EP4683743A1

EP4683743A1 - A semi-continuous method and system for the production of nucleic acids

Info

Publication number: EP4683743A1
Application number: EP24712054.6A
Authority: EP
Inventors: Camille CORREIA; Kai Jaehrling; Philipp LAU; Jens Fruendt
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2023-03-20
Filing date: 2024-03-19
Publication date: 2026-01-28
Also published as: JP2026511479A; WO2024194303A1; CN121152683A

Abstract

The disclosure herein relates to a bioreactor for large-scale production of nucleic acids using polymerase chain reaction (PCR) and methods of use thereof.

Description

A SEMI-CONTINUOUS METHOD AND SYSTEM FOR THE PRODUCTION OF NUCLEIC ACIDS

RELEVANT FIELD

[0001] Embodiments described herein relate to a method and system for large- scale production of nucleic acids using polymerase chain reaction (PCR). More specifically, some embodiments of the technology relate to a method of PCR using a semi-continuous bioreactor.

BACKGROUND

[0002] 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 doublestranded 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 plates with multiple 20 - 250 pL conical vials, which are placed in a thermal cycling PCR instrument comprising a thermal heating block.

[0003] Using microchannels based on microfluidic capillary or chip designs as a reaction vessel for amplification of DNA provides a number of advantages, such as rapid heat transfers, compact design, high-throughput for assays, minimal footprints, and ease of integration automation. Microfluidic PCR instruments include spiral and oscillating flow designs. (See, Kopp et al. 1998 Micro Total Anal. Syst. 98 7-10, which is hereby incorporated by reference in its entirety)

[0004] The chip-based designs have limited working volume and recently have been surpassed by spiral capillary designs incorporating either a metal heating block (Peltier elements) or baths containing thermal media as heat exchangers. (See, KR20050078568; US200801 5923; Park et al., 2003 Anal. Chem. 75 6029-33; and Kim et al. 2016 Bull. Korean Chem. Soc. 37 1878-81 , each of which is hereby incorporated by reference in its entirety) The heating element may be divided in multiple zones of varying sizes and temperature corresponding to the optimum conditions for the activation, denaturation, annealing, and elongation steps. DNA is amplified as a continuous process by feeding the mixture through a capillary tube wound around the heating elements. Similar to the microchannel chip PCR, multiple chips or heating elements would be required to encompass a wide portfolio of DNA targets. (See, US8163489, which is hereby incorporated by reference in its entirety) Alternatively, a multi-segmented heating block may be used. For these devices, some variability in PCR protocols is tolerated by temperature control of each section/segment. The physical scalability of the previous designs was restricted by the low contact area and heat transfers between an electric heating element and a capillary tube. The use of a bath with thermal media does not suffer this constraint as the outer tube is fully immersed in a medium.

[0005] A fully continuous PCR device employing four temperature controlled fluid chambers and capillary tubing through which the master mix is pumped has been previously described. (See, US7217699, which is hereby incorporated by reference in its entirety) To maximize yield, multiple cycles are performed by the solution exiting the final chamber reenters the first chamber and continuing along the microchannel path. In this way each cycle is represented by an additional set of capillary coils for activation, denaturing, annealing, and elongation step resulting in a large amount of tubing per PCR cycle.

[0006] Another version of a PCR device includes multiple designs for nucleic acid amplification, one of which being the pumping of a reaction mixture in a continuous and repetitive loop between two temperature-controlled fluid baths. (See, US5720923, which is hereby incorporated by reference in its entirety) The major disadvantage is the lack of variability making more complex PCR protocols impossible. Additionally, incorporation of a valve immersed in the fluid baths can lead to compromised joints and contamination.

[0007] Scalability remains the greatest limitation to industrial production of nucleic acids using PCR. 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 vessels combined with the stringent temperature and time requirements of a PCR protocol, they often suffer from low yields, and inconsistent quality.

[0008] 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.

[0009] Capillary bioreactors can act as a compromise between the “scale-up” and “numbering-up” approaches by taking advantage of the rapid heat and mass transfers on small dimensions while circumventing the redundancy of “numbering- up”. Although there has been considerable work on microfluidic PCR methods and devices, it should be noted that examples of large scale continuous PCR are limited. (See, US8163489, which is hereby incorporated by reference in its entirety) In order to perform large scale continuous PCR with acceptable levels of throughout and space/time yield, the flow rate is generally increased requiring much longer or larger internal diameter capillaries ensuring the internal volume and residence time is maintained.

[0010] The approach must remain balanced as each parameter affects the efficiency and quality of the PCR results. For example, increasing the flow rate and, consequently, the sheer stress can have detrimental effects on the polymerase enzyme. Likewise, increasing the length of the capillary increases the pressure drop and can lead to deactivation of the pressure sensitive polymerase. On the other hand, although an larger internal diameter capillary can eliminate the pressure stress, it also has an adverse effect on the mixing and turbulence within the capillary, resulting in inadequate heat and mass transfers and consequently low quality product.

[0011 ] One of the major drawbacks of a fully continuous bioreactor, capable of performing a typical number of PCR cycles (30 to 45), is the in large amount of waste, as the capillaries are often single-use and must be disposed of after each campaign.

[0012] A system and method to amplify a variety of nucleic acids targets in large volumes, specifically a method resulting in a high quality product and a high level of reproducibility, represents an inventive advance in the art.

SUMMARY

[0013] The shortcomings of the prior art are overcome by embodiments described herein.

[0014] Some embodiments provide a bioreactor for nucleic acid production using polymerase chain reaction (PCR), the bioreactor comprising: at least one fluidic pathway; at least two pumps capable of pumping an amount of a reaction mixture through the fluidic pathway; at least three temperature-controlled baths downstream of the pump, wherein the temperature-controlled bath receives the fluidic pathway, and the temperature-controlled bath is capable of heating the reaction mixture to a temperature enabling at least one PCR step selecting from the group consisting of: an activation step, a denaturation step, an annealing step, and an elongation step to occur; a central loop comprising a portion of the fluidic pathway corresponding to at least the denaturation, annealing, and elongation steps, wherein at least one pump is capable of recirculating the reaction mixture through the central loop to repeat the denaturation step, annealing step, and elongation step; and at least two valves outside of the central loop, wherein the valves are capable of controlling the amount of reaction mixture entering or exiting the central loop.

[0015] In some embodiments, the fluidic pathway comprises capillary tubing. In some embodiments, the capillary tubing comprises at least one material selected from the group consisting of: a metal, a plastic, and a silicone. In some embodiments, the capillary tubing comprises an internal diameter within the range of 0.5 mm to 10 mm. In some embodiments, the reaction mixture enters and exits the temperature-controlled bath through at least one connection selected from the group consisting of: a union joint (180 degree), an automated three-way connector, an automated four-way connector, a manual three-way connector, a manual four-way connector, and a switch valve. In some embodiments, the bioreactor comprises at least four temperature-controlled baths. In some embodiments, the temperature- controlled bath comprises a fluid. In some embodiments, the fluid is selected from the group consisting of: water or air.

[0016] In some embodiments, more than one PCR step occurs in the same temperature-controlled bath. In some embodiments, the activation step and the denaturation step occur in the same temperature-controlled bath. In some embodiments, each PCR step is performed in a different temperature-controlled bath. In some embodiments, the pump is outside the temperature-controlled bath. In some embodiments, the valve is outside the temperature controlled bath. In some embodiments, the fluidic pathway is wound around a frame more than once. In some embodiments, the pump is selected from the group consisting of: a peristaltic pump, a gear pump, a lobe pump, a membrane pump, and a syringe pump. [0017] Some embodiments provide a method of nucleic acid production using polymerase chain reaction (PCR), the method comprising: pumping reaction mixture into a fluidic pathway comprising portions corresponding to at least one of the following PCR steps: an activation step, a denaturation step, an annealing step, an elongation step, and a final elongation step; heating the reaction mixture with a temperature-controlled bath as the reaction mixture is pumped through the fluidic pathway; opening and closing at least one valve to control flow of reaction mixture into a central loop comprising at least the portions of the fluidic pathway corresponding to the following PCR steps: the denaturation step, the annealing step, and the elongation step; recirculating the reaction mixture within the central loop when at least two valves are closed to prevent flow of reaction mixture into or out of the central loop, wherein the denaturation step, the annealing step, and the elongation step are repeated for the reaction mixture within the central loop; and releasing PCR product from the central loop by opening the valve.

[0018] In some embodiments, the method further comprises pumping the reaction mixture to the portion of the fluidic pathway corresponding to the final elongation step. In some embodiments, the PCR product is in a batch within a range selected from the group consisting of: 100 to 20,000 base pairs. In some embodiments, the type of PCR is selected from the group consisting of: real-time PCR, quantitative real time PCR (Q-RT 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, variable number of tandem repeats (VNTR) PCR, asymmetric PCR, repetitive sequence-based PCR, overlap extension PCR, assemble PCR, intersequence-specific (ISSR) PCR, ligation- mediated PCR, methylation-specific PCR, and miniprimer PCR. In some embodiments, the reaction mixture is recirculated in the central loop for at least 30 cycles. In some embodiments, the reaction mixture is recirculated in the central loop for a number of cycles within the range of 30 to 40. In some embodiments, the PCR product has a volume within the range of 1 pl to 5 L. In some embodiments, heating comprises the temperature of the temperature-controlled bath for the activation step and the denaturation step being within the range of 85°C and 100°C. In some embodiments, heating comprises the temperature of the temperature-controlled bath for the annealing step being within the range of 55°C and 75°C. In some embodiments, heating comprises the temperature of the temperature-controlled bath for the elongation step and the final elongation step being within the range of 65°C and 80°C.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 provides a diagram of some embodiments of a PCR bioreactor. [0020] 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 equally 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

[0021] Some embodiments described herein provide a semi-continuous bioreactor which can be used to perform large scale PCR. Some embodiments of the bioreactor comprise three or more temperature controlled baths, capillary tubing used as a fluidic pathway for the reaction mixture flowing from one reaction step to the next, and pumps capable of driving the master mix through the tubes.

[0022] Some embodiments described herein combine the inherent advantages of capillary flow bioreactors, while also avoiding sheer and pressure stress on the polymerase in large scale, as well as significantly reducing the waste in single use systems. Flexibility for utilization of various PCR protocols may also be accomplished by the use of individual baths for each reaction step.

[0023] In some embodiments, the bioreactor 1 , as shown in FIG. 1 , may produce a PCR product with a length within the range of 100 to 20,000 base pairs. In some embodiments, the length of the PCR product is within a range selected from the group consisting of: 100 to 1000 base pairs, 500 to 5000 base pairs, 1000 to 10,000 base pairs, and 2000 to 20,000 base pairs. In some embodiments, the PCR product has a length of less than 100 base pairs. In some embodiments, the PCR has a length of greater than 20,000 base pairs.

[0024] Additionally, in some embodiments, the bioreactor 1 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-RT PCR), reverse transcriptase PCR (RT-PCR), multiplex PCR, nested PCR, long-range PCR, single-cell PCR, fast-cycling PCR, methylationspecific PCR (MSP), hot start PCR, high-fidelity PCR, In situ PCR, variable number of tandem repeats (VNTR) PCR, asymmetric PCR, repetitive sequence-based PCR, overlap extension PCR, assemble PCR, intersequence-specific (ISSR) PCR, ligation- mediated PCR, methylation-specific 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-RT 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 fastcycling 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 methylation-specific 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. Methods

[0025] Some embodiments herein describe a method of large-scale production of nucleic acids using polymerase chain reaction (PCR).

[0026] In some embodiments of a bioreactor 1 as shown in FIG. 1 , the reaction mixture may be continuously fed by Pump A from the source container 2 into a first portion of the capillary tubing 3 for the Activation step. In some embodiments, the source container 2 is a conical vial. In some embodiments, the capillary tubing 3 is completely filled with reaction mixture. Alternatively, the reaction mixture may be introduced into the capillary tubing 3 in segmented form by intercalation of an non- miscible gas, liquid, or oil forming sequential and separate segments.

[0027] Then, in some embodiments, the reaction mixture is pumped through the capillary tubing 3 for the Denaturation step, the Annealing step, and the Elongation step. In some embodiments, each portion of the capillary tubing 3 corresponding to a step is submerged in a separate temperature-controlled bath 5 at a temperature corresponding to the step.

[0028] In some embodiments, as the reaction mixture reaches the end of the portion of the capillary tubing 3 corresponding to the Elongation step, valves A and B are switched to close the central loop 4 of the bioreactor and prevent additional flow of reaction mixture in and out of the central loop 4. Simultaneously, in some embodiments, Pump B is switched into the “on” position to begin pumping and allow circulation within the central loop 4 for the predetermined number of cycles. On completion of the final cycle, in some embodiments, valves A and B are again switched to allow flow in and out of the central loop 4, and Pump A is used to allow continuous flow of the reaction mixture through the bioreactor 1 to the Final Elongation step. In some embodiments, the PCR product is continuously collected from the bioreactor 1 at the end of the PCR protocol.

[0029] 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.

[0030] In some embodiments, the Activation step is performed for 10 seconds. In some embodiments, the Activation step is performed for 11 seconds. In some embodiments, the Activation step is performed for 12 seconds. In some embodiments, the Activation step is performed for 13 seconds. In some embodiments, the Activation step is performed for 14 seconds. In some embodiments, the Activation step is performed for 15 seconds. In some embodiments, the Activation step is performed for 16 seconds. In some embodiments, the Activation step is performed for 17 seconds. In some embodiments, the Activation step is performed for 18 seconds. In some embodiments, the Activation step is performed for 19 seconds. In some embodiments, the Activation step is performed for 20 seconds. In some embodiments, the Activation step is performed for 21 seconds. In some embodiments, the Activation step is performed for 22 seconds. In some embodiments, the Activation step is performed for 23 seconds. In some embodiments, the Activation step is performed for 24 seconds. In some embodiments, the Activation step is performed for 25 seconds. In some embodiments, the Activation step is performed for 26 seconds. In some embodiments, the Activation step is performed for 27 seconds. In some embodiments, the Activation step is performed for 28 seconds. In some embodiments, the Activation step is performed for 29 seconds. In some embodiments, the Activation step is performed for 30 seconds. In some embodiments, the Activation step is performed for 31 seconds. In some embodiments, the Activation step is performed for 32 seconds. In some embodiments, the Activation step is performed for 33 seconds. In some embodiments, the Activation step is performed for 34 seconds. In some embodiments, the Activation step is performed for 35 seconds. In some embodiments, the Activation step is performed for 36 seconds. In some embodiments, the Activation step is performed for 37 seconds. In some embodiments, the Activation step is performed for 38 seconds. In some embodiments, the Activation step is performed for 39 seconds. In some embodiments, the Activation step is performed for 40 seconds. In some embodiments, the Activation step is performed for 41 seconds. In some embodiments, the Activation step is performed for 42 seconds. In some embodiments, the Activation step is performed for 43 seconds. In some embodiments, the Activation step is performed for 44 seconds. In some embodiments, the Activation step is performed for 45 seconds. In some embodiments, the Activation step is performed for 46 seconds. In some embodiments, the Activation step is performed for 47 seconds. In some embodiments, the Activation step is performed for 48 seconds. In some embodiments, the Activation step is performed for 49 seconds. In some embodiments, the Activation step is performed for 50 seconds. In some embodiments, the Activation step is performed for 51 seconds. In some embodiments, the Activation step is performed for 52 seconds. In some embodiments, the Activation step is performed for 53 seconds. In some embodiments, the Activation step is performed for 54 seconds. In some embodiments, the Activation step is performed for 55 seconds. In some embodiments, the Activation step is performed for 56 seconds. In some embodiments, the Activation step is performed for 57 seconds. In some embodiments, the Activation step is performed for 58 seconds. In some embodiments, the Activation step is performed for 59 seconds. In some embodiments, the Activation step is performed for 1 minute. In some embodiments, the Activation step is performed for 2 minutes. In some embodiments, the Activation step is performed for 3 minutes. In some embodiments, the Activation step is performed for 4 minutes. In some embodiments, the Activation step is performed for 5 minutes. In some embodiments, the Activation step is performed for 6 minutes. In some embodiments, the Activation step is performed for 7 minutes. In some embodiments, the Activation step is performed for 8 minutes. In some embodiments, the Activation step is performed for 9 minutes. In some embodiments, the Activation step is performed for 10 minutes. 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.

[0031] 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.

[0032] 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 2 minutes. In some embodiments, the Denaturation step is performed for 3 minutes. In some embodiments, the Denaturation step is performed for 4 minutes. In some embodiments, the Denaturation step is performed for 5 minutes. In some embodiments, the Denaturation step is performed for 6 minutes. In some embodiments, the Denaturation step is performed for 7 minutes. In some embodiments, the Denaturation step is performed for 8 minutes. In some embodiments, the Denaturation step is performed for 9 minutes. In some embodiments, the Denaturation step is performed for 10 minutes. In some embodiments, the Denaturation step is performed for less than 10 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. [0033] 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 time period 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.

[0034] In some embodiments, the Annealing step is performed for 1 minute. In some embodiments, the Annealing step is performed for less than one minute. In some embodiments, the Annealing step is performed for 2 minutes. In some embodiments, the Annealing step is performed for 3 minutes. In some embodiments, the Annealing step is performed for 4 minutes. In some embodiments, the Annealing step is performed for 5 minutes. In some embodiments, the Annealing step is performed for 6 minutes. In some embodiments, the Annealing step is performed for 7 minutes. In some embodiments, the Annealing step is performed for 8 minutes. In some embodiments, the Annealing step is performed for 9 minutes. In some embodiments, the Annealing step is performed for 10 minutes. 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.

[0035] 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 performed for 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 selected from 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.

[0036] 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 some embodiments, 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 some embodiments, 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. [0037] The movement of reaction mixture within the capillary tubing 3 and between the temperature-controlled baths 5 corresponds to the reaction steps and is controlled by the flow rate of the pump. In some embodiments, the PCR product produced by the method is in batch sizes selected from the range from 1 mL to 5 L. In some embodiments, the batch size was less than 1 mL. In some embodiments, the batch size is greater than 5 L. In some embodiments, the batch size is within a range selected from the group consisting of: 1 mL to 100 mL, 50 mL to 200 mL, 150 mL to 300 mL, 250 mL to 400 mL, 350 mL to 500 mL, 450 mL to 600 mL, 550 mL to 700 mL, 650 mL to 800 mL, 750 mL to 900 mL, 850 mL to 1 L, 950 mL to 1 .5 L, 1 L to 2 L, 1 .5 L to 3 L, 2 L to 3.5 L, 2.5 L to 4 L, 3 L to 4.5 L, and 3.5 L to 5 L.

II. System

[0038] Some embodiments herein describe a system for large-scale production of nucleic acids using polymerase chain reaction (PCR). In some embodiments, the system includes all connections. a. Capillary Tubing

[0039] Some embodiments of the method described herein are performed using a system comprising capillary tubing 3 as a reaction vessel.

[0040] In some embodiments, the capillary tubing 3 is wound on frames and submerged within the temperature-controlled bath 5. Alternatively, the capillary tubing 3 may be wound on frames mounted under the lid of the temperature- controlled bath 5. Additionally, in some embodiments, multiple frames with capillary tubing 3 may be prepared in advance of performing PCR and kept in storage, which allows quick changes of capillary tubing 3 between rounds of nucleic acid production using PCR. The frame may be constructed in any design and made of any material capable of supporting the type of winding and weight of the filled capillary tubing 3. In some embodiments, the capillary tubing 3 may be wound onto the frame in any design which encourages turbulent mixing of the reagents and also allows optimum contact between the thermal medium and outer surface of the capillary tubing 3. In some embodiments, capillary tubing 3 is wound in a design selected from the list consisting of: simple helical coils, 180 degree turns, and other more complex coiled flow inverters (CFI). (See, A. K. Saxena and K. D. P. Nigam , AIChE J., 1984, 30 , 363 — 368, which is hereby incorporated by reference in its entirety)

[0041] In some embodiments, the size and length of capillary tubing used for each step may be dictated by the flow rate and the reaction time required for the desired results. In some embodiments, the number of cycles for each of the Denaturation, Annealing, and Elongation steps has no influence on the length of the capillary tubing 3 of the bioreactor 1 because the reaction mixture is recirculated within the central loop 4 of the bioreactor 1.

[0042] The reaction vessel is capillary tubing 3 comprising any suitable material. In some embodiments, the capillary tubing 3 comprises at least one material selected from the group consisting of: metals, plastics, and silicones. In some embodiments, the capillary tubing 3 comprises at least one metal. In some embodiments, the capillary tubing 3 is made of a metal. In some embodiments, the capillary tubing 3 comprises a metal selected from the group consisting of: stainless steel, copper, and hastelloy. In some embodiments, the capillary tubing 3 comprises stainless steel. In some embodiments, the capillary tubing 3 comprises copper. In some embodiments, the capillary tubing 3 comprises hastelloy. In some embodiments, the capillary tubing 3 comprises at least one plastic selected from the group consisting of: perfluoroalkoxy (PFA), polysulfone, fluorinated ethylene propylene (FEP), and polyethylene (PE). In some embodiments, the capillary tubing 3 comprises PFA. In some embodiments, the capillary tubing 3 comprises polysulfone. In some embodiments, the capillary tubing 3 comprises FEP. In some embodiments, the capillary tubing 3 comprises PE.

[0043] In some embodiments, the capillary tubing 3 is sterilizable. In some embodiments, the capillary tubing 3 is single use. In some embodiments, the capillary tubing 3 is silicone tubing. In some embodiments, the capillary tubing 3 is platinum-cured silicone tubing. In some embodiments, the capillary tubing 3 is peroxide-cured silicone tubing. In some embodiments, the capillary tubing 3 is sterilizable and single use platinum cured silicone tubing.

[0044] In some embodiments, the internal diameter of the capillary tubing 3 is between 0.5 mm to 10 mm. In some embodiments, the internal diameter is within a range select from the group consisting of: 0.5 to 1 .5 mm, 1 .0 mm to 2.0 mm, 1 .5 mm to 2.5 mm, 2.0 mm to 3.0 mm, 2.5 mm to 3.5 mm, 3.0 mm to 4.0 mm, 3.5 mm to 4.5 mm, 4.0 mm to 5.0 mm, 4.5 mm to 5.5 mm, 5.0 mm to 6.0 mm, 5.5 mm to 7.0 mm, 6.0 mm to 7.0 mm, 6.5 mm to 7.5 mm, 7.0 mm to 8.0 mm, 7.5 mm to 8.5 mm, 8.0 mm to 9.0 mm, 8.5 mm to 9.5 mm, and 9.0 mm to 10.0 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 0.5 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 0.6 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 0.7 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 0.8 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 0.9 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 1.0 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 1.1 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 1 .2 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 1.3 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 1.4 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 1 .5 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 1.6 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 1.7 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 1 .8 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 1.9 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 2.0 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 2.1 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 2.2 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 2.3 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 2.4 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 2.5 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 2.6 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 2.7 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 2.8 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 2.9 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 3.0 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 3.1 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 3.2 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 3.3 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 3.4 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 3.5 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 3.6 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 3.7 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 3.8 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 3.9 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 4.0 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 4.1 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 4.2 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 4.3 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 4.4 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 4.5 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 4.6 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 4.7 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 4.8 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 4.9 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 5.0 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 5.1 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 5.2 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 5.3 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 5.4 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 5.5 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 5.6 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 5.7 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 5.8 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 5.9 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 6.0 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 6.1 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 6.2 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 6.3 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 6.4 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 6.5 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 6.6 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 6.7 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 6.8 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 6.9 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 7.0 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 7.1 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 7.2 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 7.3 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 7.4 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 7.5 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 7.6 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 7.7 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 7.8 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 7.9 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 8.0 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 8.1 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 8.2 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 8.3 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 8.4 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 8.5 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 8.6 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 8.7 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 8.8 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 8.9 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 9.0 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 9.1 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 9.2 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 9.3 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 9.4 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 9.5 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 9.6 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 9.7 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 9.8 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 9.9 mm. In some embodiments, the internal diameter of the capillary tubing 3 is 10.0 mm. In some embodiments, the diameter and length is calculated based on the volume of the batch required, as well as the rate of heat transfer required during the PCR protocol.

[0045] In some embodiments, the capillary tubing 3 can be prepared to carry out the PCR steps by first filling the capillary tubing 3 with at least one selected from the group consisting of: air, nitrogen, water, and buffer. b. Pump

[0046] In some embodiments, the flow rate of the pump is calculated based on the length and size of the capillary tubing 3, as well as the required reaction time for each reaction step of the PCR protocol.

[0047] In some embodiments, filling, emptying and/or movement of the reaction mixture through the bioreactor 1 can be performed with any type of pump. In some embodiments, the pump is a type selected from the group consisting of: a peristaltic pump, a gear pump, a lobe pump, a membrane pump, and a syringe pump. In some embodiments, at least one pump is the bioreactor 1 is a peristaltic pump. For example, the peristaltic pump may be easily disposable with sterilizable tubing. In some embodiments, at least one pump is a gear pump. In some embodiments, at least one pump is a lobe pump. In some embodiments, at least one pump is a membrane pump. In some embodiments, at least one pump is a syringe pump. In some embodiments, the bioreactor 1 comprises at least one pump. In some embodiments, the bioreactor 1 comprises at least two pumps. In some embodiments, the bioreactor 1 comprises two pumps. In some embodiments, the pumps are the same type of pump. In some embodiments, the pumps are different types of pumps. c. Temperature-Controlled Bath

[0048] In some embodiments, the temperature-controlled bath 5 may use air as the thermal medium, which is circulated within the bath, ensuring a uniform temperature profile by convection. Alternatively, the temperature-controlled bath 5 may use a fluid other than air as the thermal medium, which is circulated within the bath 5 to ensure a uniform temperature profile by convection. In some embodiments, the temperature in each bath 5 is separately controlled using electrical heating. In some embodiments, the temperature in each bath 5 is separately controlled with a thermostat. In some embodiments, any thermal fluid having suitable chemical compatibility with the capillary tubing 3 may be used as the thermal medium. In some embodiments, water is the thermal medium.

[0049] In some embodiments, the lid of each temperature-controlled bath 5 contains sealed ports for entry and exit of the capillary tubing 3. In some embodiments, connections between the capillary tubing 3 and pumps are located outside of the baths 5 to avoid contamination of the PCR product by the fluid or air in the bath. Any type of suitable connections may be used.

[0050] In some embodiments, at least one of the connections is selected from the group consisting of: a union joint (180 degree), an automated three-way connector or valve, an automated four-way connector or valve, a manual three-way connector or valve, a manual four-way connector or valve. In some embodiments, at least one connection is a union joint. In some embodiments, at least one connection is an automated three-way connector. In some embodiments, at least one connection is an automated four-way connector. In some embodiments, at least one connection is a manual three-way connector. In some embodiments, at least one connection is a manual four-way connector. In some embodiments, at least one connection is a switch valve.

[0051 ] In some embodiments, the number of temperature-controlled baths 5 in the bioreactor 1 is dependent on the complexity of the PCR protocol. In some embodiments, the number of temperature-controlled baths 5 is selected from the group consisting of: at least 1 , at least 2, at least 3, at least 4, at least 5, and at least 6. In some embodiments, the number of temperature-controlled baths 5 is within the range of 3 to 6. In some embodiments, the number of temperature-controlled baths 5 is 4. In some embodiments, the Activation and Denaturation steps may occur in the same temperature-controlled baths 5. In some embodiments, the Activation and Denaturation steps may occur in the same temperature-controlled baths 5. In some embodiments, the Activation, Denaturation, Annealing, Elongation, and Final Elongation steps each occur in separate temperature-controlled baths 5. In some embodiments, more than one PCR step is performed in each temperature-controlled bath 5. In some embodiments, each PCR step is performed in a different temperature-controlled bath 5.

[0052] In some embodiments, the temperature of each bath 5 is determined by the master mix and polymerase used. In some embodiments, the temperature for the Activation and Denaturation steps is within the range of 85°C to 100°C.

[0053] 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.

III. Definitions

[0054] 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.

[0055] As used herein, the singular forms "a", "an," and "the" include plural unless the context clearly dictates otherwise.

[0056] 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).

[0057] As used herein, the term “reaction mixture” refers to a master mix combined with the nucleic acid template.

EXAMPLES

Example 1. PCR Protocol

[0058] The following is a working example of a PCR protocol for target DNA of 2079 base pairs using the newly designed bioreactor as described in some embodiments described herein. The Master Mix was prepared in a 50 mL Eppendorf conical tube as follows: 3 mL Reaction Buffer; 7.8 mL Nuclease Free Water from VWR; 3 mL Enhancer appropriate for the polymerase; 300 pL dNTP Solution Mix (each dNTP at a concentration of 0.2mM) (1 OmM); 75 pL Primer (Forward Primer A 100pM); 75 pL Primer (Reverse Primer B 100pM); and 150 pL DNA Polymerase (2000U/ml). The master mix was combined with the 600 pL Template (1 OOpg/pl) to create the reaction mixture. The bioreactor vessel was formed using helical coils of 1.58 mm internal diameter (ID) of platinum-cured silicone tubing. The lengths of the tubing were as follows: 1. Activation: 4.5 m; 2. Denaturation: 1.5 m; 3. Annealing: 2.2 m; 4. Elongation: 3.2 m; and 5. Final Elongation: 7.7 m.

[0059] Two peristaltic pumps were equipped with a 3-stop, 2.03 mm ID pump tubing and connected to the prepared bioreactor. The first pump was used to control the filling and emptying of the bioreactor as well as movement of the reaction mixture within the Activation and Final Elongation capillary tubes. The second pump was used to control the Denaturing, Annealing, and Elongation cycles within a central loop. Four different temperatures were used for the different temperature-controlled baths 5. As Activation and Denaturation are performed at the same temperature, one temperature-controlled bath 5 was used for both capillaries at a temperature of 98°C. The Annealing step was performed at 63°C. The Elongation step was performed at 72°C. The Final Elongation step was performed at 72°C. The pump head tubing and bioreactor capillary tubing were completely filled with buffer solution and the chosen flow rates were set.

[0060] The optimum flow rate was calculated to be 7.5 mL/min for the following PCR protocol: Activation step - 71 seconds; 30 cycles of Denaturation - 24 seconds, Annealing - 34 seconds, and Elongation - 50 seconds; Additional Elongation - 120 seconds. To begin the reaction, the pump was started and the reaction mixture was transferred from the Eppendorf conical tube to the Activation portion of the capillary tubing in the bioreactor. The reaction mixture was continuously fed from one bioreactor to the next via the pumping action. As the reaction mixture reached the end of the portion of the capillary tubing used for the Elongation step, the switch valves and second pump were used to ensure only circulation within the Denaturation, Annealing and Elongation portions of the capillary tubing. The reaction mixture was continuously circulated in this loop for 29 more cycles. At the end of the 30 cycles, the switch valves were opened again used to feed the reaction mixture into the Final Elongation portion of the capillary tubing. The product exited the bioreactor and was continuously collected in a new sterile conical tube. A 2 L sample of the product was diluted to 20 pL (1 Ox) and analyzed on 1 % Agarose Gel using electrophoresis. The gel indicated the presence of the expected 2 kbp product based on the process parameters described above.

EQUIVALENTS

[0061] 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 the like, 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.

[0062] 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.

[0063] Publications of patent applications and patents and other non-patent 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

CLAIMS What is claimed is:

1 . A bioreactor for nucleic acid production using polymerase chain reaction (PCR), the bioreactor comprising: a) at least one fluidic pathway; b) at least two pumps capable of pumping an amount of a reaction mixture through the fluidic pathway; c) at least three temperature-controlled baths downstream of the pump, wherein the temperature-controlled bath receives the fluidic pathway, and the temperature- controlled bath is capable of heating the reaction mixture to a temperature enabling at least one PCR step selecting from the group consisting of: an activation step, a denaturation step, an annealing step, and an elongation step to occur; d) a central loop comprising a portion of the fluidic pathway corresponding to at least the denaturation, annealing, and elongation steps, wherein at least one pump is capable of recirculating the reaction mixture through the central loop to repeat the denaturation step, annealing step, and elongation step; and e) at least two valves outside of the central loop, wherein the valves are capable of controlling the amount of reaction mixture entering or exiting the central loop.

2. The bioreactor of claim 1 , wherein the fluidic pathway comprises capillary tubing.

3. The bioreactor of any one of claims 1 and 2, wherein the capillary tubing comprises at least one material selected from the group consisting of: a metal, a plastic, and a silicone.

4. The bioreactor of any one of claims 1 to 3, wherein the capillary tubing comprises an internal diameter within the range of 0.5 mm to 10 mm.

5. The bioreactor of any one of claims 1 to 4, wherein reaction mixture enters and exits the temperature-controlled bath through at least one connection selected from the group consisting of: a union joint (180 degree), an automated three-way connector, an automated four-way connector, a manual three-way connector, a manual four-way connector, and a switch valve.

6. The bioreactor of any one of claims 1 to 5, wherein the bioreactor comprises at least four temperature-controlled baths.

7. The bioreactor of any one of claims 1 to 6, wherein the temperature-controlled bath comprises a fluid.

8. The bioreactor of claim 7, wherein the fluid is selected from the group consisting of: water or air.

9. The bioreactor of any one of claims 1 to 8, wherein more than one PCR step occurs in the same temperature-controlled bath.

10. The bioreactor of claim 9, wherein the activation step and the denaturation step occur in the same temperature-controlled bath.

11 . The bioreactor of any one of claims 1 to 8, wherein each PCR step is performed in a different temperature-controlled bath.

12. The bioreactor of any one of claims 1 to 11 , wherein the pump is outside the temperature-controlled bath.

13. The bioreactor of any one of claims 1 to 12, wherein the valve is outside the temperature controlled bath.

14. The bioreactor of any one of claims 1 to 13, wherein the fluidic pathway is wound around a frame more than once.

15. The bioreactor of any one of claims 1 to 14, wherein the pump is selected from the group consisting of: a peristaltic pump, a gear pump, a lobe pump, a membrane pump, and a syringe pump

16. A method of nucleic acid production using polymerase chain reaction (PCR), the method comprising: a) pumping reaction mixture into a fluidic pathway comprising portions corresponding to at least one of the following PCR steps: an activation step, a denaturation step, an annealing step, an elongation step, and a final elongation step; b) heating the reaction mixture with a temperature-controlled bath as the reaction mixture is pumped through the fluidic pathway; c) opening and closing at least one valve to control flow of reaction mixture into a central loop comprising at least the portions of the fluidic pathway corresponding to the following PCR steps: the denaturation step, the annealing step, and the elongation step; d) recirculating the reaction mixture within the central loop when at least two valves are closed to prevent flow of reaction mixture into or out of the central loop, wherein the denaturation step, the annealing step, and the elongation step are repeated for the reaction mixture within the central loop; and e) releasing PCR product from the central loop by opening the valve.

17. The method of claim 16, further comprising pumping the reaction mixture to the portion of the fluidic pathway corresponding to the final elongation step.

18. The method of any one of claims 16 and 17, wherein the PCR product is in a batch within a range selected from the group consisting of: 100 to 20,000 base pairs.

19. The method of any of claims 16 to 18, wherein the type of PCR is selected from the group consisting of: real-time PCR, quantitative real time PCR (Q-RT 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, variable number of tandem repeats (VNTR) PCR, asymmetric PCR, repetitive sequence-based PCR, overlap extension PCR, assemble PCR, intersequence-specific (ISSR) PCR, ligation-mediated PCR, methylation-specific PCR, and miniprimer PCR.

20. The method of any of claims 16 to 19, wherein the reaction mixture is recirculated in the central loop for at least 30 cycles.

21 . The method of any of claims 16 to 20, wherein the reaction mixture is recirculated in the central loop for a number of cycles within the range of 30 to 40.

22. The method of any of claims 16 to 21 , wherein the PCR product has a volume within the range of 1 pl to 5 L.

23. The method of any of claims 16 to 22, wherein heating comprises the temperature of the temperature-controlled bath for the activation step and the denaturation step being within the range of 85°C and 100°C.

24. The method of any of claims 16 to 23, wherein heating comprises the temperature of the temperature-controlled bath for the annealing step being within the range of 55°C and 75°C.

25. The method of any of claims 16 to 24, wherein heating comprises the temperature of the temperature-controlled bath for the elongation step and the final elongation step being within the range of 65°C and 80°C.