A method for detecting the enzyme activity of MMLV reverse transcriptase
Through the residual dATP detection after the reverse transcription reaction, the existing MMLV reverse transcriptase enzyme activity detection methods have solved the problems of large errors and complex operations, and high-precision and simple operational enzyme activity detection is achieved, expanding the detection range and improving accuracy and stability.
Patent Information
- Application Number
- CN202411988607.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-08
AI Technical Summary
The existing MMLV reverse transcriptase enzyme activity detection methods have problems such as large errors, complex operation, long time and high cost, and it is difficult to achieve high-throughput detection.
The enzyme activity of MMLV reverse transcriptase was determined by the remaining dATP after the reverse transcription reaction. Luciferase was used to detect dATP consumption, establish a standard relationship curve, and directly detect the reverse transcription reaction substrate, eliminate the interference of subsequent qPCR reactions, and enhance the stability of the detection.
The MMLV reverse transcriptase enzyme activity detection is achieved with high accuracy, simple operation and high repeatability, expanding the linear range of enzyme activity detectable, and improving the accuracy and stability of the detection.
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Figure CN119776491A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of DNA reverse transcriptase, and in particular to a method for detecting the enzyme activity of MMLV reverse transcriptase. Background Art
[0002] Reverse transcriptase is an enzyme expressed by all retroviruses, and it plays an important role in the entire life cycle of retroviruses. Reverse transcriptase usually has DNA or RNA-dependent DNA polymerase activity and RNase H activity, and can synthesize the corresponding double-stranded DNA sequence according to the single-stranded RNA genome sequence of the retrovirus, and then integrate the DNA sequence into the host cell genome through reverse transcriptase integrase. After the integrated DNA is activated under appropriate conditions, it uses the host's own enzyme system to transcribe and generate the corresponding RNA, one part of which is used as the genetic material of the virus, and the other part is translated into the protein required by the virus as mRNA. Finally, RNA and protein can be assembled into new virus particles to achieve the reproduction of the virus. Under certain conditions, the integrated DNA can also transform cells into cancer cells. The study of reverse transcriptase is of great significance to the study of pathogenic viruses. At present, the kits and detection methods for detecting reverse transcriptase activity usually use the isotope method, but this method has the problems of easy radioactive contamination, high requirements for detection equipment, high operating costs, and difficulty in achieving high throughput.
[0003] MMLV reverse transcriptase is a mutant MMLV reverse transcriptase DNA polymerase, isolated and purified from E. coli cells expressing Moloney murine leukemia virus reverse transcriptase gene (pol gene), composed of a single subunit with a molecular weight of 76.1KDa, which can catalyze the polymerization reaction of complementary DNA chains with single-stranded DNA, RNA or DNA:RNA hybrid chains as templates. Compared with the wild type, it has no RNase H activity.
[0004] The existing method for detecting the activity of MMLV reverse transcriptase generally involves reverse transcription to form a cDNA template and then performing qPCR to detect the fluorescence value. This method has large errors, high requirements for experimental personnel, a cumbersome and time-consuming process, and high costs. Summary of the invention
[0005] In view of the above-mentioned shortcomings that currently exist, the present invention provides a method for detecting the enzyme activity of MMLV reverse transcriptase, which has the advantages of high precision, simple operation, high repeatability, and directly detecting the reverse transcription reaction substrate to determine the MMLV reverse transcriptase activity, eliminating the interference caused by the subsequent qPCR reaction in the prior art, and enhancing the stability of enzyme activity detection.
[0006] In order to achieve the above object, in a first aspect, the present invention provides a method for detecting the enzyme activity of MMLV reverse transcriptase, comprising the following steps:
[0007] S1. Based on the known concentration of luciferase, the known concentration of dATP is detected to prepare a standard relationship curve between the chemiluminescence value of luciferase and dATP consumption;
[0008] S2, using RNA MS2 as a reverse transcription template, designing a reverse transcription primer sequence, and then performing reverse transcription reaction 1 to obtain target cDNA; performing reverse transcription reaction 2 on MMLV reverse transcriptase at different concentration gradients to obtain a standard relationship curve between enzyme activity and dATP consumption;
[0009] S3, after the reverse transcription reaction 1 is completed, the dATP remaining after the reverse transcription reaction is detected by using a luciferase of known concentration, and the dATP consumption is obtained by comparing with the standard relationship curve of the chemiluminescence value of luciferase and dATP consumption;
[0010] S4. Compare the dATP consumption and enzyme activity with the standard relationship curve of dATP consumption to obtain the enzyme activity of the MMLV reverse transcriptase to be tested.
[0011] The present invention determines the enzyme activity of MMLV reverse transcriptase by the raw material dATP remaining after reverse transcription of MMLV reverse transcriptase. The present invention provides the above detection method with high precision, simple operation, high repeatability, and directly detects the reverse transcription reaction substrate to determine the MMLV reverse transcriptase activity, eliminating the interference caused by the subsequent qPCR reaction of the prior art, and enhancing the stability of enzyme activity detection. And the enzyme activity detectable linear range of the method of the present application is wider than that of the prior art (0-50U); there is a higher difference between different enzyme activity gradients, and primers with high specificity to the template RNA are screened at the same time, so it has higher accuracy (fluorescence difference value is between 0-1200RFU value) and stability.
[0012] Furthermore, the nucleotide sequence of the reverse transcription primer is the nucleotide sequence shown in any one of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.
[0013] Furthermore, in step S3, the concentration of the luciferase is 1-5 mg / mL.
[0014] Furthermore, the reaction conditions of the reverse transcription reaction are: 40-50°C, reaction time 30-60 min; 80-95°C, reaction time 1-5 min.
[0015] In a specific embodiment, the reaction conditions of the reverse transcription reaction are: 42° C., reaction time 30 min; 85° C., reaction time 1 min.
[0016] It should be noted that the temperature for the above-mentioned reverse transcriptase to perform in vitro nucleic acid synthesis and / or amplification can be 50°C, 50.5°C, 51°C, 51.5°C, 52°C, 52.5°C, 53°C, 53.5°C, 54°C, 54.5°C, or 55°C, or it can be a range consisting of any two of the above values, or any intermediate value in the range.
[0017] In a second aspect, the present invention provides a kit for detecting MMLV reverse transcriptase activity, the kit comprising a reverse transcription primer having a nucleotide sequence as shown in any one of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.
[0018] Furthermore, the concentration of the reverse transcription primer is 5-15 mM, and the volume is 1-4 μL.
[0019] Furthermore, the kit also includes at least one of dNTP, MMLV reverse transcriptase, MS2 template, RRI enzyme and MMLV reaction buffer.
[0020] Furthermore, the dNTP includes dATP, dTTP, dCTP and dGTP, the concentration of the dATP / dTTP / dCTP / dGTP is 5-15mM, and the volume of the dNTP is 1-4μL; the concentration of the RNA MS2 is 200ng / μL, and the volume is 0.5-4μL.
[0021] It should be noted that dNTP is deoxyribonucleoside triphosphate; dATP is adenine deoxyribonucleotide triphosphate; dTTP is thymine deoxyribonucleotide triphosphate; dCTP is cytosine deoxyribonucleotide triphosphate; dGTP is guanine deoxyribonucleotide triphosphate.
[0022] It should be noted that, in the above-mentioned RNA MS2 with a concentration of 200 ng / μL and a volume of 0.5 to 4 μL, the electrophoresis pattern generated by the target cDNA is as follows Figure 5 The effect of the amount of RNA MS2 added on dATP consumption is shown in Figure 6 As shown. Figure 5 It can be seen that when the volume of RNA MS2 with a concentration of 200ng / μL is 3μL, the difference is the largest. Figure 6 It can be seen that the amount of RNA MS2 added is linearly related to the amount of dATP consumed. Therefore, the best effect is achieved when the volume of 200ng / μL RNA MS2 is 3μL.
[0023] Furthermore, the Buffer of the MMLV reaction includes Tris-HCl, KCl, MgCl2 and DTT.
[0024] In a third aspect, the present invention provides use of any of the above-described detection methods or any of the above-described kits for detecting MMLV reverse transcriptase activity in detecting MMLV reverse transcriptase activity. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 Electrophoresis of the reverse transcription primers of the MMLV reverse transcriptase designed in Example 1 of the present invention;
[0026] Figure 2 The standard curve for detecting dATP in dNTP by luciferase in Example 2 of the present invention;
[0027] Figure 3 The curves of reverse transcription and dATP consumption of MMLV reverse transcriptase at different concentrations in Example 2 of the present invention;
[0028] Figure 4 The curve of reverse transcription and dATP consumption of MMLV reverse transcriptase at some concentrations in Example 2 of the present invention;
[0029] Figure 5 The electrophoresis diagram of the effect of the amount of RNA MS2 added on the generation of target cDNA of the present invention;
[0030] Figure 6 The figure shows the effect of the added amount of RNA MS2 of the present invention on the consumption of dATP. DETAILED DESCRIPTION
[0031] To make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in the field; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.
[0032] Example 1. Design of reverse transcription primers for MMLV reverse transcriptase
[0033] (1) Experimental setup:
[0034] RNA MS2 (known sequence) was selected as the reverse transcription template for primer design. A total of 12 reverse transcription primers were designed (see Table 1). All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal)) uses RNA MS2 as a reverse transcription template, and adds the 12 reverse transcription primers in Table 1 according to the steps to perform a reverse transcription reaction. After the reverse transcription reaction is completed, PCR is performed using the cDNA obtained by reverse transcription as a template to detect whether each primer can be reverse transcribed to synthesize cDNA.
[0035] Table 1:
[0036]
[0037]
[0038] (2) Reaction solution: The reverse transcription reaction system uses the Quanshijin reverse transcription kit, and the PCR reaction system uses the Takara kit.
[0039] (3) On-machine detection: After PCR is completed, nucleic acid electrophoresis is used to detect PCR products.
[0040] (4) Experimental results: The experimental results are as follows Figure 1 As shown by Figure 1 It can be seen that the bands in lanes 4, 6, and 8 (see SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 in Table 1) are in accordance with the expected sizes, which are 449, 1181, and 3246 bp, respectively.
[0041] Example 2: A method for detecting the enzyme activity of MMLV reverse transcriptase
[0042] 1. Standard relationship curve between luciferase and dATP consumption chemiluminescence value
[0043] (1) Experimental setup: The same concentration (3 mg / mL) of luciferase was used to detect substrate raw material dATP diluted to 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM.
[0044] (2) Preparation of reaction solution: Prepare an appropriate amount of dATP detection working solution according to the ratio of 100 μL of dATP detection working solution for each sample. Melt the reagent to be used in an ice bath. Take an appropriate amount of luciferase reagent and add 100 μL of the prepared dATP detection working solution to each well of a 96-well black plate. Add 20 μL of the sample to be tested to each well for chemiluminescence reaction.
[0045] (3) On-machine detection: Use an ELISA instrument to perform chemiluminescence detection on the reaction solution.
[0046] (4) Experimental results: The experimental results are as follows Figure 2 As shown. Figure 2 It can be seen that dATP can be detected with a good linear relationship using luciferase assay.
[0047] 2. Gradient curve of dATP consumption during reverse transcription by MMLV reverse transcriptase
[0048] (1) Experimental setup: MMLV reverse transcriptase standard was diluted to 5 U / μL, 10 U / μL, 15 U / μL, 20 U / μL, 25 U / μL, 35 U / μL, 45 U / μL, 50 U / μL, 100 U / μL, 200 U / μL, and 400 U / μL, and added to the reverse transcription reaction system. After the reverse transcription reaction, the chemiluminescence value of the residual dATP was detected to determine the consumption of dATP.
[0049] (2) Reaction solution: The reaction solutions of the reverse transcription reaction system are shown in Table 2:
[0050] Table 2:
[0051] Reagents Addition amount (μL) 5×MMLVBuffer 4 dNTP(10mM) 1 Reverse transcription primer (10mM) 1 MMLV reverse transcriptase (μL) 1 RRI enzyme (20U / μL) 0.5 RNAMS2 (200 ng / μL) 1 <![CDATA[ddH2O]]> To20
[0052] The reverse transcription primer in Table 2 is shown in SEQ ID NO: 8; dNTP includes dATP, dTTP, dCTP and dGTP, and the concentration of dATP / dTTP / dCTP / dGTP is 10 mM.
[0053] (3) On-machine testing: Use a PCR instrument to perform reverse transcription reaction.
[0054] (4) Experimental results: The above reverse transcription reaction system was used to react MMLV reverse transcriptase at various gradients. The results are as follows: Figure 3 and Figure 4 As shown. Figure 3 It can be seen that as the concentration of MMLV reverse transcriptase standard increases, the consumption of dATP increases. When it is 0-50U, the difference in enzyme activity is greater and in a linear relationship, such as Figure 4 As shown, our standard curve R in the range of 0-50U 2 It is equal to 0.997, which has a good linear relationship. Therefore, the dATP consumption curve can be used to detect the enzyme activity of the MMLV reverse transcriptase sample to be tested.
[0055] Example 3: A method for detecting the enzyme activity of MMLV reverse transcriptase
[0056] (1) Experimental setup: The MMLV reverse transcriptase standard was diluted to 15 U / μL, 20 U / μL, and 25 U / μL, and each group of test sample enzymes was diluted to 15 U / μL, 20 U / μL, and 25 U / μL, and added to the reverse transcription reaction system respectively. After the reverse transcription reaction, the chemiluminescence value of the residual dATP after the reaction was detected to determine the consumption of dATP.
[0057] (2) Reaction solution
[0058] The reaction solutions of the reverse transcription reaction system are shown in Table 2:
[0059] The reverse transcription primer in Table 2 is shown in SEQ ID NO:8.
[0060] (5) On-machine testing:
[0061] Perform reverse transcription reaction using a PCR instrument.
[0062] (4) Experimental results
[0063] The MMLV reverse transcriptase standard and each gradient of the test sample enzyme were reacted by the reverse transcription reaction system in Example 2, and the results are shown in Table 3. As can be seen from Table 3, the maximum deviation value measured by the three groups of test samples with different enzyme activities does not exceed 3%, indicating that the enzyme activity detection method is stable.
[0064] Table 3:
[0065]
[0066] Example 4: A method for detecting the enzyme activity of MMLV reverse transcriptase
[0067] The difference between this embodiment and embodiment 3 is that the reverse transcription primer in Table 2 is shown as SEQ ID NO: 4, and the other steps and parameters are the same as those in embodiment 3.
[0068] Example 5: A method for detecting the enzyme activity of MMLV reverse transcriptase
[0069] The difference between this embodiment and embodiment 3 is that the reverse transcription primer in Table 2 is shown as SEQ ID NO: 6, and the other steps and parameters are the same as those in embodiment 3.
[0070] Comparative Example 1
[0071] The difference between this comparative example and Example 3 is that the reverse transcription primer in Table 2 is shown as SEQ ID NO: 1, and the other steps and parameters are the same as those in Example 3.
[0072] Performance testing and result analysis:
[0073] The chemiluminescence values of Examples 3-5 and Comparative Example 1 were detected by an enzyme marker (Synergy H1) to determine the dATP consumption, and based on this, the gradient curve of the dATP consumption of the MMLV reverse transcriptase was compared to obtain the enzyme activity of the MMLV reverse transcriptase, and compared with the enzyme activity of the MMLV reverse transcriptase measured in Examples 3 to 5 and Comparative Example 1. It was found that the enzyme activity of the MMLV reverse transcriptase measured by the detection method of the present application was not much different from the enzyme activity detected by the enzyme marker (Synergy H1). Comparing the dATP consumption of Examples 3-5 and Comparative Example 1 measured by the enzyme marker (Synergy H1), it can be seen that under the same reverse transcription reaction conditions, the corresponding reverse transcription primers of Examples 3-5 can consume more dATP relative to Comparative Example 1, indicating that the reverse transcription primers of the present application can improve the accuracy of detecting the enzyme activity of the MMLV reverse transcriptase.
[0074] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.
Claims
1. A method for detecting the enzymatic activity of MMLV reverse transcriptase, characterized in that: The following steps are involved: S1. Based on the known concentration of luciferase, the known concentration of dATP is detected to prepare a standard relationship curve between the chemiluminescence value of luciferase and dATP consumption; S2, using RNA MS2 as a reverse transcription template, designing a reverse transcription primer sequence, and then performing reverse transcription reaction 1 to obtain target cDNA; performing reverse transcription reaction 2 on MMLV reverse transcriptase at different concentration gradients to obtain a standard relationship curve between enzyme activity and dATP consumption; S3, after the reverse transcription reaction 1 is completed, the dATP remaining after the reverse transcription reaction is detected by using a luciferase of known concentration, and the dATP consumption is obtained by comparing with the standard relationship curve of the chemiluminescence value of luciferase and dATP consumption; S4. Compare the dATP consumption and enzyme activity with the standard relationship curve of dATP consumption to obtain the enzyme activity of the MMLV reverse transcriptase to be tested.
2. The method for detecting the enzymatic activity of MMLV reverse transcriptase according to claim 1, characterized in that: The nucleotide sequence of the reverse transcription primer is the nucleotide sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO:
8.
3. The method for detecting the enzymatic activity of MMLV reverse transcriptase according to claim 1, characterized in that: In step S3, the concentration of the luciferase is 1-5 mg / mL.
4. The method for detecting the enzymatic activity of MMLV reverse transcriptase according to claim 1, characterized in that: The reaction conditions of the reverse transcription reaction are: 40-50° C., reaction time 30-60 min; 80-95° C., reaction time 1-5 min.
5. A kit for detecting MMLV reverse transcriptase activity, characterized in that: The kit comprises a reverse transcription primer having a nucleotide sequence as shown in any one of SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:
8.
6. The kit for detecting MMLV reverse transcriptase activity according to claim 5, characterized in that: The concentration of the reverse transcription primer is 5-15 mM, and the volume is 1-4 μL.
7. The kit for detecting MMLV reverse transcriptase activity according to claim 5, characterized in that: The kit further comprises at least one of dNTP, MMLV reverse transcriptase, RNA MS2 template, RRI enzyme and MMLV reaction buffer.
8. The kit for detecting MMLV reverse transcriptase activity according to claim 7, characterized in that: The dNTPs include dATP, dTTP, dCTP and dGTP, the concentration of the dATP / dTTP / dCTP / dGTP is 5-15 mM, and the volume of the dNTPs is 1-4 μL; the concentration of the RNA MS2 is 200 ng / μL, and the volume is 0.5-4 μL.
9. The kit for detecting MMLV reverse transcriptase activity according to claim 7, characterized in that: The MMLV reaction buffer includes Tris-HCl, KCl, MgCl2 and DTT.
10. Use of the detection method according to any one of claims 1 to 4 or the kit for detecting MMLV reverse transcriptase activity according to any one of claims 5 to 9 in detecting MMLV reverse transcriptase activity.