Methods and systems for large-scale polymerase chain reaction (PCR)

Abstract

The disclosure herein relates to a device including a thin-walled single-use bag for large-scale production of nucleic acids using polymerase chain reaction (PCR) and methods of use thereof.

Description

METHODS AND SYSTEMS FOR LARGE-SCALE POLYMERASE CHAIN REACTION (PCR)RELATED APPLICATIONS

[0001] The present application claims the benefit of priority of EP Priority patent application no. 23217861.6, filing date December 19, 2023, the entire content of which is incorporated herein in its entirety.RELEVANT FIELD

[0002] Embodiments described herein relate to a method and system for large-scale production of nucleic acids using a polymerase chain reaction (PCR) method. More specifically, some embodiments of the technology relate to a method of PCR using a system in which a thin-walled single-use bag is contacted by heating elements to form a layer of a reaction mixture with a thickness allowing the reaction mixture to be heated and cooled to a substantially uniform temperature corresponding to each step of a PCR method.BACKGROUND

[0003] Polymerase chain reaction (PCR) is a method used in molecular biology for the amplification of nucleic acids in vitro. The method relies on an initial activation step and repeated cycles of three additional steps: 1 ) denaturation of double-stranded deoxyribonucleic DNA into two accessible single strands, 2) annealing of complementary primers to each of the single strands, and 3) elongation, also referred to as extension, of nucleic acid chains using a polymerase enzyme to synthesize a new complementary DNA strand. Conventional PCR is performed in multi-well plates with multiple 20 pL - 250 pL conical vials, which are placed in a thermal cycling PCR instrument comprising a thermal heating block. Alternative to a thermal cycling PCR instrument, temperatures may be controlled by fluid baths, such as water baths.

[0004] Using a large-scale PCR method is one way to provide large amounts of DNA, also referred to herein as amplification product, to be used as a template for the production of mRNA or RNA products. Scalability remains the greatest limitation to industrial production of nucleic acids usingPCR. One reason for challenges to scaling-up PCR is that the three repeated steps of PCR are performed at different temperatures. However, thermal cyclers or similar devices are limited to production of smaller volumes. Currently, no adequate thermocycling PCR instruments suitable for large volumes which avoids significant numbering up are on the market.

[0005] Large volume production is typically performed through a “scale-up” approach or a “numbering-up” approach by combining the products obtained from multiple microliter conical tubes. A traditional “scale up” has the advantage of practicality and smaller footprint. However, due to the low heat and mass transfers within these reaction vessels combined with the stringent temperature and time requirements of a PCR protocol, they often suffer from low yields, and inconsistent quality.

[0006] Alternatively, “numbering up” takes advantage of the high surface- area-volume-ratio in microliter conical tubes for rapid heat transfers. However, there are several disadvantages, such as greater waste generated as well as larger footprint and increased cost due to additional devices required for filling and emptying of the microliter tubes.

[0007] An example of development in the diagnostic field for PCR include techniques to optically detect the presence of nucleic acids by using small detection bags containing the reaction mixture and color developing agents. The bag consists of multiple components, such as a feed port, amplification cabin, detection consumables, and disposable components. To ensure proper heating, a heating module is used to bring the consumable to the correct temperature.

[0008] In another example, a reaction vessel for amplification increases heat transfer by agitating the reaction fluid in a flow channel or sample container. The reaction mixture in the container is pushed with the use of the moving parts between two heating modules kept at a constant temperature. This ensures a rapid temperature change for nucleic acid amplification. However, the scalability of this concept is limited due to the difficulties in physically and reliably transferring larger volumes. Additionally, the movement of larger volumes can lead to stress on the enzyme lowering product quality, as well as on the reaction vessel increasing the risk of damage to the vessel.

[0009] In other techniques, nucleic acid preparation and amplification isperformed in flexible tubing in which heating and cooling elements, also referred to as thermocouples, compress the tubing to provide rapid heat delivery. Commonly, the thermocouples are electrothermal transducers, such as Peltier elements. Further embodiments also include different segments of the tubing for sample preparation . The last segment of the tubing may be used for amplification.

[0010] Another system used for PCR includes upper and lower temperature zones within a chamber for the PCR reaction. The system design is intended to allow movement of the reaction fluid through convection cells within channels. This is intended to eliminate the need to switch temperatures within a thermocouple and simplify control of the system.

[0011] Another technique describes PCR performed continuously in a tube / capillary, where the temperature of each step is provided by multiple fluid baths. A similar technique uses fluid baths at different temperatures for different PCR steps. A reaction tube can be placed in each fluid bath to provide the rapid temperature change required for the PCR reaction. Often several mitigation strategies are necessary to reduce the probability of crosscontamination.

[0012] All prior art reflects the limitations for scalable, fast heat transfer or they do not address the complexity of fluid baths in GMP manufacturing environments. There is therefore compelling need for new technologies to address the gap in the industry. A system and method to maximize the amount of amplification product and thereby maximizing the amount of mRNA or RNA products produced, while minimizing the risk of cross-contamination, represents an inventive advance in the art.SUMMARY

[0013] Shortcomings of the prior art are overcome by embodiments described herein. Some embodiments provide a system for nucleic acid production using a polymerase chain reaction (PCR) method, the system comprising: at least one thin-walled single-use bag to hold a reaction mixture; at least two heat transfer elements to transfer heat to the bag, wherein the bag is contacted by the two heat transfer elements to an internal thickness allowing the reaction mixture to heat uniformly and the temperature to bechanged at rates to perform the PCR method; and a heating device to generate a temperature in the heat transfer elements corresponding to at least one step of the PCR method.

[0014] In some embodiments, the system further comprises more than one thin-walled single-use bag. In some embodiments, the thin-walled single-use bag is flexible. In some embodiments, the system further comprises at least twice as many heat transfer elements as the number of thin-walled single-use bags. In some embodiments, the heating device comprises a double jacket or closed vessel or plate containing a thermofluid or an electrical temperature controller. In some embodiments, the electrical temperature controller is an electrical heating block. In some embodiments, the thickness of the reaction mixture is selected from the range consisting of: 0.1 mm to 20 mm when the PCR method is being performed in the system. In some embodiments, the system further comprises a fan to remove excess heat. In some embodiments, the system further comprises a heat exchanger to remove excess heat or facilitate fast cooling.

[0015] Some embodiments provide a method of producing a amplification product using a polymerase chain reaction (PCR) method using the system described herein, the method comprising: filling the at least one thin-walled single-use bag with the reaction mixture; contacting the bag with at least two heat transfer elements; moving the heat transfer elements to mix the reaction mixture in the bag; and heating the reaction mixture to a temperature corresponding to each step of the PCR method.

[0016] In some embodiments, filling occurs to a volume within the range of 5% to 100% of the maximum volume of the thin-walled single-use bag. In some embodiments, the amplification product has a length within the range of 100 to 20,000 base pairs. In some embodiments, contacting the thin-walled single-use bag between the heat transfer elements occurs before the filling step. In some embodiments, contacting the thin-walled single-use bag between the heat transfer elements occurs after the filling step. Filling may occur by any means resulting in a transfer of the reaction mixture into the thinwalled single-use bag. In some embodiments, filling comprises injecting the reaction mixture into the thin-walled single-use bag. In some embodiments, filling comprises pumping the reaction mixture into the thin-walled single-usebag. In some embodiments, filling comprises a gravity transfer of the reaction mixture into the thin-walled single-use bag. In some embodiments, the method further comprises compressing the thin-walled single-use bag to an internal thickness of within a range of 0.1 mm to 20 mm.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 provides an illustration of some embodiments of a system used for a PCR method.

[0018] FIG. 2 provides an illustration of some embodiments of a system used for a PCR method.

[0019] The appended drawings illustrate some embodiments of the disclosure herein and are therefore not to be considered limiting in scope, for the invention may admit to other egually effective embodiments. It is to be understood that elements and features of any embodiment may be found in other embodiments without further recitation and that, where possible, identical reference numerals have been used to indicate comparable elements that are common to the figures.DETAILED DESCRIPTION

[0020] Some embodiments of a system for a PCR method comprise at least one thin-walled single-use bag filled with a volume of a reaction mixture between two heat transfer elements.

[0021] In some embodiments, production of large volumes of amplification product is achieved by using multiple thin-walled single-use bags and at least two heat transfer elements for each thin-walled single-use bag in parallel. In some embodiments, the heating elements can be moved to mix the reaction mixture. In some embodiments, an amplification product has a length within the range of 100 to 20,000 base pairs. In some embodiments, the length of the amplification product is within a range of 100 base pairs to 500 base pairs. In some embodiments, the length of the amplification product is within a range of 1000 base pairs to 1500 base pairs. In some embodiments, the length of the amplification product is within a range of 2000 base pairs to 2500 base pairs. In some embodiments, the length of the amplification product is within a range of 3000 base pairs to 3500 base pairs. In some embodiments, thelength of the amplification product is within a range of 4000 base pairs to 4500 base pairs. In some embodiments, the length of the amplification product is within a range of 5000 base pairs to 5500 base pairs. In some embodiments, the length of the amplification product is within a range of 6000 base pairs to 6500 base pairs. In some embodiments, the length of the amplification product is within a range of 7000 base pairs to 7500 base pairs. In some embodiments, the length of the amplification product is within a range of 8000 base pairs to 8500 base pairs. In some embodiments, the length of the amplification product is within a range of 9000 base pairs to 9500 base pairs. In some embodiments, the length of the amplification product is within a range of 10000 base pairs to 10500 base pairs. In some embodiments, the length of the amplification product is within a range of 11000 base pairs to 11500 base pairs. In some embodiments, the length of the amplification product is within a range of 12000 base pairs to 12500 base pairs. In some embodiments, the length of the amplification product is within a range of 13000 base pairs to 13500 base pairs. In some embodiments, the length of the amplification product is within a range of 14000 base pairs to 14500 base pairs. In some embodiments, the length of the amplification product is within a range of 15000 base pairs to 15500 base pairs. In some embodiments, the length of the amplification product is within a range of 16000 base pairs to 16500 base pairs. In some embodiments, the length of the amplification product is within a range of 17000 base pairs to 17500 base pairs. In some embodiments, the length of the amplification product is within a range of 18000 base pairs to 18500 base pairs. In some embodiments, the length of the amplification product is within a range of 19000 base pairs to 19500 base pairs. In some embodiments, the length of the amplification product is within a range of 20000 base pairs to 20500 base pairs.

[0022] Additionally, in some embodiments, the system described herein can be used to perform any type of PCR protocol. In some embodiments, the type of PCR protocol is selected from the group consisting of: real-time PCR, quantitative real time PCR (Q- PCR), reverse transcriptase PCR (RT-PCR), multiplex PCR, nested PCR, long-range PCR, single-cell PCR, fast-cycling PCR, methylation-specific PCR (MSP), hot start PCR, high-fidelity PCR, In situ PCR, asymmetric PCR, overlap extension PCR, assemble PCR,intersequence-specific (ISSR) PCR, ligation-mediated PCR, and miniprimer PCR. In some embodiments, the type of PCR protocol is real-time PCR. In some embodiments, the type of PCR protocol is quantitative real time PCR (Q-PCR). In some embodiments, the type of PCR protocol is reverse transcriptase PCR (RT-PCR). In some embodiments, the type of PCR protocol is multiplex PCR. In some embodiments, the type of PCR protocol is nested PCR. In some embodiments, the type of PCR protocol is long-range PCR. In some embodiments, the type of PCR protocol is single-cell PCR. In some embodiments, the type of PCR protocol is fast-cycling PCR. In some embodiments, the type of PCR protocol is methylation-specific PCR (MSP). In some embodiments, the type of PCR protocol is hot start PCR. In some embodiments, the type of PCR protocol is high-fidelity PCR. In some embodiments, the type of PCR protocol is in situ PCR. In some embodiments, the type of PCR protocol is variable number of tandem repeats (VNTR) PCR. In some embodiments, the type of PCR protocol is asymmetric PCR. In some embodiments, the type of PCR protocol is repetitive sequence-based PCR. In some embodiments, the type of PCR protocol is overlap extension PCR. In some embodiments, the type of PCR protocol is assemble PCR. In some embodiments, the type of PCR protocol is intersequence-specific PCR(ISSR). In some embodiments, the type of PCR protocol is ligation-mediated PCR. In some embodiments, the type of PCR protocol is miniprimer PCR. In some embodiments, the type of PCR performed is reverse transcriptase (RT) PCR using RNA-directed DNA polymerases.I. System

[0023] Some embodiments herein describe a system for large-scale production of nucleic acids using polymerase chain reaction (PCR). Some embodiments are shown in FIG. 1 and FIG. 2. In some embodiments, the system includes all connections 4. In some embodiments, the thin-walled single-use bag 1 is contacted by two heat transfer elements 2 as shown in FIG. 2. In some embodiments, the heat transfer elements 2 are Peltier elements. In some embodiments, the thin-walled single-use bag 1 is held between two heat transfer elements 2 by a frame. In some embodiments, the thin-walled single-use bag 1 is contacted by the heat transfer elements 2, andfollowing contact, the reaction mixture 5 is injected into the thin-walled singleuse bag1 using a syringe 3. Alternatively, in some embodiments, the reaction mixture 5 is injected into the thin-walled single-use bag 1 using a pump or a tube assembly.A. Thin-walled single-use bag

[0024] In some embodiments, the thin-walled single-use bag 1 is disposable. In some embodiments, the thin-walled single-use bag 1 is flexible. In some embodiments, the thin-walled single-use bag 1 is sterilizable. In some embodiments, the thin-walled single-use bag 1 comprises any suitable materials. In some embodiments, the thin-walled single-use bag 1 is made of a robust material suitable for varying temperatures. In some embodiments, the thin-walled single-use bag 1 is made of a material that prevents the reaction mixture 5 from leaking out of the thin-walled single-use bag 1 under the varying temperature conditions. In some embodiments, the thin-walled single-use bag 1 is made of a polymeric material suitable for holding liquids. In some embodiments, the thin-walled single-use bag 1 comprises at least one of plastic or silicone. In some embodiments, the plastic is selected from the group consisting of: polypropylene, polyethylene, polysulfone, and fluoropolymers. In some embodiments, the thin-walled single-use bag 1 is single use.

[0025] In some embodiments, when the thin-walled single-use bag 1 is between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 0.1 mm to 20 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 0.1 mm to 2 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 1 mm to 3 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 2 mm to 4 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 3 mm to 5 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 4 mm to 6 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reactionmixture 5 is within the range of 5 mm to 7 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 6 mm to 8 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 7 mm to 9 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 8 mm to 10 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 9 mm to 11 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 10 mm to 12 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 11 mm to 13 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 12 mm to 14 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 13 mm to 15 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 14 mm to 16 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 15 mm to 17 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 16 mm to 18 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 17 mm to 19 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 18 mm to 20 mm. In some embodiments, between the heat transfer elements 2, the internal thickness of the reaction mixture 5 is within the range of 19 mm to 21 mm.

[0026] In some embodiments, the thickness of each wall of the thin-walled single-use bag is less than about 1.0 mm. In some embodiments, the thickness of each wall of the thin-walled single-use bag is within a range of between 0.1 mm to 0.3 mm. In some embodiments, the thickness of each wall of the thin-walled single-use bag is within a range of between 0.2 mm to 0.4mm. In some embodiments, the thickness of each wall of the thin-walled single-use bag is within a range of between 0.3 mm to 0.5 mm. In some embodiments, the thickness of each wall of the thin-walled single-use bag is within a range of between 0.4 mm to 0.6 mm. In some embodiments, the thickness of each wall of the thin-walled single-use bag is within a range of between 0.5 mm to 0.7 mm. In some embodiments, the thickness of each wall of the thin-walled single-use bag is within a range of between 0.6 mm to 0.8 mm. In some embodiments, the thickness of each wall of the thin-walled single-use bag is within a range of between 0.7 mm to 0.9 mm. In some embodiments, the thickness of each wall of the thin-walled single-use bag is within a range of between 0.8 mm to 1 mm.

[0027] In some embodiments, the thin-walled single-use bag 1 is filled and emptied through built-in connections 4. In some embodiments, the thin-walled single-use bag 1 is filled or emptied using a syringe 3.

[0028] In some embodiments, various thin-walled single-use bag 1 volumes can be used for the invention, which are scalable in height and length.

[0029] In some embodiments, the reaction mixture 5 for amplification is transferred to a thin, rigid thin-walled single-use bag 1 to create a thin and uniform liquid layer that can be temperature controlled using the heat transfer elements.B. Heat Transfer Elements

[0030] In some embodiments, the heat transfer elements 2 are Peltier elements. In some embodiments, the thin-walled single-use bag 1 is clamped between the heat transfer elements 2 creates a thin film of reaction mixture 5 within the thin-walled single-use bag 1 capable of experiencing the temperature changes required for PCR. In some embodiments, the temperature changes required for PCR are rapid. In some embodiments, heat transfer elements 2 are capable of heating and cooling within a range of 55°C to 100°C or can be used at a constant temperature. In some embodiments, the heating and cooling of the thin-walled single-use bag 1 can be performed by using two heat transfer elements 2. These elements can be controlled by an electrical temperature controller.

[0031] In some embodiments, the temperature of the heating elements isdetermined by the master mix and polymerase used in the PCR method. In some embodiments, the temperature for the Activation and Denaturation steps is within the range of 85°C to 100°C. In some embodiments, the temperature for the Annealing step is within the range of 50°C to 85°C. In some embodiments, the temperature for the Elongation step or the Final Elongation step is within the range of 55°C to 90°C.

[0032] In some embodiments, the electrical means powers a fan 8 to remove excess heat from a heat exchanger 9 when the heat transfer elements 2 heat as shown in FIG 1 . In some embodiments the direction of DC current is reversed to cool the heat transfer elements or cool to the temperature for a step of the PCR method as shown in FIG. 1 . In some embodiments, the thin-walled single-use bag 1 contacted by the heat transfer elements 2 is placed in or next to a heat exchanger 9. Heat transfer elements 2 are used to subsequently bring the temperature of the reaction mixture 5 to a temperature for a step of the PCR method.C. Mixing Component

[0033] In some embodiments, the amplification fluid is mixed by mechanical movement of the heat transfer elements. In some elements, the reaction mixture 5 can be mixed by convection of different temperature zones within the thin-walled single-use bag 1.II. Methods

[0034] Some embodiments herein describe a method of large-scale production of nucleic acids using polymerase chain reaction (PCR).

[0035] In some embodiments, the method of PCR comprises filling the thin-walled single-use bag 1 with a volume of a reaction mixture 5. In some embodiments, the volume of a reaction mixture 5 is between 1-100% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 5% to 7% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 6% to 8% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 7% to 9% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 8% to 10% of the maximum volume of the thin-walled single-use bag1. In some embodiments, the volume of a reaction mixture 5 is between 9% to 11 % of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 10% to 12% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 11% to 13% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 12% to 14% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 13% to 15% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 14% to 16% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 15% to 17% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 16% to 18% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 17% to 19% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is between 18% to 20% of the maximum volume of the thin-walled single-use bag 1.

[0036] In some embodiments, the volume of a reaction mixture 5 is about 5% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 6% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 7% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 8% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 9% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 10% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 11% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 12% of the maximum volume of the thin-walled single-use bag 1. Insome embodiments, the volume of a reaction mixture 5 is about 13% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 14% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 15% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 16% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 17% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 18% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 19% of the maximum volume of the thin-walled single-use bag 1. In some embodiments, the volume of a reaction mixture 5 is about 20% of the maximum volume of the thin-walled single-use bag 1.

[0037] In some embodiments, the method of PCR produces an unlimited volume of amplification product. In some embodiments, the volume of amplification product produced is between 1 ml to 10 L. In some embodiments, the amplification product can be further increased to an unlimited amount via numbering up. In some embodiments, the volume of the amplification product produced is selected from a range of 1 ml to 100 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 50 ml to 150 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 100 ml to 200 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 150 ml to 250 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 200 ml to 300 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 250 ml to 350 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 300 ml to 400 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 350 ml to 450 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 400 ml to 500 ml. In some embodiments, the volume of the amplification product produced is selectedfrom a range of 450 ml to 550 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 500 ml to 600 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 550 ml to 650 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 600 ml to 700 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 650 ml to 750 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 700 ml to 800 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 750 ml to 850 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 800 ml to 900 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 850 ml to 950 ml. In some embodiments, the volume of the amplification product produced is selected from a range of 900 ml to 1 L.

[0038] The time of each reaction step is determined by the optimum PCR protocol for each master mix. In some embodiments, the Activation step is performed for a time period selected from the range consisting of: 10 seconds to 10 minutes. In some embodiments, the Activation step is performed for a time period within in the range of 5 seconds to 1 minute. In some embodiments, the Activation step is performed for a time period within in the range of 1 minute to 2 minutes. In some embodiments, the Activation step is performed for a time period within in the range of 2 minutes to 3 minutes. In some embodiments, the Activation step is performed for a time period within in the range of 3 minutes to 4 minutes. In some embodiments, the Activation step is performed for a time period within in the range of 4 minutes to 5 minutes. In some embodiments, the Activation step is performed for a time period within in the range of 5 minutes to 6 minutes. In some embodiments, the Activation step is performed for a time period within in the range of 6 minutes to 7 minutes. In some embodiments, the Activation step is performed for a time period within in the range of 7 minutes to 8 minutes. In some embodiments, the Activation step is performed for a time period within in the range of 8 minutes to 9 minutes. In some embodiments, the Activation step is performed for a time period within in the range of 9 minutes to 10 minutes.

[0039] In some embodiments, the Activation step is performed for less than 10 minutes. In some embodiments, the Activation step is performed for greater than 10 minutes. In some embodiments, the Activation step is performed for greater than 10 seconds.

[0040] In some embodiments, the Denaturation step is performed for a time period selected from the range consisting of: 10 seconds to 10 minutes. In some embodiments, the Denaturation step is performed for a time period within in the range of 5 seconds to 1 minute. In some embodiments, the Denaturation step is performed for a time period within in the range of 1 minute to 2 minutes. In some embodiments, the Denaturation step is performed for a time period within in the range of 2 minutes to 3 minutes. In some embodiments, the Denaturation step is performed for a time period within in the range of 3 minutes to 4 minutes. In some embodiments, the Denaturation step is performed for a time period within in the range of 4 minutes to 5 minutes. In some embodiments, the Denaturation step is performed for a time period within in the range of 5 minutes to 6 minutes. In some embodiments, the Denaturation step is performed for a time period within in the range of 6 minutes to 7 minutes. In some embodiments, the Denaturation step is performed for a time period within in the range of 7 minutes to 8 minutes. In some embodiments, the Denaturation step is performed for a time period within in the range of 8 minutes to 9 minutes. In some embodiments, the Denaturation step is performed for a time period within in the range of 9 minutes to 10 minutes. In some embodiments, the Denaturation step is performed for 1 minute. In some embodiments, the Denaturation step is performed for less than 1 minutes. In some embodiments, the Denaturation step is performed for greater than 10 minutes. In some embodiments, the Denaturation step is performed for greater than 10 seconds.

[0041] In some embodiments, the Annealing step is performed for a time period selected from the range consisting of: 10 seconds to 10 minutes. In some embodiments, the Annealing step is performed for a time period within in the range of 5 seconds to 1 minute. In some embodiments, the Annealing step is performed for a time period within in the range of 1 minute to 2 minutes. In some embodiments, the Annealing step is performed for a timeperiod within in the range of 2 minutes to 3 minutes. In some embodiments, the Annealing step is performed for a time period within in the range of 3 minutes to 4 minutes. In some embodiments, the Annealing step is performed for a time period within in the range of 4 minutes to 5 minutes. In some embodiments, the Annealing step is performed for a time period within in the range of 5 minutes to 6 minutes. In some embodiments, the Annealing step is performed for a time period within in the range of 6 minutes to 7 minutes. In some embodiments, the Annealing step is performed for a time period within in the range of 7 minutes to 8 minutes. In some embodiments, the Annealing step is performed for a time period within in the range of 8 minutes to 9 minutes. In some embodiments, the Annealing step is performed for a time period within in the range of 9 minutes to 10 minutes.

[0042] In some embodiments, the Annealing step is performed for less than one minute. In some embodiments, the Annealing step is performed for less than 10 minutes. In some embodiments, the Annealing step is performed for greater than 10 minutes. In some embodiments, the Annealing step is performed for greater than 10 seconds.

[0043] In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 30 seconds to 1 minute. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 1 minute to 2 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 2 minutes to 3 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 3 minutes to 4 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 4 minutes to 5 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 5 minutes to 6 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 6 minutes to 7 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 7 minutes to 8 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 8 minutes to 9 minutes. In some embodiments, the Elongation step is performedfor a time period selected from the range consisting of: 9 minutes to 10 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 10 minutes to 11 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 11 minutes to 12 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 12 minutes to 13 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 13 minutes to 14 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 14 minutes to 15 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 15 minutes to 16 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 16 minutes to 17 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 17 minutes to 18 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 18 minutes to 19 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 19 minutes to 20 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 20 minutes to 21 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 21 minutes to 22 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 22 minutes to 23 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 23 minutes to 24 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 24 minutes to 25 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 25 minutes to 26 minutes. In some embodiments, the Elongation step is performed for a time period selectedfrom the range consisting of: 26 minutes to 27 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 27 minutes to 28 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 28 minutes to 29 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 29 minutes to 30 minutes. In some embodiments, the Elongation step is performed for a time period selected from the range consisting of: 30 minutes to 31 minutes. In some embodiments, the Elongation step is performed for less than 30 seconds. In some embodiments, the Elongation step is performed for greater than 30 minutes.

[0044] In some embodiments, the Final Elongation step is not performed. In some embodiments, the Final Elongation step is performed for less than 30 seconds. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 30 seconds to 1 minute. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 1 minute to 2 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 2 minutes to 3 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 3 minutes to 4 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 4 minutes to 5 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 5 minutes to 6 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 6 minutes to 7 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 7 minutes to 8 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 8 minutes to 9 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 9 minutes to 10 minutes. In someembodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 10 minutes to 11 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 11 minutes to 12 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 12 minutes to 13 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 13 minutes to 14 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 14 minutes to 15 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 15 minutes to 16 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 16 minutes to 17 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 17 minutes to 18 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 18 minutes to 19 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 19 minutes to 20 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 20 minutes to 21 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 21 minutes to 22 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 22 minutes to 23 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 23 minutes to 24 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 24 minutes to 25 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 25 minutes to 26 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 26 minutes to 27 minutes. In someembodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 27 minutes to 28 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 28 minutes to 29 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 29 minutes to 30 minutes. In some embodiments, the Final Elongation step is performed for a time period selected from the range consisting of: 30 minutes to 31 minutes. In some embodiments, the Final Elongation step is performed for greater than 30 minutes.III. Definitions

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0046] As used herein, the singular forms "a", "an," and "the" include plural unless the context clearly dictates otherwise.

[0047] As used herein, the phrase “large-scale” refers to volumes of amplification product produced at liter-scale.

[0048] As used herein, the term “master mix” refers to a solution including the components required for a PCR reaction, for example, a polymerase, at least one primer, and deoxynucleoside triphosphates (dNTPs).

[0049] As used herein, the term “reaction mixture” or “amplification mixture” refers to a master mix combined with the nucleic acid template.

[0050] As used herein, the term “thin-walled” refers to walls of a single-use bag with a thickness of less than or equal to one (1 ) millimeter (mm).EXAMPLESExample 1. PCR Protocol

[0051] Table 1 : The following is a working example of a PCR protocol for target DNA of 2079 base pairs using the system described in some embodiments herein. The system used in Example 1 includes a 2D polypropylene bag with a 100 ml volume. The bag was filled with 13 ml of the Master Mix. Table 1.

[0052] The Master Mix was prepared accordingly: 215 pL Reaction Buffer; 3010 pL Water ; 7826 pL Enhancer appropriate for the polymerase; 301 pL dNTP Solution Mix (each dNTP at a concentration of 0.2mM) (10mM); forward primer 100 pM (75 pL); reverse primer 100 pM (75 pL); and 150.5 pL Q5® Hot Start High Fidelity DNA Polymerase (New England Biolabs). The master mix was combined with the 602 pL Template (1 OOpg / pl) to create the reaction mixture.

[0053] The results were analyzed by gel electrophoresis and the product confirmed via comparison to the DNA ladder. The amount of PCR product measured fluorometrically was in a comparable range.EQUIVALENTS

[0054] All ranges for formulations recited herein include ranges therebetween and can be inclusive or exclusive of the endpoints. Optional included ranges are from integer values therebetween (or inclusive of one original endpoint), at the order of magnitude recited or the next smaller order of magnitude. For example, if the lower range value is 0.2, optional included endpoints can be 0.3, 0.4,... 1.1 , 1.2, and the like, as well as 1 , 2, 3 and the like; if the higher range is 8, optional included endpoints can be 7, 6, and thelike, as well as 7.9, 7.8, and the like. One-sided boundaries, such as 3 or more, similarly include consistent boundaries (or ranges) starting at integer values at the recited order of magnitude or one lower. For example, 3 or more includes 4, or 3.1 or more.

[0055] Reference throughout this specification to “one embodiment,” “certain embodiments,” “one or more embodiments,” “some embodiments,” or “an embodiment” indicates that a feature, structure, material, or characteristic described is included some embodiments of the disclosure. Therefore, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in one embodiment,” “some embodiments,” or “in an embodiment” throughout this specification are not necessarily referring to the same embodiment.

[0056] Publications of patent applications and patents and other nonpatent references, cited in this specification are herein incorporated by reference in their entirety in the entire portion cited as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in the manner described above for publications and references.

Claims

CLAIMSWhat is claimed is:1 . A system for nucleic acid production using a polymerase chain reaction (PCR) method, the system comprising: a) at least one thin-walled single-use bag to hold a reaction mixture; b) at least two heat transfer elements to transfer heat to the bag, wherein the bag is contacted with the two heat transfer elements placing the reaction mixture at an internal thickness allowing the reaction mixture to heat substantially uniformly and the temperature to be changed by the two heat transfer elements at rates to perform the PCR method; and c) a heating device to generate a temperature in the heat transfer elements corresponding to at least one step of the PCR method.

2. The system of claim 1 , further comprising more than one bag.

3. The system of any one of claims 1 and 2, the reaction vessel is flexible.

4. The system of any one of claims 1-3, further comprising at least twice as many heat transfer elements as the number of bags.

5. The system of any one of claims 1-4, wherein the heating device comprises a thermofluid or an electrical temperature controller.

6. The system of any one of claims 1-5, wherein the thermofluid is a temperature-controlled fluid bath.

7. The system of any one of claims 1-5, wherein the electrical temperature controller is an electrical heating block.

8. The system of any one of claims 1-7, wherein the thickness of the reaction mixture is selected from the range consisting of: 0.1 mm to 20 mm when the PCR method is being performed in the system.

9. The system of any one of claims 1-8, further comprising a fan to remove excess heat.

10. The system of claim 9, further comprising a heat exchanger to receive the heat from the fan.

11. A method of producing a amplification product using a polymerase chain reaction (PCR) method using the system of any one of claims 1-10, the method comprising: a) filling the at least one thin-walled single-use bag with the reaction mixture; b) contacting the bag with at least two heat transfer elements; c) heating the reaction mixture with the heat transfer elements in the bag to a temperature corresponding to each step of the PCR method; and d) removing excess heat using a fan.

12. The method of claim 11 , wherein filling occurs to a volume within the range of 5% to 20% of the maximum volume of the reaction vessel.

13. The method of any one of claims 11 and 12, wherein the amplification product has a length within the range of 100 to 20,000 base pairs.

14. The method of any one of claims 11-13, wherein contacting the bag with the heat transfer elements occurs before the filling step.

15. The method of any one of claims 11-14, wherein contacting the bag by the heat transfer elements occurs after the filling step.

16. The method of claim 15, wherein filling comprises injecting, pumping, or gravity transferring the reaction mixture into the reaction vessel.

Images