Method for analyzing random hexamer primer degradation product
Through high-performance liquid chromatography analysis technology and nuclease treatment, standard curves are constructed to analyze the degradation of random hexamer primers, solving the problem of lack of detection methods in the existing technology, achieving rapid and accurate degradation analysis, with simple operation , low cost and high sensitivity.
Patent Information
- Application Number
- CN202411305205.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-19
- Publication Date
- 2024-12-20
AI Technical Summary
The prior art lacks effective detection methods to analyze the degradation products of random hexamer primers, making it difficult to monitor their degradation in DNA replication or polymerase chain reactions.
Using high-performance liquid chromatography analysis technology, combined with nuclease to treat random hexamer primers, the concentration and peak area standard curves of deoxynucleoside monophosphate were constructed to quickly and accurately analyze their degradation. The Waters xBridge C18 chromatography column, acetonitrile and Triethylamine phosphate or triethylamine acetate is used as mobile phase and gradient elution is performed to improve resolution.
It realizes rapid and accurate analysis of the degradation of random hexamer primers, and has the technical advantages of simple operation, low cost and high sensitivity, and can quantitatively detect its degradation products.
Smart Images
Figure CN119165076A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of molecular biology, and in particular to a method for analyzing degradation products of random hexamer primers. Background Art
[0002] Random hexamer primers (6N) are primers composed of six random sequence nucleotides, which have no specificity and are widely used in biological experiments such as DNA replication or polymerase chain reaction. Currently, there is no public detection method.
[0003] In view of this, the present invention is proposed. Summary of the invention
[0004] The object of the present invention is to provide a method for analyzing degradation products of random hexamer primers to solve the above technical problems.
[0005] The present invention is achieved in that:
[0006] The present invention provides a method for analyzing random hexamer primer degradation products, which comprises the following steps:
[0007] S1: using a mixed standard solution containing four deoxynucleoside monophosphates as a control sample, preparing control sample solutions of different concentrations; treating the random hexamer primer to be tested with nuclease to obtain a reaction product;
[0008] S2: loading control sample solutions and reaction products of different concentrations respectively, and analyzing by high performance liquid chromatography, constructing a standard curve of the concentration and peak area of each deoxynucleoside monophosphate, and obtaining the concentration of each deoxynucleoside monophosphate after random hexamer primer degradation according to the standard curve;
[0009] The conditions for HPLC analysis included:
[0010] A Waters xBridge C18 (150×4.6mm, 3.5μm) chromatographic column was used; the detection wavelength was 254nm; the injection volume was 5-100μL, the mobile phase A was acetonitrile, the mobile phase B was triethylamine phosphate or triethylamine acetate with a concentration of 0.1mol / L, and the gradient elution was performed at a flow rate of 0.8-1.5mL / min and a column temperature of 30-60°C.
[0011] The present invention has the following beneficial effects:
[0012] The present invention utilizes nuclease to digest random hexamer primers, combined with high performance liquid chromatography technology, to rapidly and accurately analyze the degradation of random hexamer primers, and has the technical advantages of simple operation, strong liquid phase method specificity, low analysis cost, and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.
[0014] Figure 1 This is the HPLC chromatogram of the blank control of triethylammonium phosphate system after loading;
[0015] Figure 2 This is a high performance liquid chromatogram of the standard reference sample solution of triethylamine phosphate system after loading;
[0016] Figure 3 This is a high performance liquid chromatogram of the liquid before random hexamer primer digestion reaction in triethylamine phosphate system after loading;
[0017] Figure 4 This is a high performance liquid chromatogram of the liquid sampled after random hexamer primer digestion reaction in triethylamine phosphate system;
[0018] Figure 5 This is the dAMP standard curve after loading the triethylammonium phosphate system mixed standard;
[0019] Figure 6 This is the dCMP standard curve spectrum after loading the triethylammonium phosphate system mixed standard;
[0020] Figure 7 This is the dGMP standard curve spectrum after the triethylammonium phosphate system mixed standard is loaded;
[0021] Figure 8 This is the dTMP standard curve spectrum after loading the triethylammonium phosphate system mixed standard;
[0022] Fig. 9 This is the HPLC chromatogram of the blank control of triethylammonium acetate system after loading;
[0023] Fig.10 This is the HPLC chromatogram after loading the standard reference sample solution of triethylammonium acetate system;
[0024] Fig.11 This is a high performance liquid chromatogram of the liquid before random hexamer primer digestion reaction in triethylamine acetate system after loading;
[0025] Fig.12 This is a high performance liquid chromatogram of the liquid after random hexamer primer digestion reaction in triethylamine acetate system after loading;
[0026] Fig.13This is the dAMP standard curve spectrum after the triethylammonium acetate system mixed standard is loaded;
[0027] Fig.14 This is the dCMP standard curve spectrum after the triethylammonium acetate system mixed standard is loaded;
[0028] Fig.15 This is the dGMP standard curve spectrum after the triethylammonium acetate system mixed standard is loaded;
[0029] Fig.16 This is the dTMP standard curve after loading the triethylammonium acetate system mixed standard. DETAILED DESCRIPTION
[0030] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.
[0031] The present invention provides a method for analyzing random hexamer primer degradation products, which comprises the following steps:
[0032] S1: using a mixed standard solution containing four deoxynucleoside monophosphates as a control sample, preparing control sample solutions of different concentrations; treating the random hexamer primer to be tested with nuclease to obtain a reaction product;
[0033] S2: loading control sample solutions and reaction products of different concentrations respectively, and analyzing by high performance liquid chromatography, constructing a standard curve of the concentration and peak area of each deoxynucleoside monophosphate, and obtaining the concentration of each deoxynucleoside monophosphate after random hexamer primer degradation according to the standard curve;
[0034] The conditions for HPLC analysis included:
[0035] A Waters xBridge C18 (150×4.6mm, 3.5μm) chromatographic column was used; the detection wavelength was 254nm; the injection volume was 5-100μL, the mobile phase A was acetonitrile, the mobile phase B was triethylamine phosphate or triethylamine acetate with a concentration of 0.1mol / L, and the gradient elution was performed at a flow rate of 0.8-1.5mL / min and a column temperature of 30-60°C.
[0036] Using triethylamine phosphate or triethylamine acetate as mobile phase B can make each deoxynucleoside monophosphate have a higher separation degree on the chromatogram, which is helpful for quantitative detection and analysis of random hexamer primer degradation products.
[0037] The method for analyzing the degradation products of random hexamer primers provided by the present invention can quickly and accurately analyze the degradation of random hexamer primers, and has the technical advantages of simple operation, strong liquid phase method specificity, low analysis cost and high sensitivity.
[0038] In a preferred embodiment of the present invention, the gradient elution process comprises:
[0039]
[0040] The percentages of the mobile phase A and the mobile phase B are all volume percentages. The four deoxynucleoside monophosphates can be better separated by gradient elution, providing a basis for the detection of subsequent detection units.
[0041] In a preferred embodiment of the present invention, in step S1, the conditions for treating the random hexamer primer to be tested with nuclease include: 37°C±0.5°C, 1h; 85°C±0.5°C, 15min. The treatment at 37°C±0.5°C for 1h is the step of nuclease breaking the phosphodiester bond of the random hexamer primer to be tested. The treatment at 85°C for 15min is the step of inactivating the nuclease.
[0042] In a preferred embodiment of the present invention, the nuclease is selected from at least one of 3'→5' exonuclease, 5'→3' exonuclease and deoxyribonuclease I. Any method is feasible as long as it can break the phosphodiester bond in the random hexamer primer to be tested.
[0043] In a preferred embodiment of the present invention, the exonuclease is selected from at least one of exonuclease I or exonuclease III and deoxyribonuclease I.
[0044] In a preferred embodiment of the present invention, each 8.5×10 -3 μmol of the random hexamer primer to be tested is mixed with 1-200 U of the nuclease, for example, 1-20 U of the nuclease, or 5-50 U of the nuclease, or 80-200 U of the nuclease.
[0045] In a preferred embodiment of the present invention, each 8.5×10 -3 μmol of the random hexamer primer to be tested is mixed with 100 U of exonuclease and 5 U of deoxyribonuclease I. Under the above mixing conditions, the nuclease digestion efficiency is high.
[0046] In a preferred embodiment of the present invention, in step S1, at least 5 control sample solutions of different concentrations are prepared to ensure the linearity and accuracy of the standard curve. For example, at least 5, 6, 7, 8 or 10 control sample solutions of different concentrations are prepared.
[0047] In a preferred embodiment of the present invention, in each control sample solution, the mixing molar ratio of deoxyadenosine monophosphate, deoxycytidine monophosphate, deoxyguanosine monophosphate and deoxythymidine monophosphate is 1-1.05:1-1.05:1-1.05:1-1.05. The same mixing molar ratio helps to simplify the method.
[0048] In a preferred embodiment of the present invention, the pH of mobile phase B is 6.0-8.0.
[0049] In a preferred embodiment of the present invention, mobile phase B is triethylamine phosphate.
[0050] Triethylamine phosphate has a better separation effect.
[0051] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.
[0052] Example 1
[0053] This example provides a method for analyzing degradation products of random hexamer primers, and the specific method is as follows:
[0054] 1. The instruments and reagents involved in this method are as follows:
[0055] (1) Instruments: water bath, small centrifuge, Waters Arc high performance liquid chromatograph equipped with Waters 2698PDA detector, liquid phase pump and autosampler;
[0056] (2) Reagents: exonuclease I, 10X reaction buffer, 100 μmol / L random hexamer primers, deoxyribonuclease I, phosphoric acid (chromatographic grade), triethylamine (chromatographic grade), acetonitrile (chromatographic grade), purified water.
[0057] Preparation of 0.1 mol / L triethylamine phosphate: Measure 13.9 mL of triethylamine and add it to a 1 L mobile phase bottle containing 900 mL of purified water, then adjust the pH to 7.0 with phosphoric acid, and finally make up to 1 L with purified water.
[0058] A mixed standard solution containing four deoxynucleoside monophosphates was used as the control sample. The preparation process of the control sample stock solution was as follows: accurately weigh 33.12 mg dAMP, 30.72 mg dCMP, 34.72 mg dGMP, and 32.22 mg dTMP standards in a 10 mL volumetric flask, dissolve them to the scale with first-grade pure water, and obtain a 10 mmol / L dNMP mixed standard solution (i.e., the control sample stock solution) for use.
[0059] Preparation of 0.1mmol / L dNMP standard control sample solution: Accurately pipette 1mL of the control sample stock solution into a 100mL volumetric flask, dilute to the mark with primary pure water to obtain a 0.1mmol / L dNMP standard control sample solution.
[0060] 2. Test steps:
[0061] 1) The reaction system is as follows:
[0062]
[0063]
[0064] 2) Reaction procedure:
[0065] The above components were mixed and shaken, and after instant centrifugation, incubated at 37° C. for 1 hour and incubated at 85° C. for 15 minutes to obtain a liquid test solution 1 after random hexamer primer digestion reaction.
[0066] The reaction system without random hexamers was used as a blank control.
[0067] 3) High performance liquid chromatography analysis
[0068] Chromatographic conditions: Waters xBridge C18 (4.6×150mm.Column, id3.5μm) chromatographic column; injection volume of 10μL; detection wavelength of 254nm; acetonitrile as mobile phase A, 0.1mol / L triethylamine phosphate as mobile phase B, gradient elution at a flow rate of 1.0mL / min and a column temperature of 40°C.
[0069] The gradient elution program is as follows:
[0070]
[0071] 4) Result analysis
[0072] First, the blank control solution and the 0.1mmol / L dNMP standard control sample solution were loaded onto the machine, and then the random hexamer primers before the digestion (i.e., nuclease digestion) reaction and the liquid test solution 1 after the digestion reaction were respectively loaded onto the machine for analysis and detection to obtain the chromatograms, respectively referring to Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown, compared Figure 2 The standard reference sample spectrum can be used to determine Figure 4 The random hexamer is degraded into four nucleotides: dAMP, dCMP, dGMP, and dTMP. Figure 1 , Figure 3 as well as Figure 4It can be determined that the random hexamer is partially degraded into four nucleotides: dAMP, dCMP, dGMP, and dTMP.
[0073] Example 2
[0074] This example analyzes the degradation products of the random hexamer primer sample to be tested in Example 1, specifically including the construction and detection of a standard curve:
[0075] 1. The instruments involved in this method are the same as those in Example 1, and the reagents are as follows:
[0076] Exonuclease I, 10X reaction buffer, 100 μmol / L random hexamer primers, deoxyribonuclease I, phosphoric acid (chromatographic grade), triethylamine (chromatographic grade), acetonitrile (chromatographic grade), purified water.
[0077] Preparation of 0.1 mol / L triethylamine phosphate: Measure 13.9 mL of triethylamine and add it to a 1 L mobile phase bottle containing 900 mL of purified water, then adjust the pH to 7.0 with phosphoric acid, and finally make up to 1 L with purified water.
[0078] A mixed standard solution containing four deoxynucleoside monophosphates was used as the control sample. The preparation process of the control sample stock solution was as follows: accurately weigh 33.12 mg dAMP, 30.72 mg dCMP, 34.72 mg dGMP, and 32.22 mg dTMP standards in a 10 mL volumetric flask, dissolve them to the scale with first-grade pure water, and obtain a 10 mmol / L dNMP mixed standard solution (i.e., the control sample stock solution) for use.
[0079] Preparation of 0.1mmol / L dNMP control sample solution: Accurately pipette 1mL of control sample stock solution into a 100mL volumetric flask, dilute to the mark with primary pure water to obtain 0.1mmol / L dNMP control sample solution. In the same way, prepare 0.5mmol / L dNMP, 1.0mmol / L dNMP, 1.5mmol / L dNMP, and 2.0mmol / L dNMP control sample solutions.
[0080] 2. Test steps:
[0081] 1) The reaction system is as follows:
[0082]
[0083]
[0084] 2) Reaction procedure:
[0085] The above components were mixed and shaken, and after instant centrifugation, incubated at 37° C. for 1 hour and incubated at 85° C. for 15 minutes to obtain a liquid test solution 1 after random hexamer primer digestion reaction.
[0086] 3) High performance liquid chromatography analysis
[0087] Chromatographic conditions: Waters xBridge C18 (4.6×150mm.Column, id3.5μm) chromatographic column; injection volume of 10μL; detection wavelength of 254nm; acetonitrile as mobile phase A, 0.1mol / L triethylamine phosphate as mobile phase B, gradient elution at a flow rate of 1.0mL / min and a column temperature of 40°C.
[0088] The gradient elution program is as follows:
[0089]
[0090] 4) Result analysis
[0091] First, first-grade pure water was loaded as a blank on the machine, and then 0.1mmol / L dNMP, 0.5mmol / L dNMP, 1.0mmol / L dNMP, 1.5mmol / L dNMP, 2.0mmol / L dNMP control sample solutions and test solution 1 were respectively loaded on the machine for analysis and detection to obtain chromatograms. Based on the chromatograms of the five control samples, a standard curve of the concentration (X) and peak area (Y) of each deoxynucleoside monophosphate was drawn (Table 1). Based on the peak areas of the four deoxynucleoside monophosphates of test solution 1, the concentration of the deoxynucleoside monophosphate was calculated by substituting them into the standard curve.
[0092] Standard curves for 4 deoxynucleoside monophosphates Figure 5 , Figure 6 , Figure 7 , Figure 8 shown.
[0093] Table 1 Standard curve
[0094]
[0095] Figure 4 It is the HPLC chromatogram of the test solution 1.
[0096] According to the chromatographic conditions of the present invention, it can be confirmed that the peak order of the chromatogram is dCMP, dGMP, dTMP and dAMP.
[0097] Example 3
[0098] Compared with Example 2, the only difference is that the mobile phase B is different. In this embodiment, the mobile phase B is triethylamine acetate with a pH of 7.0 and a concentration of 0.1 mol / L.
[0099] 1. The instruments involved in this method are the same as those in Example 1, and the reagents are as follows:
[0100] Exonuclease I, 10X reaction buffer, 100 μmol / L random hexamer primers, deoxyribonuclease I, acetic acid (chromatographic grade), triethylamine (chromatographic grade), acetonitrile (chromatographic grade), purified water.
[0101] Preparation of 0.1mol / L triethylamine acetate: Measure 13.9mL of triethylamine and add it to a 1L mobile phase bottle containing 900mL of purified water, then adjust the pH to 7.0 with acetic acid, and finally make up to 1L with purified water.
[0102] A mixed standard solution containing four deoxynucleoside monophosphates was used as the control sample. The preparation process of the control sample stock solution was as follows: accurately weigh 33.12 mg dAMP, 30.72 mg dCMP, 34.72 mg dGMP, and 32.22 mg dTMP standards in a 10 mL volumetric flask, dissolve them to the scale with first-grade pure water, and obtain a 10 mmol / L dNMP mixed standard solution (i.e., the control sample stock solution) for use.
[0103] Preparation of 0.1mmol / L dNMP control sample solution: Accurately pipette 1mL of control sample stock solution into a 100mL volumetric flask, dilute to the mark with primary pure water to obtain 0.1mmol / L dNMP control sample solution. In the same way, prepare 0.5mmol / L dNMP, 1.0mmol / L dNMP, 1.5mmol / L dNMP, and 2.0mmol / L dNMP control sample solutions.
[0104] 2. Test steps:
[0105] 1) The reaction system is as follows:
[0106]
[0107] 2) Reaction procedure:
[0108] The above components were mixed and shaken, and after instant centrifugation, incubated at 37° C. for 1 hour and incubated at 85° C. for 15 minutes to obtain a liquid test solution 1 after random hexamer primer digestion reaction.
[0109] 3) High performance liquid chromatography analysis
[0110] Chromatographic conditions: Waters xBridge C18 (4.6×150mm.Column, id3.5μm) chromatographic column; injection volume of 10μL; detection wavelength of 254nm; acetonitrile as mobile phase A, 0.1mol / L triethylamine acetate as mobile phase B, gradient elution at a flow rate of 1.0mL / min and a column temperature of 40°C.
[0111] The gradient elution program is as follows:
[0112]
[0113] 4) Result analysis
[0114] First, first-grade pure water was loaded as a blank on the machine, and then 0.1mmol / L dNMP, 0.5mmol / L dNMP, 1.0mmol / L dNMP, 1.5mmol / L dNMP, 2.0mmol / L dNMP control sample solutions and test solution 1 were respectively loaded on the machine for analysis and detection to obtain chromatograms. Based on the chromatograms of the five control samples, a standard curve of the concentration (x) and peak area (y) of each deoxynucleoside monophosphate was drawn (Table 2). Based on the peak areas of the four deoxynucleoside monophosphates of test solution 1, the concentration of the deoxynucleoside monophosphate was calculated by substituting them into the standard curve.
[0115] Standard curves for 4 deoxynucleoside monophosphates Fig.13 , Fig.14 , Fig.15 as well as Fig.16 shown.
[0116] Table 2 Standard curve
[0117]
[0118] Fig. 9 , Fig.10 , Fig.11 , Fig.12 The blank control, the standard control sample solution, the liquid before the random hexamer primer digestion reaction, and the liquid after the random hexamer primer digestion reaction are HPLC chromatograms after the sample is loaded on the triethylamine acetate system. According to the chromatographic conditions of the present invention, it can be confirmed that the peak order of the chromatogram is dCMP, dGMP, dTMP and dAMP.
[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for analyzing degradation products of random hexamer primers, It is characterized in that It includes the following steps: S1: using a mixed standard solution containing four deoxynucleoside monophosphates as a control sample, preparing control sample solutions of different concentrations; treating the random hexamer primer to be tested with nuclease to obtain a reaction product; S2: loading the control sample solution and the reaction product with different concentrations respectively, analyzing them by high performance liquid chromatography, constructing a standard curve of the concentration and peak area of each deoxynucleoside monophosphate, and obtaining the concentration of each deoxynucleoside monophosphate after the random hexamer primer is degraded according to the standard curve; The conditions of the HPLC analysis include: A Waters xBridge C18 (150×4.6mm, 3.5μm) chromatographic column was used; the detection wavelength was 254nm; the injection volume was 5-100μL, the mobile phase A was acetonitrile, the mobile phase B was triethylamine phosphate or triethylamine acetate with a concentration of 0.1mol / L, and the gradient elution was performed at a flow rate of 0.8-1.5mL / min and a column temperature of 30-60°C.
2. The method for analyzing random hexamer primer degradation products according to claim 1, It is characterized in that The gradient elution process comprises:
3. The method for analyzing random hexamer primer degradation products according to claim 1, It is characterized in that In the step S1, the conditions for treating the random hexamer primers to be tested with nuclease include: 37°C±0.5°C, 1h; 85°C±0.5°C, 15min.
4. The method for analyzing random hexamer primer degradation products according to claim 3, It is characterized in that The nuclease is at least one selected from 3′→5′ exonuclease, 5′→3′ exonuclease and deoxyribonuclease I.
5. The method for analyzing random hexamer primer degradation products according to claim 4, It is characterized in that The nuclease is selected from at least one of exonuclease I, exonuclease III and deoxyribonuclease I.
6. The method for analyzing random hexamer primer degradation products according to claim 3, It is characterized in that Each 8.5×10 -3 μmol of the random hexamer primer to be tested is mixed with 1-200 U of the nuclease.
7. The method for analyzing random hexamer primer degradation products according to claim 1, It is characterized in that In the step S1, at least 5 control sample solutions with different concentrations are prepared.
8. The method for analyzing random hexamer primer degradation products according to claim 7, It is characterized in that In each of the control sample solutions, the mixing molar ratio of deoxyadenosine monophosphate, deoxycytidine monophosphate, deoxyguanosine monophosphate and deoxythymidine monophosphate is 1-1.05:1-1.05:1-1.05:1-1.
05.
9. The method for analyzing random hexamer primer degradation products according to claim 1, It is characterized in that The pH of the mobile phase B is 6.0-8.
0.
10. The method for analyzing random hexamer primer degradation products according to claim 1, It is characterized in that The mobile phase B is triethylamine phosphate.