A dna polymerase aptamer and application in qpcr

Abstract

The application discloses a DNA polymerase nucleic acid aptamer, the nucleic acid aptamer is a single-stranded nucleic acid, the nucleotide sequence of the nucleic acid aptamer is 5'-GCGGGCGGGTGTGGTAGGCGCCCGC-3'; and the DNA polymerase is Taq DNA polymerase. The nucleic acid aptamer is combined with the DNA polymerase through a non-covalent bond, and then the polymerase is inhibited from carrying out a polymerization reaction at a non-permitted temperature. The hot start Taq DNA enzyme prepared by using the nucleic acid aptamer does not need preheating, but is directly added into a PCR cycle at one time like common Taq DNA enzyme, does not cause DNA damage, does not contaminate amplification products, and shortens reaction time.

Description

Technical Field

[0001] This application relates to a DNA polymerase aptamer and its application in qPCR, specifically to C12Q, and more specifically to a method for assaying or testing containing enzymes or microorganisms. Background Technology

[0002] Quantitative Real-time PCR (qPCR) is a technique that involves adding a fluorescent dye to the PCR reaction system, using the accumulation of fluorescence signals to monitor the entire PCR process in real time, and finally performing quantitative and qualitative analysis of unknown templates using a standard curve. It has been widely applied in various fields of life sciences. Taq DNA polymerase is a key component of PCR technology. While its activity is highest at 75-80℃, it also retains some activity at lower temperatures. During heating, because the number of primers is much higher than the amount of template, single-stranded primers are prone to mismatch, forming primer dimers and non-specific products. As these primer dimers and non-specific products amplify, a large amount of primers are consumed, reducing the amplification efficiency of the target product, leading to decreased sensitivity and false positive or false negative results. Therefore, inhibiting the activity of DNA polymerase at low temperatures is crucial for increasing PCR amplification efficiency and improving reaction sensitivity.

[0003] Chinese invention patent CN202111657624.7 discloses a method for preparing hot-start Taq DNA polymerase. By modifying the DNA polymerase with an antibody, the reaction of the DNA polymerase in a low-temperature environment is inhibited, reducing primer mismatches and improving the amplification efficiency of the target analyte. However, the reaction performance of the antibody-modified DNA polymerase in a low-temperature environment cannot be completely blocked. Chinese invention patent CN202111128889.8 discloses a method for HGFR recognition based on nucleic acid aptamer probes and a kit for detecting HGFR. By inducing an immune reaction between HGFR and the nucleic acid aptamer probe, followed by PCR amplification and fluorescence detection under the action of DNA polymerase, it can rapidly identify and stably bind to the HGFR antigen protein, avoiding false positives. However, during the reaction heating process, a large number of primer mismatches occur, reducing the amplification efficiency of the target analyte and decreasing the detection sensitivity. Summary of the Invention

[0004] To inhibit the reaction of Taq DNA polymerase in a low-temperature environment and improve the amplification efficiency of PCR reaction, the first aspect of the present invention provides a DNA polymerase nucleic acid aptamer, wherein the nucleic acid aptamer is a single-stranded nucleic acid, and the nucleotide sequence of the nucleic acid aptamer is selected from any one of the following three: (a) 5'-GCGGGCGGGTGTGGTAGGCGCCCGC-3'; (b) a nucleotide sequence having more than 80% identity with the nucleotide sequence described in (a); (c) a nucleotide sequence that is complementary to the nucleotide sequence described in (a) or (b).

[0005] In a preferred embodiment, the nucleic acid aptamer is a modified nucleic acid aptamer, and the modification site is selected from one or a combination of the 5' end, 3' end, and base sequence.

[0006] In a preferred embodiment, the modification of the 5' end of the nucleic acid aptamer is selected from one or a combination of thiomodification, aminomodification, thiol modification, phosphorylation modification, and biotin modification.

[0007] In a preferred embodiment, the 3' end of the nucleic acid aptamer is modified with a small groove binder (MGB).

[0008] In a preferred embodiment, the nucleic acid aptamer has ΔG = -9.10 kcal / mol, ΔH = -79.00 kcal / mol, ΔS = -225.3 cal / (K·mol), and Tm = 77.3 °C at 37 °C.

[0009] In a preferred embodiment, the DNA polymerase is derived from the genus Thermomyces and is selected from wild type, mutant type, and modified type.

[0010] In a preferred embodiment, the nucleic acid aptamer can inhibit thermostable polymerases at 55-60°C.

[0011] In a preferred embodiment, the unit concentration ratio of the DNA polymerase to the nucleic acid aptamer is 1:(1-10).

[0012] A second aspect of the present invention provides a method for preparing a DNA polymerase nucleic acid aptamer, comprising the following steps:

[0013] (1) The nucleic acid aptamer was diluted with annealing buffer and then annealed;

[0014] (2) Mix DNA polymerase and nucleic acid aptamer and incubate them together. The incubation temperature is 4-50℃, the incubation time is 0.5-24h, and the pH environment for incubation is 8.0-9.5.

[0015] More preferably, the co-incubation temperature is 20-40℃ and the co-incubation time is 8-24h; more preferably, the co-incubation temperature is 37℃ and the co-incubation time is 10h.

[0016] In a preferred embodiment, the annealing conditions are as follows: annealing from 95°C to 4°C, with one annealing cycle every 8-10°C, each cycle lasting 1-2 minutes, to ensure that it is correctly folded into a stem-ring structure.

[0017] DNA polymerase exhibits peak activity at 75-80℃, but retains some activity at lower temperatures. While polymerase activity is low at low temperatures, the amount of primers required far exceeds the amount of template. During instrument heating, mismatches occur in single-stranded primers, forming primer dimers and non-specific products, leading to significant primer consumption and reduced amplification efficiency. Current methods to inhibit polymerase activity at low temperatures include paraffin embedding, antibody modification, and chemical modification. Paraffin embedding isolates the polymerase from the reaction system at room temperature; heating the paraffin balls at high temperatures melts and releases the polymerase. However, paraffin ball preparation is complex, and paraffin interferes with fluorescence detection. Antibody modification has limited inhibitory effects on polymerase, failing to completely inhibit activity at low temperatures. Chemical modification can cause irreversible damage to the polymerase structure, affecting sensitivity. Furthermore, chemically modified polymerases are inherently more stable, requiring significant energy and a longer thermal activation time to release enzyme activity, increasing reaction time and uncertainty. This application designs a nucleic acid aptamer with a specific configuration that can bind to DNA polymerase via non-covalent bonds, thereby inhibiting the polymerization reaction of the polymerase at unpermitted temperatures. The hot-start Taq DNAase prepared using the nucleic acid aptamer requires no preheating and can be directly added to the PCR cycle in a single step, just like ordinary Taq DNAase. This avoids DNA damage and contamination of the amplification products, and shortens the reaction time, improving amplification efficiency. Furthermore, the nucleic acid aptamer of this application introduces an MGB group at the 3' end, which can further improve the thermal stability of the nucleic acid aptamer, increasing its Tm value to above 85°C.

[0018] The nucleic acid aptamer provided by this invention can inhibit polymerase activity at room temperature and has good hot-start performance, thereby effectively controlling the generation of primer dimers or non-specific products in the initial stage of PCR reaction. Furthermore, the nucleic acid is not inactivated or degraded during the reaction and can refold during renaturation, rebinding to the thermostable polymerase to block its activity, ensuring sufficient activity of the thermostable polymerase in the system. In addition, due to the shorter and more stable structure of the nucleic acid sequence, it can be directly synthesized based on the determined nucleic acid sequence, ensuring the stability of nucleic acid between different batches. The method for preparing the nucleic acid aptamer-modified enzyme provided by this invention includes co-incubating the thermostable polymerase with nucleic acid. This method is simple, convenient, and the modification process is mild and controllable. The prepared nucleic acid-modified enzyme has good hot-start performance, and quantitative and qualitative analysis results show that it is superior to the unmodified enzyme and the control enzyme, with a significant activity blocking effect.

[0019] A third aspect of the present invention provides an application of a DNA polymerase nucleic acid aptamer in a 2X qPCR amplification system for scientific research or pathogen detection.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The DNA polymerase aptamer described in this invention binds to DNA polymerase via non-covalent bonds, thereby inhibiting the polymerization reaction of polymerase at unpermitted temperatures. The hot-start Taq DNAase prepared using the aptamer does not require preheating, but is added directly to the PCR cycle in one step, just like ordinary Taq DNAase, without causing DNA damage or contaminating the amplification product, and shortening the reaction time.

[0022] (2) The DNA polymerase nucleic acid aptamer of the present invention has an MGB group introduced at the 3' end, which can further increase its Tm value to above 85 degrees and improve its thermal stability.

[0023] (3) The DNA polymerase aptamer described in this invention has ΔG = -9.10 kcal / mol, ΔH = -79.00 kcal / mol, ΔS = -225.3 cal / (K·mol), and Tm = 77.3℃ at 37℃. It has a shorter sequence length, a more stable structure, and a better low-temperature inhibition effect on Taq DNA polymerase. Attached Figure Description

[0024] Figure 1 The neck ring structure formed by folding nucleic acid aptamers after annealing;

[0025] Figure 2 The amplification curve of Taq DNA polymerase modified with the nucleic acid aptamer of the present invention;

[0026] Figure 3 Amplification curves were obtained using unmodified Taq DNA polymerase. Detailed Implementation

[0027] Example 1

[0028] A DNA polymerase aptamer, wherein the aptamer is a single-stranded nucleic acid, and the nucleotide sequence of the aptamer is 5'-GCGGGCGGGTGTGGTAGGCGCCCGC-3', with an MGB group introduced at the 3' end. The aptamer exhibits the following properties at 37°C: ΔG = -9.10 kcal / mol, ΔH = -79.00 kcal / mol, ΔS = -225.3 cal / (K·mol), and Tm = 77.3°C. The purity is ≥99%, and it was purchased from Shanghai Sangon Biotech.

[0029] The DNA polymerase used was Taq DNA polymerase produced by Sanshi Biotechnology Co., Ltd.

[0030] A method for preparing a DNA polymerase nucleic acid aptamer includes the following steps:

[0031] (1) The nucleic acid aptamer was diluted with annealing buffer and then annealed. The annealing conditions were from 95°C to 4°C, with one round of annealing at 9°C for 2 minutes each round, so that it could be correctly folded into a stem-loop structure.

[0032] (2) Mix the DNA polymerase and the folded nucleic acid aptamer, put them into a 5mL centrifuge tube, place it on a plasma mixer, set the speed to 100rpm, and co-incubate at a temperature of 37℃ for 10h and a pH of 9 to obtain the DNA polymerase modified with the nucleic acid aptamer. Then add an equal volume of glycerol for storage.

[0033] The annealing buffer comprises 10 mM Tris, pH 8.0, 50 mM NaCl, and 1 mM EDTA, with an addition volume of 1 mL / 100 pmol nucleic acid aptamer.

[0034] The unit concentration ratio of the DNA polymerase to the nucleic acid aptamer is 1:1.

[0035] The application of a DNA polymerase nucleic acid aptamer in a qPCR reaction system includes the following steps:

[0036] Prepare the qPCR reaction system and perform qPCR amplification using porcine radioactive pleuropneumoniae DNA as a template. The amplification primer and probe sequences are as follows:

[0037] Upstream primer: 5'-AGTGCTTACCGCATGTAGTGGC-3';

[0038] Downstream primer: 5'-TTGGTGCGGACATATCAACCTTA-3';

[0039] Probe: 5'-Fam-CGATGAACCCGATGAGCCGCC-BHQ1-3

[0040] The formulation of the qPCR reaction system is shown in Table 1. The amplification program is shown in Table 2.

[0041] Compare the detection sensitivity and detection limit before and after modification. For example... Figure 2 As shown, the Taq DNA polymerase modified with the nucleic acid aptamer of the present invention has higher amplification efficiency and detection sensitivity than the unmodified Taq DNA polymerase.

[0042] The PCR instrument used for quantitative fluorescence quantification was ABI-Real-Time Quantitative PCR, model ABI-7500.

[0043] Table 1

[0044] 100mM Tris-HCl (pH 8.8) 10μL 100mMKCl 5μL <![CDATA[25mM MgCl2]]> 5μL <![CDATA[100mM(NH4)2SO4]]> 5μL 2.5mM dNTP 5μL 1% Tween-20 5μL Taq DNA polymerase 5 U / μL 2.5μL <![CDATA[ddH2O]]> 2.5μL DNA template 10μL Total reaction volume 50μL

[0045] Table 2

[0046]

[0047] Example 2

[0048] A DNA polymerase aptamer, with the same steps as in Example 1, except that the DNA polymerase is not modified with the aptamer.

[0049] Performance testing

[0050] The amplification efficiency of the qPCR reaction was obtained using a real-time quantitative PCR instrument, and the results are shown in Table 3.

[0051] Table 3

[0052] Amplification efficiency 106.9％ 76.2％

Claims

1. A DNA polymerase aptamer, characterized in that, The nucleic acid aptamer is a single-stranded nucleic acid, and the nucleotide sequence of the nucleic acid aptamer is 5'-GCGGGCGGGTGTGGTAGGCGCCCGC-3'; The DNA polymerase is Taq DNA polymerase.

2. The DNA polymerase aptamer according to claim 1, characterized in that, The nucleic acid aptamer is a modified nucleic acid aptamer, and the modification site is selected from one or a combination of the 5' end and the 3' end.

3. The DNA polymerase aptamer according to claim 2, characterized in that, The modification at the 5' end of the nucleic acid aptamer is selected from one or a combination of thiomodification, aminomodification, thiol modification, phosphorylation modification, and biotin modification.

4. The DNA polymerase aptamer according to claim 2, characterized in that, The modification of the 3' end of the nucleic acid aptamer is a minor groove conjugate modification.

5. The DNA polymerase aptamer according to claim 4, characterized in that, The nucleic acid aptamer has the following properties at 37 °C: ΔG = -9.10 kcal / mol, ΔH = -79.00 kcal / mol, ΔS = -225.3 cal / (K·mol), and Tm = 77.3 °C.

6. The DNA polymerase aptamer according to claim 2, characterized in that, The DNA polymerase is derived from the genus Thermomyces and is selected from either the wild type or the mutant type.

7. The DNA polymerase aptamer according to claim 2, characterized in that, The nucleic acid aptamer can inhibit thermostable polymerases at 55-60℃.

8. The DNA polymerase aptamer according to claim 1, characterized in that, The unit concentration ratio of the DNA polymerase to the nucleic acid aptamer is 1:(1-10).

9. A method for preparing a DNA polymerase nucleic acid aptamer as described in any one of claims 1-8 for non-disease diagnostic purposes, characterized in that, Includes the following steps: (1) The nucleic acid aptamer was diluted with annealing buffer and then annealed; (2) Mix DNA polymerase and nucleic acid aptamer and incubate them together. The incubation temperature is 4-50℃, the incubation time is 0.5-24h, and the pH environment for incubation is 8.0-9.5.

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