Method and system for amplifying nucleic acids using polymerase chain reaction (PCR)

Abstract

This disclosure relates to a system for mass-producing nucleic acids using polymerase chain reaction (PCR) and a method for using the same.

Description

Technical Field

[0004]

[0001] The embodiments described herein relate to methods and systems for mass-producing nucleic acids using polymerase chain reaction (PCR). More specifically, some embodiments relate to methods of using a robotic arm to move reaction vessels comprising capillaries between PCR steps.

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 elongating) 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 (also referred to as a thermocycler) 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 capacity, minimal installation area, and ease of integrated automation. Microfluidic PCR apparatuses 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 terms of working capacity, and recently, helical capillary designs incorporating either a metal heating block (Peltier element) or a tank containing a heat transfer medium as a heat exchanger have become more favored (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 the reaction mixture to the capillary wrapped around the heating element. Such designs have the same drawback as microfluidic chips, namely a lack of flexibility at an affordable price. Producing a wide variety of DNA targets using different protocols would require strategies such as numbering up, or the use of multiple chips or heating element designs, which would likely increase production costs (see US8163489, the entirety of which is incorporated herein by reference). Alternatively, a heating block segmented into multiple parts may be used. In these devices, some variation in the PCR protocol is tolerated by temperature control of each part / region, and limited changes in reaction time are possible by controlling the number of regions used. In these designs, the potential for physical scaling is 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 would overcome these limitations, as the outside of the tube would be completely immersed in the medium.

[0005] A fully continuous PCR apparatus employing four temperature-controlled fluid chambers and a capillary tube through 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.

[0006] Another form of PCR apparatus is a design for nucleic acid amplification in which the reaction mixture is pumped into a continuous, repeating loop between two temperature-controlled fluid baths (the whole design is incorporated herein by reference, see US5720923).

[0007] For the industrial production of nucleic acids using PCR, scalability remains the biggest constraint. Mass production is typically carried out using a "numbering-up" approach, which involves increasing the number of thermocycling reactions in microliter plates to aggregate the products. In contrast, physically increasing the size, often referred to as "sizing up," using milliliter or liter-scale conical tubes or similar biocontainers, can be used for amplification. "Numbering up" utilizes the high surface area-to-volume ratio of microliter conical tubes for rapid heat conduction. However, it has several drawbacks, including the generation of a lot of waste, a larger footprint, and increased costs due to the additional equipment required to fill and empty the microliter tubes. In comparison, the physical "sizing up" approach, while using milliliter or liter-scale conical tubes or similar biocontainers, results in low yields and inconsistent quality because mixing is inefficient and temperature control is impossible in such large containers.

[0008] Capillary bioreactors utilize rapid heat conduction and mass transfer in small dimensions while avoiding the redundancy of "increasing by number." As mentioned above, these devices typically rely on microliter substrates, and examples of scaling up are limited (see US8163489, the entire text of which is incorporated herein by reference). Furthermore, current forms of capillary bioreactors cannot be easily adapted to a wide range of reaction times without considerable effort to redesign the surface area of ​​the heating element. Moreover, larger DNA targets often require additional time between extension cycles for subsequent cycles in PCR protocols. Doing this with the continuous reactors described above is considered impossible because the flow rate, capillary size, and capillary length, which cannot be adjusted during operation, determine the reaction time.

[0009] For commercial production, it is essential that production equipment is extremely easy to modify and program, and that it can supply a wide range of PCR products by accommodating a broad range of reaction conditions.

[0010] Systems for amplifying various nucleic acid targets in large quantities, specifically methods that produce high-quality products and high levels of reproducibility, represent inventive advances in this field. [Prior art documents] [Patent Documents]

[0011] [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]

[0012] [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]

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

[0014] Some embodiments include a system for performing a polymerase chain reaction (PCR), the system comprising: a reaction vessel comprising capillaries wrapped around a frame; a circulator for circulating the reaction mixture into the reaction vessel; at least two incubators for holding a fluid for conducting heat to the reaction mixture; and at least one manipulator for moving the reaction vessel between the incubators while performing PCR.

[0015] In some embodiments, the manipulator is a multi-axis pulley or a robotic arm. In some embodiments, the manipulator is a robotic arm. In some embodiments, the robotic arm is automated. In some embodiments, the robotic arm is programmable. In some embodiments, the robotic arm is manual. In some embodiments, the robotic arm includes a rotary joint. In some embodiments, the circulator is a pump. In some embodiments, the pump is at least one type of pump selected from the group consisting of peristaltic pumps, gear pumps, lobe pumps, membrane pumps, and syringe pumps. In some embodiments, the system is a separate batch bioreactor. In some embodiments, the capillary is wound around a frame in a design selected from designs consisting of a simple helical coil, a 180-degree fold, and other more complex coiled flow inverter (CFI) designs. In some embodiments, the capillary is constructed comprising at least one material selected from the group consisting of metal, plastic, and silicone. In some embodiments, the capillary is a silicone tube. In some embodiments, the capillary is a platinum-cured silicone tube. In some embodiments, the capillary is a peroxide-cured silicone tube. In some embodiments, the capillaries are sterilizable. In some embodiments, the capillaries are single-use. In some embodiments, the circulating device generates an oscillating flow. In some embodiments, the system includes three constant-temperature baths.

[0016] Some embodiments include a method for performing a polymerase chain reaction (PCR) using the system described in claim 1 to produce a product, the method comprising: filling a reaction vessel with a reaction mixture; circulating the reaction mixture by introducing turbulence using a circulator; immersing the reaction vessel in a first constant temperature bath corresponding to reaction conditions for a denaturation step of PCR; and moving the reaction vessel from the first constant temperature bath to a second constant temperature bath corresponding to reaction conditions for different steps of PCR using a manipulator.

[0017] In some embodiments, the product is produced on a volume scale of milliliters or liters. In some embodiments, each immersion step corresponds to a step of PCR. In some embodiments, before performing PCR, the method further includes programming the movement of the robotic arm. In some embodiments, the product is produced in a batch size of 1 mL to 5 L. In some embodiments, the product is produced in a batch size 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. In some embodiments, before the filling step, the method further includes calculating the diameter and length of the capillary based on the batch volume. In some embodiments, before the filling step, the method further includes calculating the diameter and length of the capillary based on the rate of heat transfer required for PCR. In some embodiments, the method further includes filling the reaction vessel with the reaction mixture in a segmented form. In some embodiments, the method further includes filling the reaction vessel with the reaction mixture in a non-segmented form. In some embodiments, before the step of introducing turbulent flow, the method further includes calculating the flow rate of the reaction mixture from at least one characteristic selected from the group consisting of the optimal amounts of mixing and turbulent flow, the dimensions of the capillary, and the winding design of the capillary.

Brief Description of the Drawings

[0018] [Figure 1] Shows the capillary, pump, connection, and heating tank of the reactor.

Modes for Carrying Out the Invention

[0019] The accompanying drawings illustrate several embodiments disclosed in this specification and are not to be regarded as limiting the scope thereof. The present invention may recognize other equally effective embodiments. It should be understood that elements or features in any embodiment may exist in other embodiments without further explanation, and, where possible, the same reference numbers are used to indicate equivalent elements common to each figure.

[0020] This disclosure describes several embodiments of a discrete batch-type, capillary-based bioreactor capable of DNA amplification using PCR on the milliliter and liter volume scales. In some embodiments, the bioreactor includes a reaction vessel that is a capillary filled with a reaction mixture; two or more temperature-controlled baths; an automated mechanical manipulator such as a robotic arm; and a pump connected to the capillary. In some embodiments, the reaction time for each PCR step is fully controlled by a robotic arm by submerging, holding, removing, and moving the reaction mixture within the capillary to the next temperature-controlled bath. Each series of operations of submerging, holding, and removing in a temperature-controlled bath corresponds to one reaction step of a PCR protocol.

[0021] I. Method Several embodiments herein describe a method for mass-producing nucleic acids using polymerase chain reaction (PCR).

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] In some embodiments, the movement of reaction vessels between constant temperature baths corresponds to the reaction. 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.

[0031] II. Systems Some embodiments described herein describe systems for mass-producing nucleic acids using polymerase chain reaction (PCR). Some embodiments include the system shown in Figure 1.

[0032] In some embodiments, the system enables rapid heat transfer due to the high surface area-to-volume ratio of the reaction vessel 2 and the physical movement of the bioreactor between the constant temperature baths 5 by the manipulator. In some embodiments, turbulence is introduced into the reaction vessel 2 by incorporating a pump 1 to circulate the reaction mixture in the capillary tubes 2 and by selecting a capillary tube winding design.

[0033] In some embodiments, the systems described herein utilize the versatility of robotic elements as manipulators to physically move the reaction vessel 2. In some embodiments, any PCR time protocol can be programmed by modifying the software for multiple PCR protocols to avoid the limitations of all previously developed continuous capillary PCR reactors, where the reaction time is entirely dependent on the dimensions of the capillary 2. Combining improved heat conduction and mass transfer in the capillary 2 with physically movable batches between multiple heating vessels not only meets the stringent parameter control required for PCR but also provides a clear direction toward flexible and scalable nucleic acid production.

[0034] a. Capillary Some embodiments of the methods described herein are carried out using a system that includes a capillary tube 2 as a reaction vessel. In some embodiments, the flow rate and winding method affect the secondary flow (Dean vortex) within the capillary tube 2, improving heat conduction and mass transfer. In some embodiments, the flow rate and winding method affect the secondary flow (Dean vortex) within the capillary tube 2, avoiding hotspots. Overall, these characteristics improve the quality and reproducibility of the PCR product.

[0035] In some embodiments, the capillary tube 2 is wound around a frame and submerged in a constant temperature bath 5. The frame may be of any design and may be made of any material capable of supporting the winding and weight of the filled capillary tube 2. In some embodiments, the capillary tube 2 may be wound around the frame in any design that promotes turbulent mixing of the reagent and allows for optimal contact between the heat transfer medium and the outer surface of the capillary tube 2. In some embodiments, the capillary tube 2 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).

[0036] In some embodiments, the reaction vessel is a capillary tube 2 composed of any suitable material. In some embodiments, the capillary tube 2 is composed of at least one material selected from the group consisting of metals, plastics, and silicones. In some embodiments, the capillary tube 2 is composed of at least one metal. In some embodiments, the capillary tube 2 is made of metal. In some embodiments, the capillary tube 2 is composed of a metal selected from the group consisting of stainless steel, copper, and Hastelloy. In some embodiments, the capillary tube 2 is composed of stainless steel. In some embodiments, the capillary tube 2 is composed of copper. In some embodiments, the capillary tube 2 is composed of Hastelloy. In some embodiments, the capillary tube 2 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 2 is composed of PFA. In some embodiments, the capillary tube 2 is composed of polysulfone. In some embodiments, the capillary tube 2 is composed of FEP. In some embodiments, the capillary tube 2 is composed of PE.

[0037] In some embodiments, the capillary tube 2 is sterilizable. In some embodiments, the capillary tube 2 is single-use. In some embodiments, the capillary tube 2 is a silicone tube. In some embodiments, the capillary tube 2 is a platinum-cured silicone tube. In some embodiments, the capillary tube 2 is a peroxide-cured silicone tube. In some embodiments, the capillary tube 2 is a sterilizable, single-use platinum-cured silicone tube.

[0038] In some embodiments, the inner diameter of the capillary tube 2 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 2 is 0.5 mm. In some embodiments, the inner diameter of capillary tube 2 is 0.6 mm. In some embodiments, the inner diameter of capillary tube 2 is 0.7 mm. In some embodiments, the inner diameter of capillary tube 2 is 0.8 mm. In some embodiments, the inner diameter of capillary tube 2 is 0.9 mm. In some embodiments, the inner diameter of capillary tube 2 is 1.0 mm. In some embodiments, the inner diameter of capillary tube 2 is 1.1 mm. In some embodiments, the inner diameter of capillary tube 2 is 1.2 mm. In some embodiments, the inner diameter of capillary tube 2 is 1.3 mm. In some embodiments, the inner diameter of capillary tube 2 is 1.4 mm. In some embodiments, the inner diameter of capillary tube 2 is 1.5 mm. In some embodiments, the inner diameter of capillary tube 2 is 1.6 mm. In some embodiments, the inner diameter of capillary tube 2 is 1.7 mm. In some embodiments, the inner diameter of capillary tube 2 is 1.8 mm. In some embodiments, the inner diameter of capillary tube 2 is 1.9 mm. In some embodiments, the inner diameter of capillary tube 2 is 2.0 mm. In some embodiments, the inner diameter of capillary tube 2 is 2.1 mm. In some embodiments, the inner diameter of capillary tube 2 is 2.2 mm. In some embodiments, the inner diameter of capillary tube 2 is 2.3 mm. In some embodiments, the inner diameter of capillary tube 2 is 2.4 mm. In some embodiments, the inner diameter of capillary tube 2 is 2.5 mm. In some embodiments, the inner diameter of capillary tube 2 is 2.6 mm.In some embodiments, the inner diameter of capillary tube 2 is 2.7 mm. In some embodiments, the inner diameter of capillary tube 2 is 2.8 mm. In some embodiments, the inner diameter of capillary tube 2 is 2.9 mm. In some embodiments, the inner diameter of capillary tube 2 is 3.0 mm. In some embodiments, the inner diameter of capillary tube 2 is 3.1 mm. In some embodiments, the inner diameter of capillary tube 2 is 3.2 mm. In some embodiments, the inner diameter of capillary tube 2 is 3.3 mm. In some embodiments, the inner diameter of capillary tube 2 is 3.4 mm. In some embodiments, the inner diameter of capillary tube 2 is 3.5 mm. In some embodiments, the inner diameter of capillary tube 2 is 3.6 mm. In some embodiments, the inner diameter of capillary tube 2 is 3.7 mm. In some embodiments, the inner diameter of capillary tube 2 is 3.8 mm. In some embodiments, the inner diameter of capillary tube 2 is 3.9 mm. In some embodiments, the inner diameter of capillary tube 2 is 4.0 mm. In some embodiments, the inner diameter of capillary tube 2 is 4.1 mm. In some embodiments, the inner diameter of capillary tube 2 is 4.2 mm. In some embodiments, the inner diameter of capillary tube 2 is 4.3 mm. In some embodiments, the inner diameter of capillary tube 2 is 4.4 mm. In some embodiments, the inner diameter of capillary tube 2 is 4.5 mm. In some embodiments, the inner diameter of capillary tube 2 is 4.6 mm. In some embodiments, the inner diameter of capillary tube 2 is 4.7 mm. In some embodiments, the inner diameter of capillary tube 2 is 4.8 mm. In some embodiments, the inner diameter of capillary tube 2 is 4.9 mm. In some embodiments, the inner diameter of capillary tube 2 is 5.0 mm. In some embodiments, the inner diameter of capillary tube 2 is 5.1 mm. In some embodiments, the inner diameter of capillary tube 2 is 5.2 mm. In some embodiments, the inner diameter of capillary tube 2 is 5.3 mm. In some embodiments, the inner diameter of capillary tube 2 is 5.4 mm. In some embodiments, the inner diameter of capillary tube 2 is 5.5 mm. In some embodiments, the inner diameter of capillary tube 2 is 5.6 mm. In some embodiments, the inner diameter of capillary tube 2 is 5.7 mm. In some embodiments, the inner diameter of capillary tube 2 is 5.8 mm.In some embodiments, the inner diameter of capillary tube 2 is 5.9 mm. In some embodiments, the inner diameter of capillary tube 2 is 6.0 mm. In some embodiments, the inner diameter of capillary tube 2 is 6.1 mm. In some embodiments, the inner diameter of capillary tube 2 is 6.2 mm. In some embodiments, the inner diameter of capillary tube 2 is 6.3 mm. In some embodiments, the inner diameter of capillary tube 2 is 6.4 mm. In some embodiments, the inner diameter of capillary tube 2 is 6.5 mm. In some embodiments, the inner diameter of capillary tube 2 is 6.6 mm. In some embodiments, the inner diameter of capillary tube 2 is 6.7 mm. In some embodiments, the inner diameter of capillary tube 2 is 6.8 mm. In some embodiments, the inner diameter of capillary tube 2 is 6.9 mm. In some embodiments, the inner diameter of capillary tube 2 is 7.0 mm. In some embodiments, the inner diameter of capillary tube 2 is 7.1 mm. In some embodiments, the inner diameter of capillary tube 2 is 7.2 mm. In some embodiments, the inner diameter of capillary tube 2 is 7.3 mm. In some embodiments, the inner diameter of capillary tube 2 is 7.4 mm. In some embodiments, the inner diameter of capillary tube 2 is 7.5 mm. In some embodiments, the inner diameter of capillary tube 2 is 7.6 mm. In some embodiments, the inner diameter of capillary tube 2 is 7.7 mm. In some embodiments, the inner diameter of capillary tube 2 is 7.8 mm. In some embodiments, the inner diameter of capillary tube 2 is 7.9 mm. In some embodiments, the inner diameter of capillary tube 2 is 8.0 mm. In some embodiments, the inner diameter of capillary tube 2 is 8.1 mm. In some embodiments, the inner diameter of capillary tube 2 is 8.2 mm. In some embodiments, the inner diameter of capillary tube 2 is 8.3 mm. In some embodiments, the inner diameter of capillary tube 2 is 8.4 mm. In some embodiments, the inner diameter of capillary tube 2 is 8.5 mm. In some embodiments, the inner diameter of capillary tube 2 is 8.6 mm. In some embodiments, the inner diameter of capillary tube 2 is 8.7 mm. In some embodiments, the inner diameter of capillary tube 2 is 8.8 mm. In some embodiments, the inner diameter of capillary tube 2 is 8.9 mm. In some embodiments, the inner diameter of capillary tube 2 is 9.0 mm.In some embodiments, the inner diameter of capillary tube 2 is 9.1 mm. In some embodiments, the inner diameter of capillary tube 2 is 9.2 mm. In some embodiments, the inner diameter of capillary tube 2 is 9.3 mm. In some embodiments, the inner diameter of capillary tube 2 is 9.4 mm. In some embodiments, the inner diameter of capillary tube 2 is 9.5 mm. In some embodiments, the inner diameter of capillary tube 2 is 9.6 mm. In some embodiments, the inner diameter of capillary tube 2 is 9.7 mm. In some embodiments, the inner diameter of capillary tube 2 is 9.8 mm. In some embodiments, the inner diameter of capillary tube 2 is 9.9 mm. In some embodiments, the inner diameter of capillary tube 2 is 10.0 mm. In some embodiments, the diameter and length are calculated based on the required batch volume and the heat transfer rate required during the PCR protocol.

[0039] In some embodiments, the capillary tube 2 may be completely or partially filled with the reaction mixture. In some embodiments, the capillary tube 2 may be completely filled with the reaction mixture. In some embodiments, the capillary tube 2 may be partially filled with the reaction mixture. Alternatively, in some embodiments, the reaction mixture may be introduced into the capillary tube 2 in a segmented form. In some embodiments, the reaction mixture is segmented by inserting an immiscible gas, liquid, or oil to form a series of separated regions.

[0040] In some embodiments, connector 3 serves as an inlet to the reaction vessel 2 for the reaction mixture. In some embodiments, connector 3 serves as an outlet to the reaction vessel 2 for the reaction mixture.

[0041] In some embodiments, at least one of connectors 3 or 4 is selected from the group consisting of automatic three-way connectors or valves, automatic four-way connectors or valves, manual three-way connectors or valves, and manual four-way connectors or valves, automatic union connectors, and manual union connectors. In some embodiments, at least one connector 3 or 4 is an automatic union connector. In some embodiments, at least one connector 3 or 4 is an automatic three-way connector. In some embodiments, at least one connector 3 or 4 is an automatic four-way connector. In some embodiments, at least one connector 3 or 4 is a manual union connector. In some embodiments, at least one connector 3 or 4 is a manual three-way connector. In some embodiments, at least one connector 3 or 4 is a manual four-way connector.

[0042] b. Circulation of the reaction mixture In some embodiments, the reaction mixture may remain in a static environment. In some embodiments, the reaction mixture is mixed in a reaction vessel 2. In some embodiments, mixing and movement of the reaction mixture in the reaction vessel 2 may be performed using a circulation device 1. In some embodiments, the circulation device 1 is a pump or an oscillating flow.

[0043] In some embodiments, the flow rate of the reaction mixture is independent of the reaction time at each step of the PCR protocol. In some embodiments, the flow rate of the reaction mixture is determined by at least one characteristic selected from the group consisting of the optimal amount of mixing and turbulence, the dimensions of the capillary tube 2, and the winding design of the capillary tube 2. In some embodiments, the flow rate of the reaction mixture is determined by the optimal amount of mixing and turbulence. In some embodiments, the flow rate of the reaction mixture is determined by the dimensions of the capillary tube 2. In some embodiments, the flow rate of the reaction mixture is determined by the winding design of the capillary tube 2.

[0044] In some embodiments, filling, emptying, and / or moving the reaction mixture in the reaction vessel can be done using any type of pump 1. In some embodiments, pump 1 is selected from the group consisting of peristaltic pumps, gear pumps, lobe pumps, membrane pumps, impeller pumps, diaphragm pumps, and syringe pumps. In some embodiments, at least one pump is a peristaltic pump. For example, the peristaltic pump may be easily disposable along with a sterilizable tube. In some embodiments, the peristaltic pump can be easily cleaned by wiping, etc. In some embodiments, at least one pump 1 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, pump 1 is mounted on a robotic arm. For example, in some embodiments, pump 1 is mounted on a robotic arm as shown in Figure 2.

[0045] c. Constant temperature bath In some embodiments, the constant temperature bath 5 may use a gas (such as air or nitrogen) as the heat transfer medium, and a constant temperature profile is ensured by convection through circulation within the constant temperature bath 5. In some embodiments, the temperature of each bath is controlled separately using a heating element 6 for electric heating. In some embodiments, the temperature of each bath is controlled separately using a thermostat. In some embodiments, any thermal fluid having suitable chemical compatibility with the reaction vessel 2 may be used as the heat transfer medium. In some embodiments, water is the heat transfer medium.

[0046] In some embodiments, the heated fluid bath is covered with a lid. In some embodiments, the lid is a molded lid. In some embodiments, the lid functions as a drip tray. In some embodiments, the lid has an outlet for removing the collected fluid. In some embodiments, the lid of each constant temperature bath 5 has a sealed opening for the entry and exit of the capillary tube 2. In some embodiments, the connector 4 between the capillary tube and the pump is located outside the bath to avoid contamination of the PCR product by the fluid or air in the bath. In some embodiments, opening and closing the lid is performed by a combination of a vacuum lifter and a manipulator.

[0047] Any type of suitable connector may be used. In some embodiments, at least one connector 4 is a union connector.

[0048] In some embodiments, the number of incubators 5 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 2 to 6. In some embodiments, the number of incubators 5 is 2. In some embodiments, the number of incubators 5 is 3. 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. 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.

[0049] In some embodiments, the temperature of each thermostat 5 is determined by the template, primer, and polymerase used. In some embodiments, the temperature for the activation and denaturation steps is in the range of 85°C to 100°C. In some embodiments, the temperature for the annealing step is in the range of 55°C to 75°C. In some embodiments, the temperature for the extension step or final extension step is in the range of 65°C to 80°C.

[0050] d. Mobility of the reaction vessel In some embodiments, the movement of the reaction vessel 2 between the constant temperature baths 5 can be done by any suitable manipulator. In some embodiments, the manipulator is a multi-axis drive pulley or a robot. In some embodiments, the manipulator is a multi-axis drive pulley. In some embodiments, 2 to 5 manipulators are used. In some embodiments, the manipulator performs linear motion in x and y. In some embodiments, 3 manipulators are used. In some embodiments, the manipulator controls the movement of the capillary package. In some embodiments, the manipulator controls the movement of a drip tray that follows the movement of the capillary package to collect droplets between the constant temperature baths.

[0051] In some embodiments, the manipulator is a robot. In some embodiments, the movement of the manipulator is automatically controlled. In some embodiments, the movement of the manipulator is programmable. In some embodiments, the movement of the manipulator is reprogrammable. In some embodiments, the movement of the manipulator is automatically controlled. In some embodiments, the manipulator moves the reaction vessel in three or more directions. In some embodiments, the manipulator moves the reaction vessel along the x, y, and z axes.

[0052] In some embodiments, the manipulator is operated manually using a control panel. In some embodiments, the manipulator is operated remotely using a pendant or remote control. In some embodiments, the manipulator is integrated into process control automation software. In some embodiments, the manipulator is a robotic arm fixed in place. In some embodiments, the robotic arm is movable in three or more axes. In some embodiments, the position of the robotic arm is not limited. For example, in some embodiments, the robotic arm is mounted above, behind, in front of, or alongside the constant temperature bath. In some embodiments, the robotic arm is mounted above the constant temperature bath 5. In some embodiments, the robotic arm is mounted behind the constant temperature bath. In some embodiments, the robotic arm is mounted in front of the constant temperature bath 5. In some embodiments, the robotic arm is mounted alongside the constant temperature bath.

[0053] Figure 1 shows an embodiment of a robotic arm featuring a rotary joint on the arm connected to a twist joint at its base. In some embodiments, such six movable axes provide degrees of freedom for movement in the x, y, and z directions.

[0054] e. Prevention of cross-contamination When using fluid-filled tanks in a controlled Good Manufacturing Practice (GMP) production cleanroom, it is crucial to avoid cross-contamination. Common risks of cross-contamination in advanced PCR include the operation of the thermostat and moving parts in an open environment within a controlled GMP cleanroom. In some embodiments, moving parts include drip trays, multiple manipulators, suction devices, and other parts that come into contact with the thermal fluid. In some embodiments, the risk of cross-contamination is reduced by keeping other parts of the system away from the thermal fluid and the moving parts that can be thoroughly cleaned.

[0055] In some embodiments, the system includes a rear-separated technical area containing all supporting equipment such as electrical cables, heating elements, movable linear units, and a control panel. In some embodiments, the separated technical area never comes into contact with the reaction mixture, eliminating the risk of cross-contamination. In some embodiments, the enclosed and ventilated front work area consists of 2 to 5 areas, of which at least two are constant temperature chambers 5.

[0056] In some embodiments, the constant temperature bath 5 is designed in accordance with hygienic standards, such as minimizing sharp edges and avoiding hold-ups or dead volume. In some embodiments, the constant temperature bath 5 is designed to be completely emptied and cleaned.

[0057] In some embodiments, all components of the constant temperature bath 5 that are wetted with the thermal fluid, including pipes, pump heads, and heat exchangers, are resistant to thermal fluids at temperatures up to at least 98°C. In some embodiments, all components of the constant temperature bath 5 that are wetted with the thermal fluid, including pipes, pump heads, and heat exchangers, are compliant with cleaning protocols typical of biopharmaceutical production. ru.

[0058] In some embodiments, one of the areas may function as a loading / unloading docking station or as a tank filled with a cooling fluid to rapidly quench the process after final extension.

[0059] In some embodiments, at least one drip tray connected to a cleanroom suction device is placed between the constant temperature baths 5 and used to remove droplets during the movement of the capillary package.

[0060] In some embodiments, the moving parts of the manipulator are housed in a rear technical area to reduce the risk of cross-contamination by fluids or vapors. In some embodiments, the manipulator is positioned within the work area, behind cleanroom-appropriate faceplates or covers.

[0061] In some embodiments, the system is automated and controlled by FDA21 CFR Part 11 compliant software to ensure that an audit trail is provided for all changes.

[0062] 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.

[0063] As used herein, the singular form includes the plural form unless the context makes it clear otherwise.

[0064] As used herein, the term “capillary package” refers to a combination of a reaction vessel wrapped around a frame, a peristaltic pump mounted on a pump head that is easily disassembled and cleaned, and an alternative lid that securely closes the constant temperature bath during use.

[0065] 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).

[0066] As used herein, the term “reaction mixture” refers to the master mix combined with the nucleic acid template.

[0067] As used herein, the term “thermal conduction” refers to heating a mixture, such as a reaction mixture as used herein, in contact with a fluid to bring the mixture to the temperature of the fluid. [Examples]

[0068] [Example 1. PCR protocol] The following is an example of a PCR protocol for a 2079 base pair target DNA using a newly designed reaction vessel as described in some embodiments of this specification. The master mix was prepared in a 50 mL Eppendorf conical tube as follows: 5 sets of 3 mL reaction buffer; 7.8 mL of RNA water; 5 sets of 3 mL of polymerase-suitable enhancer; 300 μL of dNTPs (10 mM); 75 μL of primers (forward primer 100 μM); 75 μL of primers (reverse primer 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).

[0069] The reaction vessel was formed by preparing a 7m length of platinum-hardened silicone tubing with an inner diameter (ID) of 1.58mm and a wall thickness (WT) of 0.8mm into a helical coil. Three different temperatures were used for the constant temperature bath. Since activation and denaturation were carried out at the same temperature, one constant temperature bath was used for both reaction steps at 98°C. The annealing step was carried out at 63°C. The extension step was carried out at 72°C.

[0070] The following PCR protocol was programmed into the robotic automation software: activation step of 112 seconds; denaturation of 37 seconds; annealing of 53 seconds; extension of 66 seconds for 30 cycles; and final extension of 120 seconds.

[0071] To initiate the reaction, the pump was started and the reaction mixture (14 mL) was transferred from the Eppendorf conical tube to the reaction vessel using a peristaltic pump. The pump flow rate was set to 15 mL / min. The reaction mixture was continuously supplied through the reaction vessel by the pump. The resulting PCR product was transferred to a new, sterile conical tube using a peristaltic pump. A 2 μL sample of the resulting product was collected and diluted to 20 μL (10-fold), and the presence of the expected 2079 base pair (bp) product was confirmed by electrophoresis on a 1% agarose gel.

[0072] Equal parts 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.

[0073] 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.

[0074] 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 system for performing polymerase chain reaction (PCR), a) A reaction vessel comprising capillaries wrapped around a frame; b) A circulation device for circulating the reaction mixture in the reaction vessel; c) At least two constant temperature baths for holding a fluid for conducting heat to the reaction mixture; d) At least one manipulator for moving the reaction vessel between the constant temperature baths during PCR, A system that includes this.

2. The system according to claim 1, wherein the manipulator is a multi-axis transmission pulley or a robot arm.

3. The system according to claim 2, wherein the manipulator is a robotic arm.

4. The system according to claim 3, wherein the robotic arm is automated.

5. The system according to any one of claims 2 to 4, wherein the robot arm is programmable.

6. The system according to claim 3, wherein the robot arm is manually operated.

7. The system according to any one of claims 1 to 6, wherein the robot arm includes a rotary joint.

8. The system according to any one of claims 1 to 7, wherein the circulation device is a pump.

9. The system according to any one of claims 8, wherein the pump is at least one type of pump selected from the group consisting of a peristaltic pump, a gear pump, a lobe pump, a membrane pump, and a syringe pump.

10. The system according to at least one of claims 1 to 9, wherein the system is a separate batch bioreactor.

11. The system according to at least one of claims 1 to 10, wherein the capillary tube is wound around the frame in a design selected from designs consisting of a simple helical coil, a 180-degree fold, and other more complex coiled flow inverters (CFIs).

12. The system according to at least one of claims 1 to 11, wherein the capillary tube is composed of at least one material selected from the group consisting of metal, plastic, and silicone.

13. The system according to claim 12, wherein the capillary tube is a silicone tube.

14. The system according to claim 13, wherein the capillary tube is a platinum-cured silicone tube.

15. The system according to claim 14, wherein the capillary tube is a peroxide-cured silicone tube.

16. The system according to at least one of claims 1 to 15, wherein the capillaries are sterilizable.

17. The system according to at least one of claims 1 to 16, wherein the capillary tube is for single use.

18. The system according to at least one of claims 1 to 17, wherein the circulation device generates an oscillating flow.

19. The system according to at least one of claims 1 to 18, wherein the system includes three constant temperature baths.

20. A method for performing a polymerase chain reaction (PCR) using the system described in claim 1 to produce a product, a) The step of filling the reaction vessel with the reaction mixture; b) The step of introducing turbulence using a circulation device and circulating the reaction mixture; c) The step of immersing the reaction vessel in a first constant temperature bath corresponding to the reaction conditions for the PCR denaturation step; d) Using a manipulator, move the reaction vessel from the first constant temperature bath to a second constant temperature bath corresponding to the reaction conditions for different steps of PCR, A method that includes this.

21. The method according to claim 20, wherein the product is produced in milliliters or liters in volume.

22. The method according to any one of claims 20 and 21, wherein each immersion step corresponds to a PCR step.

23. The method according to any one of claims 20 to 22, further comprising the step of programming the movement of the robotic arm before performing PCR.

24. The method according to any one of claims 20 to 23, wherein the product is produced in a batch size of 1 mL to 5 L.

25. The method according to claim 24, wherein the product is produced in batch sizes 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.

26. The method according to any one of claims 20 to 25, further comprising the step of calculating the diameter and length of the capillary based on the batch volume before the filling step.

27. The method according to any one of claims 20 to 26, further comprising the step of calculating the diameter and length of the capillary based on the heat transfer rate required for PCR, prior to the filling step.

28. The method according to any one of claims 20 to 27, further comprising the step of filling the reaction vessel with the reaction mixture in a segmented form.

29. The method according to any one of claims 20 to 28, further comprising the step of filling the reaction vessel with the reaction mixture in an unsegmented form.

30. The method according to any one of claims 20 to 29, further comprising the step of calculating the flow rate of the reaction mixture from at least one characteristic selected from the group consisting of an optimal amount of mixing and turbulence, the dimensions of the capillary, and the winding design of the capillary, prior to the step of introducing turbulence.

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