thermocycling the volume of the PCR mixture in the volume of the sample vessel for a first number of cycles, each of the first number of cycles having a first cycle time;

(c) compressing the sample vessel, thereby reducing the volume of the sample vessel to a second volume of the sample vessel and expelling a portion of the sample from the sample vessel, thereby reducing the volume of the PCR mixture to a second volume of the PCR mixture, the second volume of the sample vessel being smaller than the volume of the sample vessel and the second volume of the PCR mixture being smaller than the volume of the PCR mixture; and

(d) thermocycling the second volume of the PCR mixture for a second number of cycles in the second volume of the sample vessel, each of the second number of cycles having a second cycle time, the second cycle time being shorter than the first cycle time.