WO2019128691A1 - Nucleotide derivative and application thereof - Google Patents

Abstract

The present invention provides a nucleotide derivative and an application thereof. The present invention also provides a nucleic acid derivative and a kit comprising the nucleotide derivative, particularly suitable for the sequencing method described in the present invention. Further, the present invention also provides applications of the nucleotide derivative and kit in sequencing or molecular diagnosis.

Description

Nucleotide derivatives and their applications

Priority information

The present application is filed on Jan. 28, 2017, the entire disclosure of which is hereby incorporated by reference.

Technical field

The invention relates to the field of nucleic acid sequencing. In particular, the present invention provides an electrical signal based sequencing method. Further, the present invention provides a nucleotide derivative and a kit comprising the nucleotide derivative, which are particularly suitable for use in the sequencing method of the present invention. Furthermore, the invention also provides the use of the nucleotide derivatives and kits for sequencing.

Background technique

Since the 1970s, gene sequencing technology has developed rapidly. It has made substantial progress in prenatal non-invasive screening and personalized diagnosis of tumors and its treatment. The application of gene sequencing technology in medicine will be A revolutionary reform of traditional medical treatment and human health management; at present, we can confirm that the future of gene sequencing will not stop there, it will have a huge impact on human cognition and lifestyle. change.

The current sequencing technology can be classified into two categories: optical signal sequencing and electrical signal sequencing according to detection methods. The second-generation sequencing technology based on optical signal represented by Illumina is mainly characterized by high throughput, low cost and high accuracy. The second generation sequencing technology based on electric signal represented by Ion Torrent is due to accuracy and flux. The market did not meet the expected requirements and the market share continued to decline. On the other hand, the new generation of single-molecule electrical signal sequencing technology represented by nanopore technology is developing rapidly. Although the accuracy is still not as good as the mainstream second-generation sequencing technology, it still has many long read lengths, fast detection and portable features. Application scenarios, such as rapid detection of pathogens, whole-genome de novo assembly. Further, considering that the electrical signal detection technology is easier to integrate with existing CMOS semiconductor technology, it is more suitable for developing a consumer-grade miniaturized fast sequencer.

The sequencing instrument of companies such as Illumina and ABI, the sequencing principle is based on optical detection means, the fluorescent molecule needs to be modified as a label to the dNTP molecule, and Roche's pyrosequencing is to detect the pyrophosphate product produced during the sequencing process. The sequencing reaction and the release coupling of the primary fluorescent signal, both of which require the use of a laser for excitation, collect the emitted fluorescent signal. For the accuracy and sensitivity of the detection, the target DNA needs to be fixed and amplified by the base; in order to achieve the purpose of fixation, the DNA sample needs to be modified, which increases the cost of sequencing; in order to improve the signalling sensitivity and sensitivity, it is necessary to Amplification of the immobilized DNA increases the time and difficulty of pre-treatment of the sample, which is not only time consuming, but also increases the operational requirements for the user of the instrument.

At present, most of the sequencing is based on optical sequencing technology, and it is necessary to excite the fluorescent molecules. The corresponding sequencer needs to be equipped with an exciter, and a device for collecting and converting signals, so that the sequencer is bulky and expensive; and in signal acquisition, only The gene chip region can be scanned sequentially for data acquisition, so that the single sequencing time is longer, and the intensity and lifetime of the fluorescent molecules are affected. The biggest advantage of the electrical signal sequencing platform is that the cost and size of the sequencer are further reduced. It is more suitable for the development of distributed sequencers for future personalized medicine and real-time diagnosis, and the formation of a central sequencer based on second-generation sequencing technology. Complementary. Therefore, considering the cost and promotion application, the better technical route is the electrical signal sequencing method based on semiconductor technology.

The key technical problem that needs to be solved for the sequencing of electrical signals is how to improve the accuracy and reach the level equivalent to the sequencing of optical signals. The core of the signal is that the signals generated by the nucleotides to be tested need to have high specificity and high sensitivity.

Summary of the invention

In order to solve the above technical problems, the present invention provides a novel sequencing method using a 5' phosphate group to which an electrochemically labeled nucleotide is attached. The electrochemical label does not affect the activity of the nucleotide, while the nucleotide inhibits the activity of the electrochemical label. After polymerization, an intermediate product is formed, and the intermediate product generates an electrochemically active substance under the action of phosphatase. An electrical signal can be generated by applying a specific voltage to cause a redox cycle reaction. Thus, the sequencing apparatus for carrying out the sequencing method of the present invention requires only a current detecting device, thereby greatly reducing the cost and volume of the sequencing device.

In one aspect, the invention provides a nucleic acid molecule sequencing method comprising the steps of:

(1) designing a channel having a support and two electrodes, providing a nucleic acid molecule to be sequenced on the support, or attaching the nucleic acid molecule to be sequenced to the support;

(2) adding a primer, a polymerase, and a nucleotide derivative for polymerization;

(3) the primer and the nucleic acid molecule to be sequenced are annealed, and the primer serves as an initial growing nucleic acid strand, and together with the nucleic acid molecule to be sequenced, forms a duplex attached to the support;

(4) performing a nucleotide polymerization reaction under the reaction conditions of the polymerase to thereby incorporate the nucleotide derivative into the 3' end of the elongated nucleotide strand;

(5) adding a phosphatase, and then applying a specific voltage to the two electrodes (the voltage interval is selected to cover the oxidation and reduction potential), and detecting the change of current on the two electrodes with time (I-t), that is, an electrical signal;

(6) removing the solution phase of the reaction system of the previous step, leaving the duplex attached to the support row;

(7) A polymerase for performing nucleotide polymerization, and a second nucleotide derivative are added, followed by (4) to (6).

(8) adding a polymerase for performing nucleotide polymerization, and a third nucleotide derivative, and then performing (4)-(6);

(9) adding a polymerase for performing nucleotide polymerization, and a fourth nucleotide derivative, and then performing (4)-(6);

(10) Repeat steps (4)-(9).

Wherein the first, second, third and fourth nucleotide derivatives described above are selected from the derivatives of nucleotides A, (T/U), C and G, and are different, four The phosphate group of the nucleotide is modified by an electrochemical tag and has a base pairing ability.

In another aspect, the invention provides a nucleotide derivative molecule having the structure:

B-S-(P)n-P-R

Wherein: B is a nitrogen-containing heterocyclic base; S is a carbocyclic ring; P = phosphate (PO ₄ ^3- ) and a derivative thereof; n ≥ 3; R is an electrochemical tag, and the electrochemical tag is suitable for the A phosphate or phosphate derivative forms a phosphate. The electrochemical tag can be 4-dimethylaminophenol or 4-nitroaminophenol.

Specifically, a deoxyribonucleotide for sequencing DNA molecules, S is a five-carbon ring, R is 4-dimethylaminophenol, n=4, and the structure is as follows:

The derivative, after carrying out the polymerization reaction, produces an intermediate 4-aminophenyl phosphate group:

This intermediate product is not electrochemically active. The intermediate product forms an electrochemically active 4-dimethylaminophenol under the action of phosphatase, and the structure is as follows:

After a specific voltage is applied to the electrochemically active 4-dimethylaminophenol, when the voltage applied to one of the electrodes is greater than the oxidation potential thereof by the semiconductor parameter meter and the probe, the target molecule can undergo an oxidation reaction under the electrode; While a voltage lower than its reduction potential is applied to the other electrode, the molecules in the oxidation state can undergo a reduction reaction at the voltage. The target molecule generates an electron gain and loss due to a redox reaction between the two metal electrodes, thereby causing a change in the current value, so that an electric signal different from the background current can be obtained.

Beneficial effect

The nucleotide derivative obtained by the present invention has a very high purity, which is distinguished from the conventional optical signal detection, and the invention realizes electrical signal detection, and the stability of the substance is high. The nucleotide derivative can be used for real-time sequencing, that is, directly immobilizing a DNA fragment to be detected on the surface of the electrode, and performing reaction and signal acquisition on the surface of the electrode by adding a primer, a polymerase, and the nucleotide derivative. Since the sample itself is fixed on the surface of the electrode, the obtained product gathers on the surface of the electrode, so that the concentration of the sample near the electrode is high, thereby increasing the sensitivity and accuracy of the detection. Due to the characteristics of the label molecules, the detection of electrical signals can only be achieved by simultaneously performing two steps of polymerization and dephosphorization, which greatly increases the controllability of the detection experiment.

In the application of the third-generation sequencer, nanochannels were introduced for detection. The advantage with respect to the nanopore is that the nanochannel acts as an electrode and has a large area. The time for the molecule to be detected to pass through the channel is long, and the corresponding detected electrical signal is increased, so that the error rate of detection can be greatly reduced.

Based on the detection mechanism of the electrical signal, a great advantage of the present invention is that the sequencing technology can be combined with the semiconductor chip technology and the microfluidic technology to realize real-time fast and high-throughput sequencing. The electrode diameter of the semiconductor chip is only a few micrometers, the spacing of the electrodes is much larger than the electrode area, and the electrodes are not affected, ensuring the independence of each sequencing reaction; the microelectrode directly converts the encoded information into digital information, greatly shortening the single The signal acquisition time of the second time does not require additional signal acquisition devices and data conversion devices, which can realize miniaturization of the instrument and reduce the cost of the instrument. Since most of the experiments can be performed on the gene semiconductor chip, the operator's requirements are reduced. In the true sense, it meets the requirements for the future of the sequencer: simple, fast and low-cost; for every researcher, the sequencer is within reach.

DRAWINGS

Figure 1 is an LC-MS diagram of four deoxyribonucleotides with electrochemical tags.

Figure 2 is a cyclic voltammogram of 4-dimethylaminophenyl phosphate before and after the action of phosphatase (CIAP) and 4-dimethylaminophenol.

Figure 3 is a schematic diagram of a single base sequencing reaction on an electrochemical workstation using a gold electrode as a working electrode.

Figure 4 is a graph of the results of a single base sequencing using electrochemical tagged deoxyribonucleotides on an electrochemical workstation.

Detailed ways

In order to improve the accuracy of electrical signal sequencing, the present invention provides a nucleotide derivative having the following structure:

B-S-(P)n-P-R

Wherein: B is a nitrogen-containing heterocyclic base; S is a carbocyclic ring; P is a phosphate (PO ₄ ^3- ) or a derivative thereof; n ≥ 3; R is an electrochemical tag, and the electrochemical tag is suitable for The phosphate or its derivative forms a phosphate. The electrochemical tag can be 4-dimethylaminophenol or 4-nitroaminophenol.

Using the above nucleotide derivative, the present invention provides a method for sequencing a nucleic acid molecule comprising the steps of: (1) designing a channel having a support and two electrodes to provide a nucleic acid molecule to be sequenced attached to a support (2) adding a primer, a polymerase, and a first nucleotide derivative for polymerization; (3) annealing the primer and the nucleic acid molecule to be sequenced, the primer as a starting nucleic acid strand for growth Forming a duplex attached to the support together with the nucleic acid molecule to be sequenced; (4) performing a nucleotide polymerization reaction under the reaction conditions of the polymerase to thereby incorporate the nucleotide derivative into the growth The 3' end of the nucleic acid strand; (5) adding a phosphatase, then applying a specific voltage to the two electrodes to detect the electrical signal; (6) removing the solution phase of the previous reaction system, leaving the double attached to the support a chain; (7) adding a polymerase and a second nucleotide derivative, repeating steps (4)-(6); the first nucleotide derivative and the second nucleotide derivative are as described above Nucleotide derivatives.

In at least some embodiments of the invention, the first nucleotide derivative and the second nucleotide derivative are each independently selected from a core comprising bases A, C, G, and T or U. One of the glycosidic acid derivatives. Using a nucleotide derivative containing a base A, a base A can be attached to the 3' end of the growing nucleic acid strand, and then a phosphatase is added, and a voltage is applied to detect a corresponding electrical signal, thereby obtaining a nucleic acid to be sequenced. Information about the base A in the molecule. Similarly, by using a nucleotide derivative containing the bases C, G, and T/U, respectively, information of the corresponding base C, G, T or U in the nucleic acid molecule to be sequenced can be obtained, thereby realizing the nucleic acid molecule to be sequenced. Sequencing. The determination of nucleic acid sequences, such as DNA and RNA, can be achieved using this method. It is also possible to analyze information on specific bases in a nucleic acid sequence.

In at least some embodiments of the invention, the first nucleotide derivative and the second nucleotide derivative carry the same electrochemical tag. During the sequence determination of the nucleic acid molecule to be tested, the first nucleotide derivative and the second nucleotide derivative have the same electrochemical label, which can avoid the change of the electrical signal due to the change of the electrochemical label itself. Thereby affecting the measurement result of the base sequence.

In at least some embodiments of the invention, the first nucleotide derivative and the second nucleotide derivative carry different electrochemical labels. Different nucleotide derivatives can also be detected according to the difference of electrical signals according to the electrical signals brought about by each electrochemical label during the experiment.

Of course, in the process of detecting different electrical signals, different channels on the sequencing platform can be used for detection, and the test can simultaneously detect a plurality of electrical signals, thereby simultaneously measuring a plurality of nucleotides. For example, only one nucleotide derivative can be added at a time, and then single-channel detection can be performed; four nucleotides can also be added at a time, and then four-channel detection is performed, and detection of four nucleotides is simultaneously performed. Nucleotide sequence determination is achieved by means of a sequencing platform that relies on electrical signals for sequencing, such as the Ion Torrent high throughput sequencing platform or nanopore sequencing platform.

In at least some embodiments of the invention, the electrical signal is determined by detecting a change in current across the two electrodes over time. Using the cyclic voltammetry for electrical signal detection, the scanning rate can be set to 0.2V/s and the scanning interval is set to 1mV. The nucleotide information is obtained by the electrical signal value, thereby realizing the determination of the nucleotide.

In at least some embodiments of the invention, the voltage is between -300 mV and 300 mV. At this voltage, the obtained electrical signal has a high intensity and is suitable for detection and measurement.

Example 1 Preparation of Nucleotide Derivatives

The synthesis of this study was carried out by chemical methods. The raw materials were natural dNTPs and 4-dimethylaminophenol. The phosphoric acid was linked to the phosphoric acid of dNTP by the phosphoric acid interconnection. Electrochemical label dNTPs of phosphoric acid. The specific synthetic path takes dATP as an example, as shown in the following reaction:

The specific step of the synthesis is as follows: dimethylaminophenol is first substituted with phosphorus oxychloride under the action of pyridine to form dimethylaminophenol phosphate, and then N,N-dimethylformamide is sequentially added. (DMF), triphenylphosphine (PPh3), 1-methylimidazole (Melm), 2,2'-dithiodipyridine (DPDS), activation of dimethylaminophenol phosphate, reaction after one hour The product was precipitated by diethyl ether, and after centrifugation, dimethylaminophenol phosphoramide having higher reactivity was collected. The product was then dissolved in a solution of dimethyl sulfoxide (DMSO), followed by the addition of triethylamine and adenine nucleoside triphosphate (ATP) triammonium salt. After reacting at 20 ° C for 8 hours, the product was precipitated with diethyl ether. The product was purified by silica gel thin layer chromatography using 1,4-dioxane/isopropanol separation/water/25% ammonium hydroxide 1:1:1:1 (V/V) as eluent. Finally, chromatographic purification by ion exchange chromatography gave the final product. The molecular weight and purity of the product were identified using LC-MS. The result is shown in Figure 1. Since the molecule has two methyl groups at the amino terminus, it is not necessary to protect the amino group during the synthesis step, making the synthesis reaction easier to handle. The portion enclosed by the dotted line in Fig. 1 shows the peak position of the main product and the corresponding purity.

1(A) represents the LC-MS spectrum and area normalization result of the product formed by the above reaction using dATP and 4-dimethylaminophenol; the result shows that the purity of the product is 99.75%, and the MS result indicates that the substance is produced. The ratio is 689.34. Figure 1 (A) corresponds to the product target peak and retention time and peak area as shown in Table 1:

Table 1 target peak and its retention time and peak area

Peak No. Retention time (min) Peak area Peak area(%) 1 0.36 6466.94 99.75 2 0.76 16.01 0.25

Figure 1 (B) represents the LC-MS spectrum and area normalization result of the product formed by the above reaction using dTTP and 4-dimethylaminophenol; the result shows that the purity of the product is 99.67%, and the MS result indicates the material charge. The ratio is 680.34. Figure 2 (B) corresponds to the target peak and retention time and peak area as shown in Table 2:

Table 2 target peaks and their retention time and peak area

Peak No. Retention time (min) Peak area Peak area(%) 1 0.36 5165.58 99.67 2 0.76 17.26 0.33

Figure 1 (C) represents the LC-MS spectrum and area normalization result of the product formed by the above reaction using dCTP and 4-dimethylaminophenol; the result shows that the purity of the product is 99.34%, and the MS result indicates the material charge. The ratio is 665.33. Figure 1 (C) corresponds to the target peak and retention time and peak area as shown in Table 3:

Table 3 target peaks and their retention time and peak area

Peak No. Retention time (min) Peak area Peak area(%) 1 0.36 2452.16 99.34 2 0.76 16.32 0.66

Figure 1 (D) represents the LC-MS spectrum and area normalization result of the product formed by the above reaction using dGTP and 4-dimethylaminophenol; the result shows that the purity of the product is 99.75%, and the MS result indicates the material charge. The ratio is 705.35. Figure 1 (D) corresponds to the target peak and the retention time and peak area are shown in Table 4:

Table 4 target peaks and their retention time and peak area

Peak No. Retention time (min) Peak area Peak area(%) 1 0.36 7338.48 99.75

2 0.76 18.72 0.25

It can be seen from Fig. 1 that the purity of the nucleotide derivative is above 99%, and the molecular weight thereof also indicates that 4-dimethylaminophenyl phosphate has been attached to the dNTP.

Example 2 Determination of the Properties of 4-Dimethylaminophenyl Phosphate Formed by Phosphatase to 4-Dimethylaminophenol

The properties of 4-dimethylaminophenyl phosphate to 4-dimethylaminophenol under the action of phosphatase were determined by cyclic voltammetry. Cyclic voltammetry is to control the potential of the electrode at different rates. It is scanned one or more times with a triangular waveform over time. The potential range is such that different reduction and oxidation reactions can alternately occur on the electrode, and the current-potential curve is recorded. The peak position on the upper side can judge the reversibility of redox and the corresponding potential. The potentiostatic sweep is mainly to set the initial potential and the termination potential, with a certain potential as the starting potential, and apply to a terminal potential at a fixed rate, and then change back to the starting potential at the same rate, which is counted as one cycle and can be Get a CV chart. When scanning from a low potential to a high potential, the analyte will generate an oxidation peak of the oxidation current at a certain potential. When scanning from a high potential to a low potential, the oxidation state of the oxidation state after the oxidation reaction occurs. , thereby producing a reduction current peak.

The device for detecting electrochemical activity is shown in Fig. 3. The surface-plated silicon wafer is used as a working electrode, and a reservoir is formed on the surface thereof, and a counter electrode (Pt) and a reference electrode (Ag/AgCl) are connected in the reservoir. The three electrodes are respectively connected to an electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., CHI 630E) for detecting the reaction product of 4-dimethylaminophenyl phosphate under the action of phosphatase and with 4-dimethyl The properties of aminophenol were compared.

During the detection process, our voltage range is selected (-600mV, 400mV), where -600mV is the lowest potential and the starting potential, 400mV is the highest potential and the termination potential, the scanning rate is set to 0.2V/s, and the forward scanning is performed. The scanning interval is 1 mV, the number of cycles is 3, and the limiting current is selected from -100 μA to 100 μA. The following groups of solutions were each subjected to cyclic voltammogram scanning, and each solution was injected into the reservoir according to the following concentrations and formulations:

(1) Buffer (formulated as Tris 50 mM + 50 mM KCl + 70 mM (NH ₄ ) ₂ SO ₄ + 0.1 mM EDTA + 3 mM MgSO ₄ , pH = 8.5); the reference numeral 1 in Fig. 2 represents the scanning result of the buffer;

(2) 1 mM 4-dimethylaminophenyl phosphate; reference numeral 2 in Fig. 2 represents a scan result of 1 mM 4-dimethylaminophenyl phosphate;

(3) 1 mM 4-dimethylaminophenyl phosphate + CIAP solution, reaction for 20 min; reference numeral 3 in Figure 2 represents a mixed solution of 1 mM 4-dimethylaminophenyl phosphate and CIAP for 20 minutes. Scan result

(4) 1 mM 4-dimethylaminophenyl phosphate + CIAP solution was reacted for 50 min; reference numeral 4 in Fig. 2 represents a mixed solution of 50 mM reaction of 1 mM 4-dimethylaminophenyl phosphate and CIAP. Scan result

(5) 1 mM 4-dimethylaminophenol; the reference numeral 5 in Fig. 2 represents the result of description for 1 mM 4-dimethylaminophenol.

At the same time, in order to verify the stability of the electrochemical activity of the sample, each sample was subjected to 3 cycles of measurement. The results are shown in Fig. 2. It can be seen from the figure that the individual 4-dimethylaminophenyl phosphate does not have redox activity (showing a similar scan result as the buffer), but the effect of phosphatase (CIAP). It shows a redox peak position similar to that of 4-dimethylaminophenol, which has the same electrochemical properties, and shows that 4-dimethylaminophenyl phosphate can be converted into a phosphatase. Electrochemically active 4-dimethylaminophenol. Further, as compared with the result of the reaction for 20 minutes, the scanning reaction of the mixed solution for 50 minutes resulted in a higher redox current.

The results in Figure 2 show that dimethylaminophenol phosphate can exhibit stable electrochemical activity in the presence of phosphatase, and its redox peak position is consistent with dimethylaminophenol, indicating that under the action of phosphatase The reacted dNTP with an electrochemical tag produces dimethylaminophenol. Moreover, although some samples have poor curve coincidence in the first cycle (due to the influence of concentration polarization, that is, the electrode potential deviates from the equilibrium potential due to the difference in the ion concentration of the electrode interface layer solution in the electrolytic cell and the concentration of the bulk solution), However, the latter two cycle curves in cyclic voltammetry show good repeatability. It is also illustrated that the electrochemical label material has stable electrochemical activity.

Example 3 application of sequencing

Add 2 ul of template (100 uM) and 4 ul of primer (100 uM) (Shanghai Shenggong) to a 0.2 ml centrifuge tube and add 14 uL of buffer (formulated as Tris 50 mM + 50 mM KCl + 70 mM (NH ₄ ) ₂ SO ₄ + 0.1 mM EDTA) +3 mM MgSO ₄ , pH=8.5), hybridization at room temperature for 0.5 h. Three of the template sequences and primer sequences are shown in Table 5:

table 5

The hybridized products were grouped into a control group and an experimental group, wherein the control group used ordinary dNTPs (i.e., the phosphate group was not modified), and the experimental group used a nucleotide derivative for polymerization.

Cyclic voltammetry is also used for electrical signal detection. During the detection process, our voltage range is selected (-300mV, 300mV), the scan rate is set to 0.2V/s, and the scan interval is 1mV. The following groups of solutions are respectively scanned by cyclic volt-ampere curve to detect the corresponding electrical signals. :

(1) buffer, as a blank control, the scan result corresponds to the reference 1 in Figure 4;

(2) the control solution, that is, the unmodified natural dNTP, without alkaline phosphatase, the scan result corresponds to the reference 2 in Figure 4;

(3) control group +1 ul alkaline phosphatase (CIAP), that is, the reaction product of unmodified natural dNTP and alkaline phosphatase, the scan result corresponds to the reference 3 in Figure 4;

(4) the experimental group solution, that is, the dNTP solution with the electrochemical label, the scan result corresponds to the reference 4 in Figure 4;

(5) Experimental group +1 ul alkaline phosphatase (CIAP), using an electrochemically labeled dNTP and an alkaline phosphatase reaction, the scan result corresponds to the reference 5 in FIG.

Three nucleotide derivatives (i.e., dGTP, dATP, dTTP nucleotide derivatives obtained in Example 1) were used as an experimental group and three normal dNTP pair template nucleic acids (sequences shown in Table 5 above) were used as a control group. The sequencing reactions are performed separately, and the steps are as follows:

(1) providing a nucleic acid molecule to be sequenced on the support or attaching the nucleic acid molecule to be sequenced to the support;

(2) adding a primer, a polymerase, and a first nucleotide derivative (one of the four nucleotide derivatives) or a corresponding normal nucleotide (that is, the phosphate group is not modified) for the polymerization reaction;

(3) the primer and the nucleic acid molecule to be sequenced are annealed, and the primer serves as an initial growing nucleic acid strand, and together with the nucleic acid molecule to be sequenced, forms a duplex attached to the support;

(4) performing a nucleotide polymerization reaction under the reaction conditions of a polymerase to thereby incorporate the nucleic acid derivative into the 3' end of the elongated nucleotide chain;

(5) adding a phosphatase, and then applying a specific voltage (-300 mV, 300 mV) to detect an electrical signal;

(6) Repeat steps (1)-(5) to detect the second and third nucleotide derivatives.

Wherein the first, second and third nucleotide derivatives described above are selected from the group consisting of nucleotides A, (T/U) and G derivatives, and are different, four nucleotide phosphate groups The cluster is modified by an electrochemical tag and has the ability to base complementary pairs. During the experiment, the first nucleotide derivative is a derivative of nucleotide A, the second nucleotide derivative is a derivative of nucleotide T, and the third nucleotide derivative is nucleotide G. derivative. The corresponding normal nucleotides are nucleotide A, nucleotide T, and nucleotide G, respectively. The electrochemical tag in the nucleotide derivative is 4-dimethylaminophenol.

Experiments were carried out using dTTP derivatives, dATP derivatives and dGTP derivatives, respectively. The buffer was subjected to cyclic voltammetry scanning to verify that there was no electrochemically active substance in the buffer participating in the reaction; the control solution (ie, unmodified natural dNTP) was subjected to cyclic voltammetry scanning to illustrate the natural The dNTP does not produce an electrochemically active substance; the reaction product of the control and alkaline phosphatase is subjected to a cyclic voltammogram scan to illustrate the natural absence of an electrochemical label even in the presence of alkaline phosphatase. The dNTP did not produce an electrochemically active substance after the reaction; the experimental group solution (ie, the electrochemically labeled dNTP solution) was subjected to cyclic voltammetry scanning to indicate that the electrochemical group was not added to the base phosphatase. The labeled dNTPs are also not electrochemically active after the reaction; the cyclic voltammetry scan of the reaction product of the experimental group and alkaline phosphatase (ie, the reaction product of the electrochemically labeled dNTP and the base phosphatase) is performed. Under the action of the base phosphatase, an electrochemically active substance is generated and an electrical signal is generated.

The experimental results are shown in Fig. 4. The nucleotides of the electrochemical tag can be modified under the guidance of the primers, and the template sequence is used as a template to polymerize and generate an electrochemically active substance under the action of alkaline phosphatase. . It can be seen from the figure that the dNTP modified by 4-dimethylaminophenol has no electrochemical activity under the action of polymerase, but has normal polymerization ability, can carry out template extension reaction, and after alkaline phosphatase reaction The electrochemical activity of the tag molecule can be restored. Based on this, the identification of the nucleotide species on the template can be accomplished by adjusting the nucleotide derivative addition sequence. The nucleotide identification results were consistent with the sequencing results obtained by the BGISEQ-500 sequencing platform.

Since the synthesis of a plurality of G is unstable (since multiple Gs will produce tetramers), a template containing a plurality of Gs is not specifically synthesized in this experiment to perform experimental detection on dNTPs. However, according to the principle, the molecule can be modified for four nucleotides and used in sequencing technology, as well as other molecular diagnostic fields.

Claims (12)

A nucleotide derivative characterized by the following formula I: B-S-(P)n-P-R(Formula I) among them: B is a nitrogen-containing heterocyclic base; S is a carbon ring; P is a phosphate or a derivative thereof; N≥3 R is an electrochemical tag suitable for forming a phosphate with the phosphate or its derivative. The nucleotide derivative according to claim 1, wherein the electrochemical label is selected from the group consisting of 4-dimethylaminophenol and/or 4-nitroaminophenol. The nucleotide derivative according to claim 1, wherein n = 3 and S is deoxyribose or ribose. The nucleotide derivative according to claim 1, wherein the nucleotide derivative is at least one selected from the group consisting of:

A nucleic acid sequencing method comprising the following steps: (1) designing a channel having a support and two electrodes to provide a nucleic acid molecule to be sequenced attached to the support; (2) adding a primer, a polymerase, and a first nucleotide derivative for polymerization; (3) annealing the primer and the nucleic acid molecule to be sequenced, the primer as an initial growing nucleic acid strand, together with the nucleic acid molecule to be sequenced, forming a duplex attached to a support; (4) performing a nucleotide polymerization reaction under the reaction conditions of a polymerase to thereby incorporate the nucleotide derivative into the 3' end of the elongated nucleic acid strand; (5) adding a phosphatase, then applying a specific voltage to the two electrodes to detect an electrical signal; (6) removing the solution phase of the reaction system of the previous step, leaving the duplex attached to the support; (7) adding a polymerase and a second nucleotide derivative, repeating steps (4)-(6); The first nucleotide derivative, the second nucleotide derivative, the nucleotide derivative according to any one of claims 1 to 4, the first nucleotide derivative and the second The nucleotide derivatives are different, each independently selected from one of the nucleotide derivatives containing the bases A, C, G and T or U. The method of claim 5 wherein said electrical signal is determined by detecting a change in current across the two electrodes over time. The method of claim 5 wherein said voltage is between -300 mV and 300 mV. The method according to claim 5, wherein the first nucleotide derivative and the second nucleotide derivative carry the same electrochemical tag. A kit comprising at least one of the following compounds:

Use of the nucleotide derivative according to any one of claims 1 to 4 for sequencing of a nucleic acid molecule. Use of the nucleotide derivative according to any one of claims 1 to 4 for molecular diagnosis. Use of the kit of claim 9 for sequencing or molecular diagnostics of nucleic acid molecules.

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