High-throughput screening system and screening method of sequencing enzyme

Abstract

The invention relates to the technical field of biology, in particular to a high-throughput screening system and method for sequencing enzymes. Fluorescence pairs CY3 and CY5 are connected to a DNA template chain and dATP respectively, a DNA polymerase screening system is constructed, and on the basis of an automatic platform and a microplate reader, the speed, efficiency and catalytic activity of doping a modified single basic group into the DNA chain by the DNA polymerase are evaluated. Experimental results show that the screening system and the matched screening method can achieve high-throughput screening of DNA polymerase, the screening time is saved, the screening cost is reduced, and the screening system and the screening method can be applied to other related projects and have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a high-throughput screening system and screening method for sequencing enzymes. Background Art

[0002] FRET refers to the situation where, among two different fluorescent groups, if the emission spectrum of one fluorescent group (donor) overlaps with the absorption spectrum of another fluorescent group (acceptor), when the two fluorescent groups are close enough, the donor molecule is excited to a higher electronic energy state after absorbing photons of a certain frequency. Before the electron returns to the ground state, energy is transferred to the adjacent acceptor molecule through dipole interaction. The FRET luminescence principle is as follows Figure 1 , excite the donor fluorescence and when the distance between the fluorescent donor molecule and the fluorescent acceptor molecule is close enough, energy resonance transfer can occur, realizing the phenomenon of acceptor luminescence, and then fluorescence detection is performed through a fluorescence capture device, such as an enzyme marker:

[0003] FRET conditions:

[0004] 1) Energy matching: The emission spectrum of the donor molecule can be absorbed by the acceptor molecule and produce a fluorescent signal. The emission spectrum of the donor molecule must overlap significantly with the excitation spectrum of the acceptor molecule (>30%).

[0005] 2) Interaction distance: The interaction distance between donor molecules and acceptor molecules is 1 to 10 nm;

[0006] 3) FRET efficiency formula: used to calculate the interaction distance R between molecules / groups

[0007]

[0008] R0=const·κ 2 ·n -4 ·φ D ·J DA

[0009] 4) Dipole-dipole interaction: The vectors when the donor molecule interacts with the acceptor molecule must meet certain conditions.

[0010] FRET technology has a wide range of applications. It has a history of 25 years since its discovery. The technology is widely used in the fields of biology and biochemistry. FRET technology can be used to obtain spatial information about the interaction between two protein molecules (action distance, action direction, energy transfer efficiency). It is often used to solve the following problems: co-localization of protein molecules; protein molecule aggregation; transcription mechanism; transformation pathway; molecular movement; protein folding and other biological problems.

[0011] Traditional methods for detecting the incorporation of single bases into DNA chains generally require electrophoresis analysis, which is time-consuming, has low throughput, requires more time and costs more; moreover, the electrophoresis analysis method can only check the incorporation of single bases by the endpoint method, and cannot detect the incorporation of single bases in real time. Therefore, it is time-consuming and labor-intensive to evaluate the speed, efficiency and catalytic activity of enzymes for the incorporation of single bases into DNA chains or to screen for highly active sequencing enzymes by electrophoresis. The first version of sequencing enzyme screening technology also used the resonance energy transfer (FRET) technology, but due to the complicated operation process and the lack of an automatic workstation, it was not possible to perform high-throughput screening. Summary of the invention

[0012] In view of this, the technical problem to be solved by the present invention is a high-throughput screening system and screening method for enzymes used for DNA manipulation.

[0013] The present invention provides a nucleotide analog having a structure as shown in the following formula (I):

[0014]

[0015] in,

[0016] Blocker is a blocking group;

[0017] P is a triphosphate group;

[0018] X is a nitrogenous base, and the nitrogenous base is selected from any one of adenine, guanine, uracil, cytosine or thymine;

[0019] Y is a polypeptide;

[0020] R is -OH or -H;

[0021] Preferably, the structure of the blocking group Blocker is as follows:

[0022]

[0023] Furthermore, the nucleotide analogues of the present invention have a structure as shown in formula (II):

[0024] The nucleotide analogs described in the present invention may include deoxyribonucleotide analogs and / or ribonucleotide analogs; the deoxyribonucleotide analogs can be incorporated into DNA chains without interfering with the synthesis of DNA chains and the function of DNA polymerase; the ribonucleotide analogs can be incorporated into RNA single chains without interfering with the synthesis of RNA and the activity of RNA polymerase.

[0025] The present invention provides a sequencing enzyme screening system, which comprises a nucleotide analog connected to a first detection group, a template DNA connected to a second detection group, and a DNA polymerase.

[0026] Furthermore, in the sequencing enzyme screening system of the present invention,

[0027] The 3' end and / or 5' end of the template DNA is connected to a second detection group;

[0028] The template DNA has at least one of a 3' single-stranded region or a 5' single-stranded region and a middle double-stranded region;

[0029] In some embodiments of the present invention,

[0030] The 3' end of the template DNA is connected to a second detection group;

[0031] The 5' end of the template DNA is connected to a second detection group;

[0032] The 3' end and the 5' end of the template DNA are both connected with a second detection group.

[0033] In the present invention, the template DNA may have a 3' single-stranded region and a middle double-stranded region, a 5' single-stranded region and a middle double-stranded region, or a 3' single-stranded region, a 5' single-stranded region and a middle double-stranded region.

[0034] Furthermore, in some specific embodiments of the present invention, the template DNA has a 3' single-stranded region, a 5' single-stranded region and a middle double-stranded region.

[0035] The 3' single-stranded region or 5' single-stranded region of the template DNA described in the present invention is used to incorporate nucleotides into the DNA chain under the action of DNA polymerase, and then the polymerase efficiency is evaluated according to the incorporation speed; the 3' single-stranded region and the 5' single-stranded region can exist at the same time, or only one of them can exist, which has no effect on the detection result; and the nucleotide sequence composition of the 3' single-stranded region and the 5' single-stranded region has no effect on the detection result; the DNA template can be replaced according to the needs of technical personnel in the field.

[0036] However, the template DNA design described in the present invention is suitable for the screening system described in the present invention, and its synthesis is simple, screening is convenient, and material consumption is economical.

[0037] In some specific embodiments of the present invention, the nucleotide sequences of the 3' single-stranded region and the 5' single-stranded region of the template DNA are opposite.

[0038] In the present invention, the template DNA can also be prepared by annealing two primers with identical or different 3' end sequences and reverse complementary 5' end sequences; the number of nucleotides at the 5' end is ≥ the number of nucleotides at the 3' end.

[0039] In the present invention, the emission spectrum of the first detection group overlaps with the absorption spectrum of the second detection group.

[0040] Furthermore, the emission spectrum of the first detection group and the absorption spectrum of the second detection group have an overlap of ≥30.

[0041] Specifically, the first detection group and the second detection group may include any combination as shown below:

[0042] The first detection group is B-Phycoerythrin; the second detection group is Cy5; or

[0043] The first detection group is Rhodamine 6G; the second detection group is Cy5; or

[0044] The first detection group is GFP; the second detection group is YFP; or

[0045] The first detection group is Cy3; the second detection group is Cy5; or

[0046] The first detection group is Fluorescein; the second detection group is Tetramethylrhodamine; or

[0047] The first detection group is CFP; the second detection group is GFP; or

[0048] The first detection group is BFP; the second detection group is DsRFP; or

[0049] The first detection group is Tryptophan; the second detection group is Dansyl.

[0050] Furthermore, in the present invention,

[0051] The first detection group is cy3;

[0052] The second detection group is cy5.

[0053] The present invention provides a kit, which comprises auxiliary materials and at least one of the following I) to II):

[0054] 1), the nucleotide analogues of the present invention;

[0055] II), the sequencing enzyme screening system described in the present invention.

[0056] Furthermore, the auxiliary material is a solid or liquid reagent commonly used in the field of biotechnology or a combination thereof;

[0057] Furthermore, the auxiliary material includes one or a combination of two or more of a lysis solution, a cleaning solution, a purification reagent or a buffer solution.

[0058] The present invention provides a method for screening a sequence enzyme, comprising: screening a sequence enzyme using the nucleotide analogs described in the present invention and / or the sequence enzyme screening system described in the present invention and / or the kit described in the present invention.

[0059] Furthermore, the screening method comprises the following steps:

[0060] A mixture of a nucleotide analog connected to a first detection group, a nucleotide, a template DNA connected to a second detection group, a sequencing enzyme, a buffer and water is reacted and detected, and the DNA polymerase is screened after obtaining a single base incorporation rate based on an activity curve of the detection signal changing over time;

[0061] The nucleotides include a mixture of nucleotides having different nitrogenous bases and nucleotide analogs.

[0062] In the screening method of the present invention,

[0063] The concentration of the nucleotide analogue is 0.25 μM;

[0064] The concentration of the nucleotide is 0.25 μM;

[0065] The concentration of the template DNA connected with the second detection group is 0.1 μM;

[0066] The concentration of the sequencing enzyme is 0.5 mg / mL.

[0067] In the screening method of the present invention,

[0068] The reaction conditions are 40°C for 2h;

[0069] The detection conditions are: excitation wavelength of 530nm, emission wavelengths of 568nm and 676nm; detection once every 1 to 3 minutes;

[0070] The incorporation rate of the single base is the slope of the activity curve.

[0071] The screening method of the present invention can also be carried out in the case of normal DNA chains or RNA chains through the pre-denaturation, annealing and extension procedures of ordinary PCR.

[0072] The sequencing enzyme described in the present invention is obtained through induced expression, and the step of induced expression can be performed with the aid of automated equipment or manually; the induced expression described in the present invention is performed with the aid of a Tecan automatic workstation, and the small-volume culture and rapid production of the sequencing enzyme are completed using automated instruments and equipment while meeting the requirements for subsequent detection, which is conducive to shortening the process and saving time and cost.

[0073] The present invention provides the use of at least one of the following i) to iv) in preparing sequencing enzyme screening products, testing sequencing enzyme reaction kinetics, evaluating catalytic activity and / or evaluating the speed and efficiency of enzyme base incorporation into template chains:

[0074] i), the nucleotide analogues of the present invention;

[0075] ii), the sequencing enzyme screening system of the present invention;

[0076] iii), the kit of the present invention;

[0077] iv) The screening method of the present invention.

[0078] Furthermore, the sequencing enzyme includes at least one of a DNA polymerase, a reverse transcriptase, a tailing enzyme or an RNA polymerase.

[0079] The screening method described in the present invention can evaluate the performance of sequencing enzyme screening products.

[0080] The present invention connects the fluorescent pair CY3 and CY5 to the DNA template chain and dATP respectively, constructs a DNA polymerase screening system, and realizes the evaluation of the speed, efficiency and catalytic activity of DNA polymerase incorporating modified single bases into the DNA chain based on the automation platform and the microplate reader. The experimental results show that the screening system and the matching screening method of the present invention can realize high-throughput screening of DNA polymerase, save the screening time, reduce the screening cost, and the screening system and screening method can be applied to other related projects, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Schematic diagram of the principle of resonance energy transfer (FRET);

[0082] Figure 2 Spectra of CY3 and CY5 are shown;

[0083] Figure 3 Shows the structure of Modified dATP;

[0084] Figure 4 .Structural diagram of P / T-2CY5;

[0085] Figure 5 The figure shows the results of crude enzyme activity screening of mutants;

[0086] Figure 6 The reaction kinetic curves of the three mutants to the DNA template are shown;

[0087] Figure 7 The kinetic curves of the three mutants' reactions to modified dATP are shown;

[0088] Figure 8 Shown is the blocking nucleotidase activity test of SSEB blocking nucleotides and N3. DETAILED DESCRIPTION

[0089] The present invention provides a high-throughput screening system, screening method and application of DNA polymerase. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0090] The present invention is based on the FRET technology and selects two fluorescent groups CY5 and CY3 with spectral overlap. This fluorescent pair is widely used in the FRET technology. The spectral overlap diagram of CY5 and CY3 is shown in FIG. Figure 2 The fluorescent group CY3 is connected as a fluorescent donor group to the nitrogenous base side chain of the 3'-OH blocked modified dATP. The modified nucleotide (modified dATP) is as follows Figure 3 (For details, see patent PCT / CN2022 / 084601); the fluorescent group CY5 is connected to the 5' end of the oligonucleotide chain as a fluorescent receptor group, and the two oligonucleotide chains are annealed to form a DNA double strand and serve as template primers for each other (P / T-2CY5). Figure 4. Modified dATP, P / T-2CY5 and DNA polymerase are present in the reaction system at the same time. When modified dATP is polymerized and incorporated into P / T-2CY5 by DNA polymerase, the distance between CY5 and CY3 is about 5nm, which meets the distance requirement for FRET to occur. The Cy3 (Excitation 530nm / emission 568nm) and FRET Cy5 (Excitation 530nm / emission 676nm) signals are detected simultaneously by an ELISA reader. According to the collected FRET Cy5 signal, the maximum slope of the signal trend line can be calculated to compare the reaction speeds of different mutants, so as to screen the mutants. The validity of the experiment must meet the following conditions: the trend line of the Cy3 signal in the positive control group shows a downward trend, the trend line of the FRET Cy5 signal shows an upward trend, and the trend lines of the Cy3 and FRET Cy5 signals in the negative control group both show a downward trend or stabilize after a downward trend, indicating that this experiment is effective and modified dATP is successfully incorporated into the DNA chain.

[0091] In the enzymatic reaction, the concentrations of the substrates P / T-2CY5 and Modified dATP are much greater than the enzyme concentration. At this time, the maximum reaction rate (Vmax) is close to a constant value, and the Km value is the substrate concentration corresponding to half the maximum reaction rate. The Michaelis-Menten equation is as follows.

[0092]

[0093] For detailed experimental steps, please refer to Example 1, Example 2 and Example 3.

[0094] In the present invention, for a library of sequencer mutants constructed by site-directed mutagenesis and multi-point mutagenesis, after inducing protein expression, this method is used to screen the incorporation activity of sequencer mutants to non-natural nucleotides, screen the kinetic activity, and evaluate the catalytic efficiency. The mutant screening method of the present invention has high throughput, high detection sensitivity, and simple operation. It can well evaluate the incorporation ability of DNA polymerase to non-natural nucleotides, and reflect the incorporation of non-natural nucleotides into DNA template chains over time in real time. The raw experimental data can be exported by an ELISA instrument, and the data can be quantified and convenient for processing and analysis.

[0095] The sequencing enzyme screening technology involved in this invention is the second version. Compared with the first version of the sequencing enzyme screening technology, it is equipped with an automatic workstation, optimizes the operating process, realizes high-throughput screening, improves screening efficiency, is easier to use, and saves time and manpower and material resources.

[0096] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0097] The present invention will be further described below in conjunction with embodiments:

[0098] Example 1 Screening of high-throughput sequencing enzyme mutant activity

[0099] The experiment in this example is mainly to evaluate and screen the ability of sequencing enzyme mutants to polymerize modified nucleotides. A total of 30 mutants were screened in this experiment, and each mutant had three parallel experiments.

[0100] 1. Inducible expression of mutants

[0101] The sequencing enzyme mutant plasmid or PCR product was subjected to a BL21 competent cell plate transformation experiment. After overnight culture at 37°C, a single clone was picked out for culture and induction expression in a 96-well plate liquid LB medium.

[0102] The experimental operation is as follows:

[0103] 1) Using Tecan automatic workstation (company brand: Tecan / Diken; specification model: Freedom ) was used to dispense LB medium in a 96-well plate (costar REF 3596), with 150 μL LB (Kan + , 50μg / mL), seal the 96-well plate and store it in a 4℃ refrigerator for later use. This operation requires a sterile environment.

[0104] 2) Use the Tecan automatic workstation to perform a single colony selection experiment on the transformed plate. Each selected pipette tip is blown and aspirated three times in the corresponding well of the 96-well plate that has been pre-packed with LB to ensure that the bacteria are added to the culture medium. This operation requires a sterile environment.

[0105] 3) The inoculated 96-well plate was re-sealed and placed in a 37°C incubator with shaking at 150 rpm for 6 h.

[0106] 4) Take 3 μL (diluted 1:50) of the above culture medium and transfer it to 150 μL LB (Kan + , 50 μg / mL) in a new 96-well plate and culture at 37°C, 150 rpm for 2-2.5 h until OD600 = 0.8.

[0107] 5) Use a Tecan automated workstation to add IPTG to each well to a final concentration of 0.5 mM, and induce expression at 25°C and 220 rpm for 12 to 16 hours.

[0108] 6) Add 2 μL 100 mM PMSF and 15 μL 10 mg / mL lysozyme to the expression bacterial solution, react at 37°C for 10 min, centrifuge, take the supernatant and place on ice for later use.

[0109] 2. Sequencing enzyme mutant activity screening

[0110] (I) Preparation of screening reaction solution: Prepare the reaction system according to the experimental conditions in Table 1. The crude enzyme solution is the bacterial solution containing the culture medium. (If the effectiveness of the system is tested with pure enzyme, the pure enzyme needs to be diluted 10 times with 1× buffer (buffer: 20mM Tris-HCl, 10mM (NH4)2SO4, 10mM KCl, 4mM MgSO4, 0.1vol% Triton X100) and 0.1% BSA protein protection is added. After dilution, 10μL of the diluted enzyme can be added to the 50μL system)

[0111] Table 1. Experimental setup and reaction system

[0112] Experimental components Mutants Positive control Negative control 10×buffer 5μL 5μL 5μL dATP-Cy3 0.25μM 0.25μM 0.25μM dCTP+dTTP+dGTP 0.25μM 0.25μM 0.25μM P / T-2Cy5 0.1μM 0.1μM 0.1μM Crude enzyme solution 20μL 20μL - <![CDATA[H2O]]> Make up to 50 μL Make up to 50 μL Make up to 50 μL

[0113] The enzyme of PC (positive control) is KOD159, sourced from CN 110818757.

[0114] Take a 384-well reaction plate (Corning REF 3573), mark it, pre-cool it on ice, and use a Tecan automatic workstation to accurately add samples.

[0115] Accurately add each prepared reaction system into the marked pre-cooled 384-well plate

[0116] (II) Enzyme activity detection

[0117] Detection of Cy3 and FRET Cy5 fluorescence signals using an ELISA reader

[0118] The reaction conditions are as follows:

[0119] 1) The reaction temperature was 40°C for 2 hours, and the fluorescence signal was collected every 1 to 3 minutes;

[0120] 2) Simultaneous detection of Cy3 (Excitation 530nm / emission 568nm) and FRET Cy5 (Excitation 530nm / emission 676nm) signals.

[0121] Analysis of experimental results: A total of 30 mutants were screened in this experiment. The single base incorporation reaction rate V (RFU / min) was defined as the slope of the activity curve. Mut_0 was used as the control, and the relative activity was defined as 1. The relative activities of the other 30 mutants were obtained by the ratio of the reaction rates V of the mutants and Mut_0. Among them, 26 mutants had polymerization activity for Modified dATP, and the other 4 mutants had no activity. Through the analysis of the experimental results, this screening method can well distinguish mutants that can polymerize a single modified base from mutants that cannot polymerize modified bases. The experimental results are shown in Figure 2. Figure 5 and Table 2.

[0122] Table 2. Relative activities of mutants

[0123]

[0124] In this experiment, there were 9 mutants with relative activities above 2.0 and 15 mutants with activities above 1.0. The comparison of relative activities can well distinguish mutants with faster and slower modified dATP polymerization rates, indicating that the method of screening sequencing enzyme mutants is very efficient, and superior mutants can be selected for further experiments based on the screening results of crude enzyme activity.

[0125] Example 2 Kinetic Test of Sequencing Enzyme Mutants on DNA Template Strand (P / T)

[0126] 1. Inducible expression of mutants

[0127] The sequencing enzyme mutant plasmid or PCR product was transformed into BL21 competent cells, cultured overnight at 37°C, and single colonies were selected for culture and induced expression in sterile culture tubes. In this experiment, three mutants KOD_M1, KOD_M2, KOD_M3, and KOD were screened. The experimental operation is as follows:

[0128] 1) Pick a single colony and place it in 5 ml LB (Kan + , 50 μg / mL) in sterile culture tubes at 37°C, 220 rpm, and culture overnight.

[0129] 2) Take 50 μL (diluted 1:100) of the above culture medium and transfer it to 5 mL LB (Kan + ,50μg / mL) in a sterile culture tube at 37℃, 220rpm until OD600=0.8.

[0130] 3) Add IPTG to a final concentration of 0.5 mM and induce expression at 30°C and 220 rpm for 4 h.

[0131] 4) Take 1 mL of the induction culture medium and centrifuge at 4°C and 12,000 rpm / min for 1 min to collect the bacteria.

[0132] 5) Add 100 μL 10× buffer to resuspend the cells.

[0133] 6) Add 1.25 μL 100 mM PMSF and 10 μL 10 mg / mL lysozyme.

[0134] 7) Incubate at 37°C for 10 min, then at 80°C for 30 min. Centrifuge at 4°C and 12,000 rpm / min for 5 min, and collect the supernatant for crude protein concentration determination.

[0135] 8) The crude protein concentration was determined using Bradford kit.

[0136] 2. Preparation of reaction solution for kinetic test of sequencing enzyme mutants on DNA template chain (P / T)

[0137] The reaction solution for the kinetic test of the sequencer mutant on the DNA template chain (P / T) is prepared in two parts, as shown in Table 3. First, the premix solution is prepared and placed on ice. Then, a series of P / T solutions with gradient concentrations are prepared and placed on ice. They are mixed according to the proportions, as shown in Table 4. The mixed reaction solution is placed on ice or at -20°C for standby use. The preparation conditions are shown in Tables 3 and 4:

[0138] Table 3. Preparation of premix

[0139]

[0140]

[0141] Table 4. Concentration gradient reaction solution preparation

[0142] Group P / T-2Cy5(μM) P / T(μM) Total P / T(μM) 1 0.2 0.2 0.4 2 0.4 0.4 0.8 3 0.6 0.6 1.2 4 1 1 2 5 1.5 1.5 3 6 2 2 4

[0143] Analysis of experimental results: Three mutants were tested in this experiment to evaluate their catalytic efficiency on modified dATP. The kinetic curves are shown in Figure 2. Figure 6 and Table 5 below:

[0144] Table 5. Kinetic parameters

[0145] Vmax Km Vmax / Km Relative Vmax / Km KOD_M1 448.8 1.4 311.0 1.0 KOD_M2 840.3 1.3 636.6 2.0 KOD_M3 519.3 0.8 680.7 2.2

[0146] The results of this experiment were analyzed. The reaction system can simulate the kinetic experiment well. Under different concentrations of DNA template, the corresponding reaction rate was calculated. GraphPad software was used for nonlinear fitting, and V was obtained by fitting. max , K m Then calculate Vmax / K m When the same enzyme concentration is added to each reaction, V max / K m The value of K cat / K m The values ​​of V max / K m The catalytic efficiency of different mutants on the substrate can be evaluated. As shown in Table 5, when the template DNA chain is kinetically evaluated, KOD_M3 and KOD_M2 have higher catalytic efficiency than KOD_M1.

[0147] Example 3 Kinetic test of sequence enzyme mutants on modified dATP

[0148] The mutant crude enzyme solutions KOD_M1, KOD_M2 and KOD_M3 in Example 2 were used to perform a modified dATP kinetic test. The preparation conditions of the reaction solution for the modified dATP kinetic test of the mutants of the sequencing enzyme are shown in Table 6:

[0149] Table 6. Preparation of premix for enzyme mutants to modified dATP kinetic test

[0150] Component Sample Negative control 10×buffer 5μL 5μL P / T-2CY5 1μM 1μM P / T 1μM 1μM dC+T+GTP 2μM 2μM Total P / T x x Enzyme 0.1μM 0.1μM BSA 0.1mg / mL 0.1mg / mL <![CDATA[H2O]]> To 50μL To 50μL

[0151] Table 7. Concentration gradient preparation of reaction solution for enzyme mutants to modified dATP kinetic test

[0152] Group Cold dATP(μM) Modified dATP(μM) Total dATP (μM) 1 0.2 0.2 0.4 2 0.4 0.4 0.8 3 0.6 0.6 1.2 4 1 1 2 5 1.5 1.5 3 6 2 2 4

[0153] Analysis of experimental results: Three mutants were tested in this experiment, and the kinetic curves are shown in Figure 7 and Table 8:

[0154] Table 8. Kinetic parameters

[0155] Vmax Km Vmax / Km Relative Vmax / Km KOD_M1 304.4 2.763 110.2 1.0 KOD_M2 297.6 1.615 184.3 1.7 KOD_M3 345.6 0.8932 386.9 3.5

[0156] The results of this experiment were analyzed. The reaction system can simulate the kinetic experiment well. Under the conditions of different concentrations of modified dATP, the corresponding reaction rate was calculated. The calculation method is as in Example 2. It was found that when the modified dATP was evaluated kinetically, the data under this reaction system could be well fitted, and KOD_M3 and KOD_M2 had higher catalytic efficiency than KOD_M1. Comprehensive analysis of the DNA template and modified dATP kinetic results showed that KOD_M3 had better catalytic efficiency.

[0157] Example 4 Comparison of blocking nucleotidase activity test between SSEB blocking nucleotide and N3

[0158] The enzyme activity test method of SSEB blocking nucleotide (CN110818757A) is consistent with the enzyme activity test reaction system of N3 blocking nucleotide (just replace the substrate of N3 with the substrate of SSEB), and the experimental method is also consistent. The experimental results are as follows Figure 8 .

[0159] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nucleotide analogue, characterized in that It has the structure shown in the following formula (I): in, Blocker is a blocking group; P is a triphosphate group; X is a nitrogenous base, and the nitrogenous base is selected from any one of adenine, guanine, uracil, cytosine or thymine; Y is a polypeptide; R is -OH or -H; Preferably, the structure of the blocking group Blocker is as follows:

2. The nucleotide analogue according to claim 1, characterized in that The nucleotide analog has a structure as shown in formula (II):

3. A sequencing enzyme screening system, characterized in that: It comprises the nucleotide analogue as claimed in claim 1 or 2 connected with a first detection group, a template DNA connected with a second detection group and a DNA polymerase; The emission spectrum of the first detection group overlaps with the absorption spectrum of the second detection group.

4. The sequencing enzyme screening system according to claim 3, characterized in that: The emission spectrum of the first detection group and the absorption spectrum of the second detection group have an overlap of ≥30; The 3' end and / or 5' end of the template DNA is connected to a second detection group; The template DNA has at least one of a 3' single-stranded region or a 5' single-stranded region and a middle double-stranded region; The 3' end and 5' end of the template DNA are connected to a second detection group; The template DNA connected with the second detection group has a 3' single-stranded region, a 5' single-stranded region and a middle double-stranded region; The first detection group is cy3; The second detection group is cy5.

5. A kit comprising auxiliary materials and at least one of the following I) to II): 1), a nucleotide analogue as claimed in claim 1 or 2; II), a sequencing enzyme screening system as described in claim 3 or 4.

6. The kit according to claim 5, characterized in that The auxiliary materials include one or a combination of two or more of a lysate, a cleaning solution, a purification reagent or a buffer.

7. A method for screening a sequencing enzyme, characterized in that: include: Sequenase is screened using the nucleotide analogs as described in claim 1 or 2 and / or the sequenase screening system as described in claim 3 or 4 and / or the kit as described in claim 5 or 6.

8. The screening method according to claim 7, characterized in that The steps include: A mixture of a nucleotide analog connected to a first detection group, a nucleotide, a template DNA connected to a second detection group, a sequencing enzyme, a buffer and water is reacted and detected, and the DNA polymerase is screened after obtaining a single base incorporation rate based on an activity curve of the detection signal changing over time; The nucleotides include a mixture of nucleotides having different nitrogenous bases than nucleotide analogs; The concentration of the nucleotide analogue is 0.25 μM; The concentration of the nucleotide is 0.25 μM; The concentration of the template DNA connected with the second detection group is 0.1 μM; The concentration of the sequencing enzyme is 0.5 mg / mL; The reaction conditions are 40°C for 2h; The detection conditions are: excitation wavelength of 530nm, emission wavelengths of 568nm and 676nm; detection once every 1 to 3 minutes; The incorporation rate of the single base is the slope of the activity curve.

9. Application of at least one of the following i) to iv) in the preparation of sequencing enzyme screening products, sequencing enzyme reaction kinetics testing, evaluation of catalytic activity and / or evaluation of the speed and efficiency of enzyme base incorporation into template chains: i), a nucleotide analogue as claimed in claim 1 or 2; ii) The sequencing enzyme screening system according to claim 3 or 4; iii), a kit as claimed in claim 5 or 6; iv) The screening method according to claim 7 or 8.

10. The use according to claim 9, characterized in that: The sequencing enzyme includes at least one of a DNA polymerase, a reverse transcriptase, a tailing enzyme or an RNA polymerase.

Images