Ready-to-use PCR (Polymerase Chain Reaction) tube preloaded with Taq polymerase and reaction components and stable at room temperature

Abstract

The invention provides a ready-to-use PCR tube preloaded with Taq polymerase and reaction components and stable at room temperature, and belongs to the field of molecular biology and nucleic acid diagnosis. The invention provides a ready-to-use PCR (polymerase chain reaction) tube which can be stored at room temperature and is pre-filled with a premix for carrying out polymerase chain reaction (PCR) amplification on all necessary components in advance. The kit is characterized in that each tube is pre-filled with locally prepared Taq polymerase, a PCR buffer solution, dNTPs, MgCl and target specific primers (only a customized PCR kit-ready-to-use PCR plus tube contains the primers), and all the components are dried and stored in a dry powder form, so that the shelf life can be prolonged, and the tedious steps of on-ice operation can be avoided. According to the method, the error rate in the PCR preparation process is reduced to the maximum extent, and the diagnosis and research work flow is simplified.

Description

[0001] This invention claims priority to the earlier application filed on January 28, 2025 with the Pakistan Patent Office, patent number PK 38 / 2025, entitled "Ready-to-use PCR tube pre-loaded with Taq polymerase and reaction components and stable at room temperature"; The entire contents of the prior application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the fields of molecular biology and nucleic acid diagnostics, and mainly relates to ready-to-use PCR tubes that are pre-loaded with Taq polymerase and reaction components and are stable at room temperature. Background Technology

[0003] Polymerase chain reaction (PCR) is one of the fundamental and basic techniques in molecular biology and biotechnology. It can amplify specific DNA sequences for detection, examination, diagnosis, and other applied scientific purposes. Developed by Kary Mullis in 1983, PCR revolutionized research, diagnostics, and genetics by enabling the exponential replication of specific DNA sequences with high precision and efficiency. PCR technology has found wide applications in life sciences, industrial operations, healthcare, agriculture, forensic science, and environmental science.

[0004] Typically, PCR involves using Taq polymerase, MgCl2, PCR buffer, dNTPs, primers, and a DNA sample / template. After several cycles of heating and cooling, the double-stranded DNA is opened, allowing the primers to bind to the target region. Then, a new DNA strand is created using a thermostable DNA polymerase (such as Taq polymerase).

[0005] Conventional PCR workflows require storing various liquid reagents at refrigerated or frozen temperatures. Preparing each reaction is very time-consuming and prone to errors or contamination due to the reproducibility of the steps. Therefore, there is an urgent need for a Taq polymerase that is stable at room temperature to eliminate the need for low-temperature transport and storage. Summary of the Invention

[0006] Ready-to-use PCR tubes and premixed solutions offer a cost-effective, stable, and scalable solution for high-throughput clinical laboratories requiring streamlined processes, as well as for field or resource-constrained settings where refrigeration is not feasible. The drying formulation of this invention enhances overall enzyme stability and PCR sensitivity, thereby ensuring reliable nucleic acid amplification on a variety of detection platforms.

[0007] A ready-to-use PCR tube, wherein the PCR reagents contained are Taq polymerase, MgCl2, buffer, and dNTPs; the reagents are in the form of dry powder.

[0008] A ready-to-use PCR PLUS tube, wherein the ready-to-use PCR PLUS tube contains customized primers, Taq polymerase, MgCl2, buffer, and dNTPs; the reagents are in dry powder form.

[0009] A ready-to-use premix containing PCR reagents including Taq polymerase, MgCl2, buffer, and dNTPs; the premix is ​​suitable for PCR reaction concentrations of 10X, 50X, or 96X.

[0010] A method for preparing a ready-to-use premix, wherein Taq polymerase, buffer, MgCl2, dNTPs and primers are mixed and placed at the bottom of a tube; wherein the Taq polymerase, buffer, MgCl2, dNTPs and primers are all dry powders; the premix is ​​used for single-reaction PCR or multiple-reaction PCR.

[0011] A Taq polymerase, wherein the Taq polymerase protein is expressed and purified by transformation of a pAKTaq plasmid containing the Taq polymerase gene into Escherichia coli.

[0012] Beneficial effects The ready-to-use PCR tubes provided by this invention eliminate or significantly reduce cold chain procedures and related costs; ensure consistency and reproducibility of results through standardized reagent concentrations; support a wide range of diagnostic and research applications, including barcode analysis, pathogen screening, and disease diagnosis; and reduce the rate of operational errors by end users during PCR experimental setup. This invention maintains stability without refrigeration, which is particularly helpful for remote or resource-limited areas, significantly reduces PCR costs, enables research institutions with limited budgets to use the technology, alleviates the economic burden on end users in the industrial and diagnostic fields, and provides convenience to the general public. Attached Figure Description

[0013] Figure 1 Flowchart for ready-to-use PCR tubes; Figure 2 The images show the PCR results of the ready-to-use PCR tube plus and the ready-to-use PCR tube, where L is the standard molecular weight of 1kb, AG is the PCR product result using the ready-to-use PCR tube plus, HJ is the PCR product result using the ready-to-use PCR tube, and -ve is the negative control. Figure 3This image shows a comparison of PCR products of different amplified fragment sizes using the Taq polymerase produced in this application and commercially available Taq enzyme. Lane L represents the standard molecular weight in kb. Lanes S1 and +ve represent the PCR product amplified using the Taq polymerase produced in this application and commercially available Taq enzyme, resulting in a 124 bp PCR product. Lanes S2 and +ve represent the PCR product amplified using the Taq polymerase produced in this application and commercially available Taq enzyme, resulting in a 500 bp PCR product. Lanes S3 and +ve represent the PCR product amplified using the Taq polymerase produced in this application and commercially available Taq enzyme, resulting in a 1 kb PCR product. Lanes S4 and +ve represent the PCR product amplified using the Taq polymerase produced in this application and commercially available Taq enzyme, resulting in a 1.7 kb PCR product. Lanes S5 and +ve represent the PCR product amplified using the Taq polymerase produced in this application and commercially available Taq enzyme, resulting in a 2.6 kb PCR product. Lane -ve represents the negative control. Detailed Implementation

[0014] Ready-to-use PCR tubes are an innovative and fully functional product designed for molecular biology laboratories to optimize workflows and enhance the efficiency of polymerase chain reaction (PCR). Each tube is pre-loaded with high-quality reagents for PCR in a precise and balanced manner to ensure optimal performance, eliminating the tedious steps of manually preparing and dispensing master mixes. These ready-to-use PCR tubes contain Taq DNA polymerase, PCR buffer, MgCl2, and deoxyribonucleoside triphosphates (dNTPs); the Plus version also includes primers. This design ensures precise operation, minimizes preparation time, and significantly reduces the risk of contamination, making them ideal for diagnostic applications.

[0015] Users only need to add high-quality DNA template to these tubes (for basic tubes without primers, primers must also be added). This method ensures consistency of results across multiple reactions, eliminating the need to add reaction components one by one, thus minimizing errors that may occur during manual operation. These PCR tubes are compatible with standard PCR instruments and suitable for high-throughput applications.

[0016] Example 1. Ready-to-use PCR tube.

[0017] The ready-to-use PCR tubes come pre-filled with a ready-to-use Master Mix, containing all the reagent components required for a standard PCR reaction, specifically natural Taq polymerase, PCR buffer, MgCl2, and dNTPs. Add all the reagents to the centrifuge tubes in precise and balanced quantities as needed, then aliquot them into each PCR tube. Place these PCR tubes in a 60°C incubator for 45 minutes to dry. After heat drying, continue drying at 45°C for another 15 minutes to ensure complete dehydration. Perform PCR reactions on each batch of PCR tubes to assess efficiency and performance. Results are shown below. Figure 2 Each batch is properly labeled and recorded.

[0018] Example 2. Ready-to-use PCR tube Plus.

[0019] It includes a pre-mixed reagent for routine PCR processes, including: domestically produced Taq DNA polymerase, PCR buffer, magnesium chloride (MgCl2), deoxynucleoside triphosphates (dNTPs), and primers (customized).

[0020] All reagents were precisely proportioned and added to the EP tubes as required, thoroughly mixed, and then dispensed into individual PCR tubes. The PCR tubes were then dried in a 60°C oven for 45 minutes. After heat drying, the tubes were maintained at 45°C for another 10 minutes to ensure complete dehydration. The efficiency and performance of the PCR tubes were checked by performing PCR testing on each batch of products. The results are as follows: Figure 2 All batches are strictly labeled and have complete traceability records established.

[0021] Example 3: Preparation of Taq polymerase.

[0022] Taq polymerase is a core component of ready-to-use PCR tubes, and its domestic production is achieved using the following methods: 1. pAKTaq plasmid in DH5-α Escherichia coli strain.

[0023] Order pAKTaq plasmids containing the Taq polymerase gene through the Addgene platform; The presence of the Taq gene was verified by restriction enzyme digestion analysis using double enzyme digestion method (EcoRI, BamHI) and single enzyme digestion method (HindIII). The plasmid was stored in 50% glycerol cryovials and streaked onto LB agar plates.

[0024] 2. Protein production and extraction.

[0025] Primary cultures of Escherichia coli were incubated overnight at 37°C. DH5-α strain containing Taq polymerase expression plasmid was cultured overnight in LB medium containing ampicillin. Inoculate 2.5 mL of the culture into 250 mL of LB medium and incubate at 37°C in a shaker until the early exponential growth phase (A600nm 0.20, approximately 4 hours). After 4 hours, check the OD value, add promoter enhancer to a final concentration of 0.5 mM (using 10 mL of 100 mM stock solution), and incubate at 37°C in a shaker for 16–20 hours.

[0026] 3. Cell harvesting and lysis.

[0027] Collect cells by centrifugation at 5,000 xg for 5 minutes, retain the pellet, and discard the LB medium; Resuspend the cells in 40 ml of buffer A, centrifuge at 5000 xg for 5 minutes to collect the washed cells, and discard the supernatant; Resuspend the cells in 10 ml buffer A, transfer them to a 50 ml Falcon tube, add 4 mg / ml lysozyme powder to the suspension, and incubate at room temperature for 15 minutes. Add 10 ml of buffer B, mix thoroughly, and incubate at 75°C for 1 hour; Precipitated proteins and cell debris were removed by centrifugation for 15 minutes at a rate of 10,000 x g.

[0028] Be careful not to remove any sediment; remove the supernatant.

[0029] 4. Protein purification.

[0030] The crude Taq polymerase was added to a dialysis bag and dialyzed in storage buffer for 2 days to obtain purified Taq polymerase protein for subsequent operations.

[0031] 5. Detection of Taq polymerase.

[0032] PCR amplification of different target fragments was performed using the Taq polymerase produced in this application and commercially available Taq polymerase, respectively. The results are as follows: Figure 3 .

Claims

1. A ready-to-use PCR tube, characterized in that, The PCR tube contains PCR reagents including Taq polymerase, MgCl2, buffer, and dNTPs; the reagents are in dry powder form.

2. A ready-to-use PCR PLUS tube, characterized in that, The ready-to-use PCR PLUS tube contains custom-designed primers, Taq polymerase, MgCl2, buffer, and dNTPs; the reagents are in dry powder form.

3. A ready-to-use premixed liquid, characterized in that, The premix contains PCR reagents including Taq polymerase, MgCl2, buffer, and dNTPs; the premix is ​​suitable for PCR reaction concentrations of 10X, 50X, or 96X.

4. A method for preparing the ready-to-use premixed liquid according to claim 3, characterized in that, The method involves mixing Taq polymerase, buffer, MgCl2, dNTPs, and primers and placing the mixture at the bottom of a tube; all components, including Taq polymerase, buffer, MgCl2, dNTPs, and primers, are dry powders; the premixed solution is used for single-reaction PCR or multiple-reaction PCR.

5. A Taq polymerase, characterized in that, The Taq polymerase was expressed and purified by transforming the pAKTaq plasmid containing the Taq polymerase gene into E. coli.

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