Semicontinuous method and system for producing nucleic acids

Abstract

This disclosure relates to a bioreactor for mass production of nucleic acids using polymerase chain reaction (PCR) and a method for using the same.

Description

Technical Field

[0001] The embodiments described herein relate to methods and systems for mass-producing nucleic acids using polymerase chain reaction (PCR). More specifically, some embodiments of the present technology relate to a PCR method using a semi-continuous bioreactor.

Background Art

[0002] Polymerase chain reaction (PCR) is a method used to amplify nucleic acids in vitro in molecular biology. This method consists of an initial activation step and a cycle that repeats the following three additional steps: 1) a step of denaturing double-stranded deoxyribonucleic acid DNA into two accessible single strands, 2) a step of annealing primers complementary to each single strand, and 3) a step of extending (also referred to as elongation) the nucleic acid strand using a polymerase enzyme to synthesize a new complementary DNA strand. Conventional PCR is performed by setting it in a thermal cycling PCR apparatus including a heating block, on a plate equipped with a plurality of 20-250 μL conical vials.

[0003] By using a microfluidic capillary or a microchannel based on a chip design as a reaction vessel for DNA amplification, many advantages are brought, such as rapid heat conduction, compact design, high throughput for analysis, minimal installation area, and ease of integrated automation. Microfluidic PCR devices have swirl flow and oscillatory flow designs (see Kopp et al., 1998, Micro Total Anal. Syst., 98, 7-10, which is incorporated herein by reference in its entirety).

[0004] Chip-type designs have limitations in their working capacity and have recently been surpassed by helical capillary designs incorporating either a metal heating block (Peltier element) or a tank containing a heat transfer medium as a heat exchanger (these are all incorporated herein by reference, KR20050078568; US20080145923; Park et al. 2003, Anal. Chem., 75, 6029-33; and Kim et al. 2016, Bull. Korean Chem. Soc., 37, 1878-81). Such heating elements may be divided into multiple regions of varying sizes and temperatures to accommodate optimal conditions for the activation, denaturation, annealing, and extension steps. DNA is amplified as a continuous process by supplying a mixture to a capillary wound around the heating element. Similar to microfluidic chip PCR, multiple chips or heating elements are likely to be required to cover a wide range of DNA targets (see US8163489, the entire structure of which is incorporated herein by reference). Alternatively, a heating block segmented into multiple sections may be used. In these devices, some variation in the PCR protocol is tolerated by temperature control of each section / region. In previous designs, the possibility of physical scaling was limited by the small contact area between the electric heating element and the capillary tube and the low thermal conductivity. Using a bath with a heat transfer medium eliminates these limitations, as the outside of the tube is completely immersed in the medium.

[0005] A fully continuous PCR apparatus employing four temperature-controlled fluid chambers and capillary tubes into which the master mix is ​​pumped has already been described (the entire apparatus is incorporated herein by reference, see US7217699). To maximize yield, multiple cycles are performed by having the solution exit the final chamber re-enter the first chamber and proceed along the microfluidic pathway. Thus, each cycle corresponds to an additional set of capillary coils for the activation, denaturation, annealing, and extension steps, resulting in a large number of tubes per PCR cycle.

[0006] Another form of PCR apparatus is one of several designs for nucleic acid amplification, one of which pumps the reaction mixture into a continuous, repeating loop between two temperature-controlled fluid baths (the entire design is incorporated herein by reference, see US5720923). Its major drawback is its lack of variability, making more complex PCR protocols impossible. Furthermore, the incorporation of valves immersed in the fluid baths can lead to damage or contamination of the connections.

[0007] For the industrial production of nucleic acids using PCR, scalability remains the biggest constraint. Mass production is typically achieved through either a "scale-up" approach or a "numbering-up" approach, which involves combining products obtained from multiple microliter conical tubes. Traditional "scale-up" offers advantages such as practicality and smaller footprint. However, the low thermal conductivity and mass transfer within these containers, combined with the stringent temperature and time conditions required for PCR protocols, often leads to low yields and inconsistent quality.

[0008] Alternatively, a "scaling up" approach utilizes the high surface area / volume ratio of microliter conical tubes for rapid heat transfer. However, this also has some drawbacks, such as generating more waste, requiring a larger footprint, and increasing costs due to the additional equipment needed to fill and empty the microliter tubes.

[0009] Capillary bioreactors can serve as a compromise between the "scaling up" and "increasing by numbers" approaches by utilizing rapid heat conduction and mass transfer in small dimensions while avoiding the redundancy of "increasing by numbers." It should be noted that while considerable research has been done on microfluidic PCR methods and apparatus, examples of large-scale, continuous PCR are limited (see US8163489, the entire case of which is incorporated herein by reference). To perform large-scale, continuous PCR at acceptable levels of throughput and space-time yield, the flow rate is generally increased, which requires much longer and larger diameter capillaries that can reliably maintain internal volume and residence time.

[0010] Since each parameter affects the efficiency and quality of the PCR process, such approaches must be kept in balance. For example, increasing the flow rate can increase shear stress, which can have a detrimental effect on the polymerase enzyme. Furthermore, a longer capillary increases pressure loss, which can inactivate pressure-sensitive polymerase. On the other hand, while a larger capillary diameter can eliminate pressure-induced stress, it also negatively impacts mixing and turbulence within the capillary, resulting in insufficient heat conduction and mass transfer, and ultimately degrading product quality.

[0011] One major drawback of fully continuous bioreactors capable of performing a typical number of PCR cycles (30-45) is the large amount of waste generated, as capillaries are often single-use and must be discarded after each operation.

[0012] Systems and methods for amplifying various nucleic acid targets in large quantities, specifically methods that yield high-quality products and a high level of reproducibility, constitute inventive advances in the field. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] Korean Patent Application Publication No. 2005 / 0078568 [Patent Document 2] U.S. Patent Application Publication No. 2008 / 0145923 [Patent Document 3] U.S. Patent No. 8163489 [Patent Document 4] U.S. Patent No. 7217699 [Patent Document 5] U.S. Patent No. 5720923 [Non-patent literature]

[0014] [Non-Patent Document 1] Kopp et al.,1998,Micro Total Anal.Syst.,98,7-10 [Non-Patent Document 2] Park et al.,2003,Anal.Chem.,75,6029-33 [Non-Patent Document 3] Kim et al.,2016,Bull.Korean Chem.Soc.,37,1878-81 [Non-Patent Document 4] AKSaxena and KDPNigam, AIChE J., 1984, 30, 363-368 [Overview of the project] [Problems that the invention aims to solve]

[0015] The shortcomings of the prior art are overcome by the embodiments described herein. [Means for solving the problem]

[0016] Some embodiments provide a bioreactor for producing nucleic acids using polymerase chain reaction (PCR), wherein the bioreactor has at least one fluid pathway; The invention comprises: at least two pumps capable of supplying a constant amount of reaction mixture into a fluid pathway; at least three constant temperature baths located downstream of the pumps, the constant temperature baths receiving the fluid pathway, and the constant temperature baths capable of heating the reaction mixture to a temperature that allows at least one PCR step, selected from the group consisting of an activation step, a denaturation step, an annealing step, and an extension step, to occur; a central loop including a portion of the fluid pathway corresponding to at least the denaturation step, the annealing step, and the extension step, the central loop capable of recirculating the reaction mixture in the central loop so as to repeat the denaturation step, the annealing step, and the extension step; and at least two valves located outside the central loop, the valves capable of controlling the amount of reaction mixture entering and leaving the central loop.

[0017] In some embodiments, the fluid path is configured to include capillaries. In some embodiments, the capillaries are composed of at least one material selected from the group consisting of metal, plastic, and silicone. In some embodiments, the capillaries have an inner diameter in the range of 0.5 mm to 10 mm. In some embodiments, the reaction mixture enters and leaves the constant temperature bath through at least one connection selected from the group consisting of union fittings (180 degrees), automatic three-way connectors, automatic four-way connectors, manual three-way connectors, manual four-way connectors, and switching valves. In some embodiments, the bioreactor includes at least four constant temperature baths. In some embodiments, the constant temperature baths contain a fluid. In some embodiments, the fluid is selected from the group consisting of water or air.

[0018] In some embodiments, two or more PCR steps occur in the same thermostatic chamber. In some embodiments, the activation step and the denaturation step occur in the same thermostatic chamber. In some embodiments, each PCR step is performed in a different thermostatic chamber. In some embodiments, the pump is located outside the thermostatic chamber. In some embodiments, the valve is located outside the thermostatic chamber. In some embodiments, the fluid path is wound around the 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.

[0019] Some embodiments provide a method for producing nucleic acids using polymerase chain reaction (PCR), the method comprising pumping a reaction mixture into a fluid path comprising a portion corresponding to at least one of the following PCR steps: an activation step, a denaturation step, an annealing step, an extension step, and a final extension step; heating the reaction mixture in a thermostatic chamber when pumping the reaction mixture into the fluid path; controlling the inflow of the reaction mixture into a central loop comprising at least a portion of the fluid path corresponding to the following PCR steps: a denaturation step, an annealing step, and an extension step, by opening and closing at least one valve; recirculating the reaction mixture in the central loop when at least two valves are closed to prevent inflow into or outflow from the central loop, repeating the denaturation step, the annealing step, and the extension step on the reaction mixture in the central loop; and releasing the PCR product from the central loop by opening a valve.

[0020] In some embodiments, the method further includes pumping the reaction mixture into a portion of the fluid path corresponding to the final extension 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, high-speed cycling PCR, methylation-specific PCR (MSP), hot-start PCR, high-fidelity PCR, in situ PCR, variable number tandem repeat (VNTR) PCR, asymmetric PCR, repetitive extragenic palindromic (rep) PCR, overlap extension (OE) PCR, assembly PCR, inter-simple sequence repeat (ISSR) PCR, ligation-mediated (LM) 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 μL to 5 L. In some embodiments, by heating, the temperature of the thermostatic bath for the activation step and the denaturation step is within the range of 85°C to 100°C. In some embodiments, by heating, the temperature of the thermostatic bath for the annealing step is within the range of 55°C to 75°C. In some embodiments, the temperature of the thermostatic bath for the extension step and the final extension step is within the range of 65°C to 80°C.

Brief Description of the Drawings

[0021] [Figure 1] Shows some embodiments of the PCR bioreactor.

Modes for Carrying Out the Invention

[0022] The accompanying drawings illustrate some embodiments disclosed herein and are not intended to limit their scope, and the present invention may allow for other equally effective embodiments. Elements and features in any embodiment may be present in other embodiments without further description, and it should be understood that, where possible, the same reference numerals are used to indicate equivalent elements common to each figure.

[0023] Some embodiments described herein provide semicontinuous bioreactors that can be used for performing large-scale PCR. Some embodiments of such bioreactors include three or more incubators, capillaries used as fluid pathways for flowing reaction mixtures from one reaction step to the next, and pumps that can pass a master mix through the capillaries.

[0024] Some embodiments described herein combine the inherent advantages of capillary flow bioreactors with the avoidance of shear and pressure stress on polymerases in large-scale applications, and significantly reduce waste in single-use systems. Flexibility for utilizing various PCR protocols can also be achieved by using separate vessels for each reaction step.

[0025] In some embodiments, a bioreactor 1, as shown in Figure 1, can produce a PCR product having a length in 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 1,000 base pairs, 500 to 5,000 base pairs, 1,000 to 10,000 base pairs, and 2,000 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 more than 20,000 base pairs.

[0026] Furthermore, 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, methylation-specific PCR (MSP), hot-start PCR, high-fidelity PCR, in situ PCR, variable repeat polymorphism (VNTR) PCR, asymmetric PCR, repeat-type (rep) PCR, overlap extension (OE) PCR, assembly PCR, simple repeat-to-repeater specific (ISSR) PCR, ligation-mediated (LM) PCR, methylation-specific PCR, and mini-primer 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 PCR protocol is long-range PCR. In some embodiments, the PCR protocol is single-cell PCR. In some embodiments, the PCR protocol is fast cycling PCR. In some embodiments, the PCR protocol is methylation-specific PCR (MSP). In some embodiments, the PCR protocol is hot-start PCR. In some embodiments, the PCR protocol is high-fidelity PCR. In some embodiments, the PCR protocol is in situ PCR. In some embodiments, the PCR protocol is variable repeat polymorphism (VNTR) PCR. In some embodiments, the PCR protocol is asymmetric PCR.In some embodiments, the PCR protocol is repetitive sequence-type (rep) PCR. In some embodiments, the PCR protocol is overlap extension (OE) PCR. In some embodiments, the PCR protocol is assembly PCR. In some embodiments, the PCR protocol is simple repetitive sequence-specific (ISSR) PCR. In some embodiments, the PCR protocol is ligation-mediated (LM) PCR. In some embodiments, the PCR protocol is methylation-specific PCR. In some embodiments, the PCR protocol is miniprimer PCR. In some embodiments, the type of PCR performed is reverse transcriptase (RT) PCR using RNA-directed DNA polymerase.

[0027] I. Method Some embodiments described herein describe methods for mass-producing nucleic acids using polymerase chain reaction (PCR).

[0028] In some embodiments of the bioreactor 1, as shown in Figure 1, the reaction mixture may be continuously supplied by pump A from the raw material container 2 to the first portion of the capillary tube 3 for the activation step. In some embodiments, the raw material container 2 is a conical vial. In some embodiments, the capillary tube 3 is completely filled with the reaction mixture. Alternatively, the reaction mixture may be introduced into the capillary tube 3 in a segmented form by introducing an immiscible gas, liquid, or oil to form continuously separated regions.

[0029] Subsequently, in some embodiments, the reaction mixture is pumped into the capillary tube 3 for the denaturation step, the annealing step, and the extension step. In some embodiments, each portion of the capillary tube 3 corresponding to each step is immersed in a separate constant-temperature bath 5 at the temperature corresponding to that step.

[0030] In some embodiments, when the reaction mixture reaches the end of the portion of the capillary tube 3 corresponding to the extension step, valves A and B are switched to close the central loop 4 of the bioreactor, preventing further inflow and outflow of the reaction mixture into and out of the central loop 4. Simultaneously, in some embodiments, pump B is switched to the ON position and begins pumping, allowing a predetermined number of cycles of circulation within the central loop 4. Upon completion of the final cycle, in some embodiments, valves A and B are switched again to allow inflow and outflow into and out of the central loop 4, and pump A is used to allow the reaction mixture to flow continuously through the bioreactor 1 to the final extension step. In some embodiments, the PCR product is continuously recovered from the bioreactor 1 at the end of the PCR protocol.

[0031] The time for each reaction step is determined by the PCR protocol best suited to each master mix. In some embodiments, the activation step is performed for a time selected from the range of 10 seconds to 10 minutes. In some embodiments, the activation step is performed for a time in the range of 5 seconds to 1 minute. In some embodiments, the activation step is performed for a time in the range of 1 minute to 2 minutes. In some embodiments, the activation step is performed for a time in the range of 2 minutes to 3 minutes. In some embodiments, the activation step is performed for a time in the range of 3 minutes to 4 minutes. In some embodiments, the activation step is performed for a time in the range of 4 minutes to 5 minutes. In some embodiments, the activation step is performed for a time in the range of 5 minutes to 6 minutes. In some embodiments, the activation step is performed for a time in the range of 6 minutes to 7 minutes. In some embodiments, the activation step is performed for a time in the range of 7 minutes to 8 minutes. In some embodiments, the activation step is performed for a time in the range of 8 minutes to 9 minutes. In some embodiments, the activation step is performed for a time in the range of 9 minutes to 10 minutes.

[0032] 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 more than 10 minutes. In some embodiments, the activation step is performed for more than 10 seconds.

[0033] In some embodiments, the modification step is performed for a time selected from the range of 10 seconds to 10 minutes. In some embodiments, the modification step is performed for a time within the range of 5 seconds to 1 minute. In some embodiments, the modification step is performed for a time within the range of 1 minute to 2 minutes. In some embodiments, the modification step is performed for a time within the range of 2 minutes to 3 minutes. In some embodiments, the modification step is performed for a time within the range of 3 minutes to 4 minutes. In some embodiments, the modification step is performed for a time within the range of 4 minutes to 5 minutes. In some embodiments, the modification step is performed for a time within the range of 5 minutes to 6 minutes. In some embodiments, the modification step is performed for a time within the range of 6 minutes to 7 minutes. In some embodiments, the modification step is performed for a time within the range of 7 minutes to 8 minutes. In some embodiments, the modification step is performed for a time within the range of 8 minutes to 9 minutes. In some embodiments, the modification step is performed for a time within the range of 9 minutes to 10 minutes.

[0034] In some embodiments, the denaturation step is performed for 1 minute. In some embodiments, the denaturation step is performed for less than 1 minute. 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 more than 10 minutes. In some embodiments, the denaturation step is performed for more than 10 seconds.

[0035] In some embodiments, the annealing step is performed for a time selected from a range of 10 seconds to 10 minutes. In some embodiments, the annealing step is performed for a time in the range of 5 seconds to 1 minute. In some embodiments, the annealing step is performed for a time in the range of 1 minute to 2 minutes. In some embodiments, the annealing step is performed for a time in the range of 2 minutes to 3 minutes. In some embodiments, the annealing step is performed for a time in the range of 3 minutes to 4 minutes. In some embodiments, the annealing step is performed for a time in the range of 4 minutes to 5 minutes. In some embodiments, the annealing step is performed for a time in the range of 5 minutes to 6 minutes. In some embodiments, the annealing step is performed for a time in the range of 6 minutes to 7 minutes. In some embodiments, the annealing step is performed for a time in the range of 7 minutes to 8 minutes. In some embodiments, the annealing step is performed for a time in the range of 8 minutes to 9 minutes. In some embodiments, the annealing step is performed for a time in the range of 9 minutes to 10 minutes.

[0036] In some embodiments, the annealing step is performed for 1 minute. In some embodiments, the annealing step is performed for less than 1 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 more than 10 minutes. In some embodiments, the annealing step is performed for more than 10 seconds.

[0037] In some embodiments, the stretching step is performed for a time selected from a range of 30 seconds to 1 minute. In some embodiments, the stretching step is performed for a time selected from a range of 1 minute to 2 minutes. In some embodiments, the stretching step is performed for a time selected from a range of 2 minutes to 3 minutes. In some embodiments, the stretching step is performed for a time selected from a range of 3 minutes to 4 minutes. In some embodiments, the stretching step is performed for a time selected from a range of 4 minutes to 5 minutes. In some embodiments, the stretching step is performed for a time selected from a range of 5 minutes to 6 minutes. In some embodiments, the stretching step is performed for a time selected from a range of 6 minutes to 7 minutes. In some embodiments, the stretching step is performed for a time selected from a range of 7 minutes to 8 minutes. In some embodiments, the stretching step is performed for a time selected from a range of 8 minutes to 9 minutes. In some embodiments, the stretching step is performed for a time selected from a range of 9 minutes to 10 minutes. In some embodiments, the stretching step is performed for a time selected from a range of 10 minutes to 11 minutes. In some embodiments, the stretching step is performed for a time selected from a range of 11 minutes to 12 minutes. In some embodiments, the extension step is performed for a time selected from the range of 12 to 13 minutes. In some embodiments, the extension step is performed for a time selected from the range of 13 to 14 minutes. In some embodiments, the extension step is performed for a time selected from the range of 14 to 15 minutes. In some embodiments, the extension step is performed for a time selected from the range of 15 to 16 minutes. In some embodiments, the extension step is performed for a time selected from the range of 16 to 17 minutes. In some embodiments, the extension step is performed for a time selected from the range of 17 to 18 minutes. In some embodiments, the extension step is performed for a time selected from the range of 18 to 19 minutes. In some embodiments, the extension step is performed for a time selected from the range of 19 to 20 minutes. In some embodiments, the extension step is performed for a time selected from the range of 20 to 21 minutes.In some embodiments, the extension step is performed for a time selected from the range of 21 to 22 minutes. In some embodiments, the extension step is performed for a time selected from the range of 22 to 23 minutes. In some embodiments, the extension step is performed for a time selected from the range of 23 to 24 minutes. In some embodiments, the extension step is performed for a time selected from the range of 24 to 25 minutes. In some embodiments, the extension step is performed for a time selected from the range of 25 to 26 minutes. In some embodiments, the extension step is performed for a time selected from the range of 26 to 27 minutes. In some embodiments, the extension step is performed for a time selected from the range of 27 to 28 minutes. In some embodiments, the extension step is performed for a time selected from the range of 28 to 29 minutes. In some embodiments, the extension step is performed for a time selected from the range of 29 to 30 minutes. In some embodiments, the extension step is performed for a time selected from the range of 30 to 31 minutes. In some embodiments, the extension step is performed for less than 30 seconds. In some embodiments, the extension step is performed for more than 30 minutes.

[0038] In some embodiments, the final extension step is omitted. In some embodiments, the final extension step is performed for less than 30 seconds. In some embodiments, the final extension step is performed for a time selected from the range of 30 seconds to 1 minute. In some embodiments, the final extension step is performed for a time selected from the range of 1 minute to 2 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 2 minutes to 3 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 3 minutes to 4 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 4 minutes to 5 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 5 minutes to 6 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 6 minutes to 7 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 7 minutes to 8 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 8 minutes to 9 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 9 minutes to 10 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 10 to 11 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 11 to 12 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 12 to 13 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 13 to 14 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 14 to 15 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 15 to 16 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 16 to 17 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 17 to 18 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 18 to 19 minutes.In some embodiments, the final extension step is performed for a time selected from the range of 19 to 20 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 20 to 21 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 21 to 22 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 22 to 23 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 23 to 24 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 24 to 25 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 25 to 26 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 26 to 27 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 27 to 28 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 28 to 29 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 29 to 30 minutes. In some embodiments, the final extension step is performed for a time selected from the range of 30 to 31 minutes. In some embodiments, the final extension step is performed for more than 30 minutes.

[0039] The movement of the reaction mixture between the capillary tube 3 and the constant temperature bath 5 corresponds to each reaction step and is controlled by the flow rate of the pump. In some embodiments, the PCR product produced by the method of the present invention has a batch size selected from the range of 1 mL to 5 L. In some embodiments, the batch size is less than 1 mL. In some embodiments, the batch size is greater than 5 L. In some embodiments, the batch size is within the 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.

[0040] II. Systems Some embodiments described herein describe systems for mass production of nucleic acids using polymerase chain reaction (PCR). In some embodiments, such systems include all connecting parts.

[0041] a. Capillary Some embodiments of the methods described herein are carried out using a system that includes a capillary tube 3 as a reaction vessel.

[0042] In some embodiments, the capillary tubes 3 are wrapped around a frame and submerged in a constant temperature bath 5. Alternatively, the capillary tubes 3 may be wrapped around a frame provided on the inside of the lid of the constant temperature bath 5. Furthermore, in some embodiments, multiple frames having capillary tubes 3 may be prepared and stored before PCR is performed, thereby allowing for rapid replacement of the capillary tubes 3 after each cycle of nucleic acid production using PCR. The frame may be of any design and may be made of any material that can support the way the filled capillary tubes 3 are wrapped and their weight. In some embodiments, the capillary tubes 3 may be wrapped around the frame in any design that promotes turbulent mixing of reagents and allows for optimal contact between the heat transfer medium and the outer surface of the capillary tubes 3. In some embodiments, the capillary tube 3 is wound in a design selected from a list consisting of a simple helical coil, a 180-degree fold, and other more complex coiled flow inverters (CFIs) (the whole of which is incorporated herein by reference, see AKSaxena and KDPNigam, AIChE J., 1984, 30, 363-368).

[0043] In some embodiments, the size and length of the capillaries used in each step may be determined by the flow rate and reaction time required for the desired result. In some embodiments, the number of cycles for each denaturation, annealing, and extension step does not affect the length of the capillaries 3 in the bioreactor 1, as the reaction mixture is recirculated within the central loop 4 of the bioreactor 1.

[0044] The reaction vessel is a capillary tube 3 composed of any suitable material. In some embodiments, the capillary tube 3 is composed of at least one material selected from the group consisting of metals, plastics, and silicones. In some embodiments, the capillary tube 3 is composed of at least one metal. In some embodiments, the capillary tube 3 is made of metal. In some embodiments, the capillary tube 3 is composed of a metal selected from the group consisting of stainless steel, copper, and Hastelloy. In some embodiments, the capillary tube 3 is composed of stainless steel. In some embodiments, the capillary tube 3 is composed of copper. In some embodiments, the capillary tube 3 is composed of Hastelloy. In some embodiments, the capillary tube 3 is composed of 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 tube 3 is composed of PFA. In some embodiments, the capillary tube 3 is composed of polysulfone. In some embodiments, the capillary tube 3 is composed of FEP. In some embodiments, the capillary tube 3 is composed of PE.

[0045] In some embodiments, the capillary tube 3 is sterilizable. In some embodiments, the capillary tube 3 is single-use. In some embodiments, the capillary tube 3 is a silicone tube. In some embodiments, the capillary tube 3 is a platinum-cured silicone tube. In some embodiments, the capillary tube 3 is a peroxide-cured silicone tube. In some embodiments, the capillary tube 3 is a sterilizable, single-use platinum-cured silicone tube.

[0046] In some embodiments, the inner diameter of the capillary tube 3 is 0.5 mm to 10 mm. In some embodiments, the inner diameter is within a range selected from the group consisting of 0.5 mm 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 inner diameter of the capillary tube 3 is 0.5 mm. In some embodiments, the inner diameter of capillary tube 3 is 0.6 mm. In some embodiments, the inner diameter of capillary tube 3 is 0.7 mm. In some embodiments, the inner diameter of capillary tube 3 is 0.8 mm. In some embodiments, the inner diameter of capillary tube 3 is 0.9 mm. In some embodiments, the inner diameter of capillary tube 3 is 1.0 mm. In some embodiments, the inner diameter of capillary tube 3 is 1.1 mm. In some embodiments, the inner diameter of capillary tube 3 is 1.2 mm. In some embodiments, the inner diameter of capillary tube 3 is 1.3 mm. In some embodiments, the inner diameter of capillary tube 3 is 1.4 mm. In some embodiments, the inner diameter of capillary tube 3 is 1.5 mm. In some embodiments, the inner diameter of capillary tube 3 is 1.6 mm. In some embodiments, the inner diameter of capillary tube 3 is 1.7 mm. In some embodiments, the inner diameter of capillary tube 3 is 1.8 mm. In some embodiments, the inner diameter of capillary tube 3 is 1.9 mm. In some embodiments, the inner diameter of the capillary tube 3 is 2.0 mm. In some embodiments, the inner diameter of the capillary tube 3 is 2.1 mm. In some embodiments, the inner diameter of the capillary tube 3 is 2.2 mm. In some embodiments, the inner diameter of the capillary tube 3 is 2.3 mm. In some embodiments, the inner diameter of the capillary tube 3 is 2.4 mm. In some embodiments, the inner diameter of the capillary tube 3 is 2.5 mm. In some embodiments, the inner diameter of the capillary tube 3 is 2.6 mm.In some embodiments, the inner diameter of capillary tube 3 is 2.7 mm. In some embodiments, the inner diameter of capillary tube 3 is 2.8 mm. In some embodiments, the inner diameter of capillary tube 3 is 2.9 mm. In some embodiments, the inner diameter of capillary tube 3 is 3.0 mm. In some embodiments, the inner diameter of capillary tube 3 is 3.1 mm. In some embodiments, the inner diameter of capillary tube 3 is 3.2 mm. In some embodiments, the inner diameter of capillary tube 3 is 3.3 mm. In some embodiments, the inner diameter of capillary tube 3 is 3.4 mm. In some embodiments, the inner diameter of capillary tube 3 is 3.5 mm. In some embodiments, the inner diameter of capillary tube 3 is 3.6 mm. In some embodiments, the inner diameter of capillary tube 3 is 3.7 mm. In some embodiments, the inner diameter of capillary tube 3 is 3.8 mm. In some embodiments, the inner diameter of capillary tube 3 is 3.9 mm. In some embodiments, the inner diameter of capillary tube 3 is 4.0 mm. In some embodiments, the inner diameter of capillary tube 3 is 4.1 mm. In some embodiments, the inner diameter of capillary tube 3 is 4.2 mm. In some embodiments, the inner diameter of capillary tube 3 is 4.3 mm. In some embodiments, the inner diameter of capillary tube 3 is 4.4 mm. In some embodiments, the inner diameter of capillary tube 3 is 4.5 mm. In some embodiments, the inner diameter of capillary tube 3 is 4.6 mm. In some embodiments, the inner diameter of capillary tube 3 is 4.7 mm. In some embodiments, the inner diameter of capillary tube 3 is 4.8 mm. In some embodiments, the inner diameter of capillary tube 3 is 4.9 mm. In some embodiments, the inner diameter of capillary tube 3 is 5.0 mm. In some embodiments, the inner diameter of capillary tube 3 is 5.1 mm. In some embodiments, the inner diameter of capillary tube 3 is 5.2 mm. In some embodiments, the inner diameter of capillary tube 3 is 5.3 mm. In some embodiments, the inner diameter of capillary tube 3 is 5.4 mm. In some embodiments, the inner diameter of the capillary tube 3 is 5.5 mm. In some embodiments, the inner diameter of the capillary tube 3 is 5.6 mm. In some embodiments, the inner diameter of the capillary tube 3 is 5.7 mm. In some embodiments, the inner diameter of the capillary tube 3 is 5.8 mm.In some embodiments, the inner diameter of capillary tube 3 is 5.9 mm. In some embodiments, the inner diameter of capillary tube 3 is 6.0 mm. In some embodiments, the inner diameter of capillary tube 3 is 6.1 mm. In some embodiments, the inner diameter of capillary tube 3 is 6.2 mm. In some embodiments, the inner diameter of capillary tube 3 is 6.3 mm. In some embodiments, the inner diameter of capillary tube 3 is 6.4 mm. In some embodiments, the inner diameter of capillary tube 3 is 6.5 mm. In some embodiments, the inner diameter of capillary tube 3 is 6.6 mm. In some embodiments, the inner diameter of capillary tube 3 is 6.7 mm. In some embodiments, the inner diameter of capillary tube 3 is 6.8 mm. In some embodiments, the inner diameter of capillary tube 3 is 6.9 mm. In some embodiments, the inner diameter of capillary tube 3 is 7.0 mm. In some embodiments, the inner diameter of capillary tube 3 is 7.1 mm. In some embodiments, the inner diameter of capillary tube 3 is 7.2 mm. In some embodiments, the inner diameter of capillary tube 3 is 7.3 mm. In some embodiments, the inner diameter of capillary tube 3 is 7.4 mm. In some embodiments, the inner diameter of capillary tube 3 is 7.5 mm. In some embodiments, the inner diameter of capillary tube 3 is 7.6 mm. In some embodiments, the inner diameter of capillary tube 3 is 7.7 mm. In some embodiments, the inner diameter of capillary tube 3 is 7.8 mm. In some embodiments, the inner diameter of capillary tube 3 is 7.9 mm. In some embodiments, the inner diameter of capillary tube 3 is 8.0 mm. In some embodiments, the inner diameter of capillary tube 3 is 8.1 mm. In some embodiments, the inner diameter of capillary tube 3 is 8.2 mm. In some embodiments, the inner diameter of capillary tube 3 is 8.3 mm. In some embodiments, the inner diameter of capillary tube 3 is 8.4 mm. In some embodiments, the inner diameter of capillary tube 3 is 8.5 mm. In some embodiments, the inner diameter of capillary tube 3 is 8.6 mm. In some embodiments, the inner diameter of the capillary tube 3 is 8.7 mm. In some embodiments, the inner diameter of the capillary tube 3 is 8.8 mm. In some embodiments, the inner diameter of the capillary tube 3 is 8.9 mm. In some embodiments, the inner diameter of the capillary tube 3 is 9.0 mm.In some embodiments, the inner diameter of capillary tube 3 is 9.1 mm. In some embodiments, the inner diameter of capillary tube 3 is 9.2 mm. In some embodiments, the inner diameter of capillary tube 3 is 9.3 mm. In some embodiments, the inner diameter of capillary tube 3 is 9.4 mm. In some embodiments, the inner diameter of capillary tube 3 is 9.5 mm. In some embodiments, the inner diameter of capillary tube 3 is 9.6 mm. In some embodiments, the inner diameter of capillary tube 3 is 9.7 mm. In some embodiments, the inner diameter of capillary tube 3 is 9.8 mm. In some embodiments, the inner diameter of capillary tube 3 is 9.9 mm. In some embodiments, the inner diameter of capillary tube 3 is 10.0 mm. In some embodiments, the diameter and length are calculated based on the required batch volume and the rate of heat transfer required during the PCR protocol.

[0047] In some embodiments, the capillary tube 3 can be prepared for the PCR step by first filling it with at least one selected from the group consisting of air, nitrogen, water, and buffer solution.

[0048] b. pump In some embodiments, the pump flow rate is calculated based on the length and size of the capillary tube 3, as well as the reaction time required for each reaction step of the PCR protocol.

[0049] In some embodiments, filling, emptying, and / or moving the reaction mixture in the bioreactor 1 can be done using any type of pump. In some embodiments, the pump is of a type selected from the group consisting of peristaltic pumps, gear pumps, lobe pumps, membrane pumps, and syringe pumps. In some embodiments, at least one pump in the bioreactor 1 is a peristaltic pump. For example, the peristaltic pump may be easily disposable together with a sterilizable tube. 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 includes at least one pump. In some embodiments, the bioreactor 1 includes at least two pumps. In some embodiments, the bioreactor 1 includes two pumps. In some embodiments, the multiple pumps are of the same type. In some embodiments, the multiple pumps are of different types.

[0050] c. Constant temperature bath In some embodiments, the constant temperature bath 5 may use air as the heat transfer medium, and a constant temperature profile is ensured by convection through circulation within the bath. Alternatively, the constant temperature bath 5 may use a fluid other than air as the heat transfer medium, and a constant temperature profile is ensured by convection through circulation within the bath 5. In some embodiments, the temperature of each bath 5 is controlled separately by electric heating. In some embodiments, the temperature of each bath 5 is controlled separately using a thermostat. In some embodiments, any thermal fluid having good chemical compatibility with the capillary tube 3 may be used as the heat transfer medium. In some embodiments, water is the heat transfer medium.

[0051] In some embodiments, the lid of each constant temperature bath 5 has a sealed opening for the entry and exit of the capillary tubes 3. In some embodiments, the connection between the capillary tubes 3 and the pump is located outside the bath 5 to avoid contamination of the PCR product by the fluid or air in the bath. Any type of suitable connection can be used.

[0052] In some embodiments, at least one of the connectors is selected from the group consisting of a union joint (180 degrees), an automatic three-way connector or valve, an automatic four-way connector or valve, a manual three-way connector or valve, or a manual four-way connector or valve. In some embodiments, at least one connector is a union joint. In some embodiments, at least one connector is an automatic three-way connector. In some embodiments, at least one connector is an automatic four-way connector. In some embodiments, at least one connector is a manual three-way connector. In some embodiments, at least one connector is a manual four-way connector. In some embodiments, at least one connector is a switching valve.

[0053] In some embodiments, the number of incubators 5 in the bioreactor 1 depends on the complexity of the PCR protocol. In some embodiments, the number of incubators 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 incubators 5 is in the range of 3 to 6. In some embodiments, the number of incubators 5 is 4. In some embodiments, the activation step and the denaturation step may occur in the same incubator 5. In some embodiments, the activation step and the denaturation step may occur in the same incubator 5. In some embodiments, the activation, denaturation, annealing, extension, and final extension steps each occur in separate incubators 5. In some embodiments, two or more PCR steps are performed in each incubator 5. In some embodiments, each PCR step is performed in a different incubator 5.

[0054] In some embodiments, the temperature of each tank 5 is determined by the master mix and polymerase used. In some embodiments, the temperature for the activation and denaturation steps is in the range of 85°C to 100°C.

[0055] In some embodiments, the temperature for the annealing step is in the range of 50°C to 85°C. In some embodiments, the temperature for the extension step or final extension step is in the range of 55°C to 90°C.

[0056] III. Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this invention pertains.

[0057] As used herein, the singular form includes the plural form unless the context makes it clear otherwise.

[0058] As used herein, the term “master mix” refers to a solution containing the components necessary for a PCR reaction, such as polymerase, at least one primer, and deoxynucleoside triphosphates (dNTPs).

[0059] As used herein, the term “reaction mixture” refers to the master mix combined with the nucleic acid template. [Examples]

[0060] [Example 1. PCR protocol] The following is an example of a PCR protocol for a 2079 base pair target DNA using a newly designed bioreactor as described in some embodiments of this specification. The master mix was prepared in a 50 mL Eppendorf conical tube as follows: 3 mL of reaction buffer; 7.8 mL of VWR nuclease-free water; 3 mL of polymerase-suitable enhancer; 300 μL of dNTP mixture (0.2 mM concentration of each dNTP) (10 mM); 75 μL of primers (forward primer A 100 μM); 75 μL of primer (reverse primer B 100 μM); and 150 μL of DNA polymerase (2000 U / mL). The reaction mixture was prepared by combining the master mix with 600 μL of template (100 pg / μL). The bioreactor vessel was formed using a helical coil of platinum-hardened silicone tubing with an inner diameter (ID) of 1.58 mm. The lengths of the tubes are as follows: 1. Activation: 4.5m; 2. Degeneration: 1.5m; 3. Annealing: 2.2m; 4. Elongation: 3.2m; and 5. Final elongation: 7.7m.

[0061] Two peristaltic pumps were fitted with 3-stop, 2.03 mm inner diameter pump tubing and connected to the prepared bioreactor. The first pump was used to fill and empty the bioreactor, as well as to control the movement of the reaction mixture in the capillaries for activation and final extension. The second pump was used to control the denaturation, annealing, and extension cycles in the central loop. Four different temperatures were employed in different thermostats 5. Since activation and denaturation were performed at the same temperature, one thermostat 5 was used for both capillaries at a temperature of 98°C. The annealing step was performed at 63°C. The extension step was performed at 72°C. The final extension step was performed at 72°C. The pump head tubing and bioreactor capillaries were completely filled with buffer and set to the selected flow rate.

[0062] The following PCR protocol was used: 30 cycles of activation (71 seconds), denaturation (24 seconds), annealing (34 seconds), and extension (50 seconds); and an additional extension (120 seconds). The optimal flow rate was calculated to be 7.5 mL / min. To initiate the reaction, the pump was started to transfer the reaction mixture from the Eppendorf conical tube to the activated capillary portion in the bioreactor. The reaction mixture was continuously supplied by pumping from one bioreactor to the next. Once the reaction mixture reached the end of the capillary portion to be used for the extension step, a switching valve and a second pump were used to ensure circulation only within the capillary portion for denaturation, annealing, and extension. The reaction mixture was continuously circulated within this loop for a further 29 cycles. After 30 cycles were completed, the switching valve was opened again and used to supply the reaction mixture to the final extension portion of the capillary. The resulting product was exited the bioreactor and continuously collected into a new, sterile conical tube. A 2 μL sample of the obtained product was diluted to 20 μL (10-fold) and analyzed on a 1% agarose gel by electrophoresis. The gel showed the presence of a 2 kbp product, as expected based on the above process parameters.

[0063] Equal portions All component ranges described herein include the ranges between them, and boundary values ​​may be included or excluded. Any included range is defined by the number of digits specified or one less than the integer value between the ranges (or including one of the original boundary values). For example, if the lower limit is 0.2, the optional included boundary values ​​may be 0.3, 0.4, ... 1.1, 1.2, as well as 1, 2, 3, etc. If the upper limit is 8, the optional included boundary values ​​may be 7, 6, as well as 7.9, 7.8, etc. For one boundary, such as 3 or greater, it includes the same boundary (or range) starting from the number of digits specified or one less than the integer value. For example, if it is 3 or greater, it includes 4 or greater and 3.1 or greater.

[0064] Throughout this specification, any reference to “one embodiment,” “a certain embodiment,” “one or more embodiments,” “several embodiments,” or “embodiments” indicates that the described features, structures, materials, or properties are included in some embodiments of the present disclosure. Therefore, any expressions throughout this specification such as “in one or more embodiments,” “in a certain embodiment,” “in one embodiment,” “several embodiments,” or “in embodiments” do not necessarily refer to the same embodiments.

[0065] Patent applications and patent publications, as well as other non-patent literature cited herein, are incorporated herein by reference in whole or in their entirety, as if each individual publication or document were specifically and individually indicated to be incorporated herein by reference as being fully contained herein. The patent applications on which this application claims priority are also incorporated herein by reference in the same manner as the aforementioned publications and documents.

Claims

1. A bioreactor for producing nucleic acids using polymerase chain reaction (PCR), a) with at least one fluid path; b) At least two pumps capable of supplying a certain amount of reaction material to the fluid path; c) At least three constant temperature baths located downstream of the pump, the constant temperature baths receiving the fluid path, and the constant temperature baths capable of heating the reaction mixture to a temperature that allows at least one PCR step, selected from the group consisting of an activation step, a denaturation step, an annealing step, and an extension step, to occur; d) A central loop comprising a portion of the fluid path corresponding to at least the modification step, the annealing step, and the extension step, wherein at least one pump can recirculate the reaction mixture in the central loop so as to repeat the modification step, the annealing step, and the extension step; e) At least two valves located outside the central loop, the valves being capable of controlling the amount of the reaction mixture entering and leaving the central loop, A bioreactor that includes a bioreactor.

2. The bioreactor according to claim 1, wherein the fluid path is configured to include capillaries.

3. The bioreactor according to any one of claims 1 and 2, wherein the capillary tube is composed of at least one material selected from the group consisting of metal, plastic, and silicone.

4. The bioreactor according to any one of claims 1 to 3, wherein the capillary tube has an inner diameter in the range of 0.5 mm to 10 mm.

5. The bioreactor according to any one of claims 1 to 4, wherein the reaction mixture enters and leaves the constant temperature bath through at least one connecting part selected from the group consisting of a union fitting (180 degrees), an automatic three-way connector, an automatic four-way connector, a manual three-way connector, a manual four-way connector, and a switching valve.

6. The bioreactor according to any one of claims 1 to 5, wherein the bioreactor includes at least four constant temperature baths.

7. The bioreactor according to any one of claims 1 to 6, wherein the constant temperature bath contains a fluid.

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

9. A bioreactor according to any one of claims 1 to 8, wherein two or more PCR steps are performed in the same constant temperature bath.

10. The bioreactor according to claim 9, wherein the activation step and the denaturation step occur in the same constant temperature bath.

11. A bioreactor according to any one of claims 1 to 8, wherein each PCR step is performed in a different constant temperature bath.

12. The bioreactor according to any one of claims 1 to 11, wherein the pump is located outside the constant temperature bath.

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

14. The bioreactor according to any one of claims 1 to 13, wherein the fluid path is wrapped around the frame more than once.

15. The bioreactor according to 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 for producing nucleic acids using polymerase chain reaction (PCR), a) The reaction mixture is pumped into a fluid pathway that includes a portion corresponding to at least one of the following PCR steps: activation step, denaturation step, annealing step, extension step, and final extension step; b) When the reaction mixture is pumped into the fluid path, the reaction mixture is heated in a constant temperature bath; c) a step of opening and closing at least one valve to control the flow of the reaction mixture into the central loop which includes at least the portion of the fluid pathway corresponding to the following PCR steps: the denaturation step, the annealing step, and the extension step; d) Recirculating the reaction mixture within the central loop while at least two valves are closed to prevent the reaction mixture from flowing into or out of the central loop, the step of repeating the modification step, the annealing step, and the extension step with respect to the reaction mixture within the central loop; e) The step of releasing the PCR product from the central loop by opening the valve, A method that includes this.

17. The method according to claim 16, further comprising the step of pumping the reaction mixture into the portion of the fluid path corresponding to the final extension step.

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

19. The method according to any one 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 repeat polymorphism (VNTR) PCR, asymmetric PCR, repeat-type (rep) PCR, overlap extension (OE) PCR, assembly PCR, simple repeat-to-repeater specific (ISSR) PCR, ligation-mediated (LM) PCR, methylation-specific PCR, and mini-primer PCR.

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

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

22. The method according to any one of claims 16 to 21, wherein the PCR product has a volume in the range of 1 μL to 5 L.

23. The method according to any one of claims 16 to 22, wherein heating brings the temperature of the constant temperature bath for the activation step and the denaturation step within the range of 85°C to 100°C.

24. The method according to any one of claims 16 to 23, wherein heating brings the temperature of the constant temperature bath for the annealing step within the range of 55°C to 75°C.

25. The method according to any one of claims 16 to 24, wherein heating brings the temperature of the constant temperature bath for the extension step and the final extension step to within the range of 65°C to 80°C.

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