CN110951852A

CN110951852A - Single-base continuous extension flow type target sequencing method

Info

Publication number: CN110951852A
Application number: CN201911165464.7A
Authority: CN
Inventors: 兰文军; 张静; 胡岳
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2019-11-25
Filing date: 2019-11-25
Publication date: 2020-04-03
Anticipated expiration: 2039-11-25
Also published as: WO2021103695A1; CN110951852B

Abstract

The invention discloses a single-base continuous extension flow-type targeted sequencing method, which utilizes a single-stranded nucleotide fragment containing a complementary sequence of a nucleic acid fragment to be detected, a primer, microspheres, nucleic acid polymerase, a fluorescence-labeled ddNTP, a ribose 3' -OH protected fluorescence-labeled dNTP, a ribose 3' -OH protected dNTP, a ribose 3' -OH deprotecting agent and the like to obtain a series of sequencing microspheres suitable for detecting a nucleic acid base sequence by a flow cytometer through single-base extension of the primer, and the sequencing microspheres are further detected by the flow cytometer to obtain the base sequence of the nucleic acid fragment to be detected. Compared with the existing sequencing method, the method has the obvious advantages of simplicity, accuracy, easy data interpretation and the like, and can be widely applied to the nucleic acid sequencing fields of gene detection, microbial examination, genetics, exons, Single Nucleotide Polymorphisms (SNPs), genomics, proteomics and the like.

Description

Single-base continuous extension flow type target sequencing method

Technical Field

The invention relates to a single-base continuous extension flow-type targeted sequencing method, in particular to a preparation method of sequencing microspheres suitable for flow cytometry detection and a method for detecting nucleic acid base sequences by adopting the sequencing microspheres, belonging to the technical field of gene sequencing.

Background

The genome carries all genetic information of a life individual, and gene sequencing not only can deepen the understanding of molecular mechanisms of diseases, particularly malignant tumors, but also plays an important role in guiding targeted drug delivery. After the human genomics plan is completed, the gene sequencing technology is developed more rapidly and is applied more widely in clinical practice and basic research. At present, the nucleic acid sequencing method has been developed to three generations, from the first generation of direct detection technology represented by Sanger sequencing and indirect sequencing technology represented by linkage analysis, to the second generation sequencing (NGS) marked by Solexa technology of illumina and SOLID technology of ABI in 2005, to the third generation sequencing represented by SMRT and Nanopore methods, which continuously improve the efficiency and accuracy of gene sequencing, but still have the problems of complicated operation and difficult data interpretation (such as gene structure rearrangement and repeated regions), and the like, and restrict the large-scale clinical application thereof.

The flow cytometry is a technology for carrying out multi-parameter and rapid quantitative analysis on single cells or particles by using a flow cytometer, has the advantages of high speed, high precision and good accuracy, and is one of advanced biosensing technologies. Existing flow cytometry based on primer or probe coated microspheres can only be used for recognition of single base and nucleotide fragments, but not for base sequencing. Therefore, the development of a single-base continuous extension flow-type targeted sequencing method suitable for a flow cytometer has great value.

Disclosure of Invention

The invention aims to provide a single-base continuous extension flow-type targeted sequencing method suitable for detecting a nucleic acid base sequence by a flow cytometer.

Currently, gene detection has important applications in the medical fields of genetics, prenatal diagnosis, companion diagnosis, and the like. For example: the mutation site of the specific gene segment of the patient is determined through gene detection, so that targeted drug delivery can be guided, and the treatment effect of the tumor is improved. However, the existing flow cytometry technology can only identify single bases and oligonucleotide fragments and cannot be used for base sequencing. The invention provides a single-base continuous extension flow-type targeted sequencing method suitable for nucleic acid sequencing by a flow cytometer, wherein the method is used for preparing sequencing microspheres and detecting the sequencing microspheres by the flow cytometer, so that a specific nucleic acid base sequence can be obtained, and further, the information of gene mutation can be read, and the method has important significance for pathogenesis explanation and diagnosis and treatment of sick individuals.

The technical scheme of the invention is as follows:

a single base continuous extension flow-type targeted sequencing method, comprising the steps of:

(1) coating a modified unidirectional primer for guiding the synthesis of a nucleic acid fragment to be detected or a single-stranded nucleotide fragment containing a complementary sequence of the nucleic acid fragment to be detected on the surface of the microsphere to obtain a coated microsphere;

(2) mixing the coated microspheres, the single-stranded nucleotide fragment containing the complementary sequence of the nucleic acid fragment to be detected or a modified unidirectional primer (a unidirectional primer or a modified unidirectional primer for short, the same below) for guiding the synthesis of the nucleic acid fragment to be detected and a buffer solution, and carrying out incubation hybridization on the obtained mixture;

(3) washing and suspending the microspheres by buffer solution, dividing the microspheres into two parts, mixing one part of microspheres with nucleic acid polymerase, buffer solution and dNTP (deoxynucleoside triphosphate) with protected ribose 3' -OH, and incubating the obtained mixture; mixing the other microsphere with nucleic acid polymerase, buffer solution, fluorescently-labeled ddNTP (dideoxynucleoside triphosphate) or/and fluorescently-labeled dNTP (deoxyriboside triphosphate) with protected ribose 3' -OH, and incubating the obtained mixture;

(4) washing the microsphere incubated after mixing the previous step with the dNTP with protected ribose 3'-OH, adding a ribose 3' -OH deprotecting agent, and uniformly mixing;

(5) washing and suspending the microspheres in the previous step by using a buffer solution, dividing the microspheres into two parts, mixing one part of microspheres with nucleic acid polymerase, the buffer solution and the dNTP with protected ribose 3' -OH, and incubating the obtained mixture; mixing the other microsphere with nucleic acid polymerase, buffer solution, fluorescence-labeled ddNTP or/and ribose 3' -OH protected fluorescence-labeled dNTP, and incubating the obtained mixture;

(6) continuously repeating the steps (4) and (5), continuously preparing n groups of microspheres by sequencing n bases of the nucleic acid fragment to be detected, and obtaining n groups of microspheres which are mixed with the fluorescence-labeled ddNTP or/and the fluorescence-labeled dNTP with protected ribose 3' -OH and then incubated;

(7) if all bases cannot be detected by using the n groups of microspheres in the step (6), replacing the fluorescently-labeled ddNTP or/and ribose 3' -OH protected fluorescently-labeled dNTP in the steps (3) and (5), replacing the container, and continuously preparing the microspheres by using the steps (2) to (6) to obtain 4 Xn or 2 Xn groups of microspheres;

(8) and (4) washing and suspending the microspheres of 4 xn, 2 xn or n groups obtained in the steps (6) and (7), and then detecting by using a flow cytometer to obtain the base sequence of the nucleic acid fragment to be detected.

Further, in the above method, n is the number of bases of the nucleic acid fragment to be detected.

Further, in the above method, when the types of the fluorescently labeled ddNTP or/and ribose 3' -OH protected fluorescently labeled dNTP added in steps (3) and (5) are 4, all base sequences of the nucleic acid fragment to be detected can theoretically be obtained by using the obtained sequencing microsphere. Depending on the type of fluorescently labeled ddNTP or/and ribose 3' -OH protected fluorescently labeled dNTP added in steps (3) and (5), the number of populations of sequencing microspheres obtained finally differs, which means that not one, but a plurality of microspheres are obtained at a time, and thus are referred to as a population. Depending on the manner of adding the fluorescently labeled ddNTP or/and the ribose 3' -OH protected fluorescently labeled dNTP, the number of the finally obtained sequencing microspheres can be n groups, 2n groups or 4n groups.

Further, when the microspheres are coated with modified unidirectional primers for guiding the synthesis of the nucleic acid fragment to be detected, the single-stranded nucleotide fragment containing the complementary sequence of the nucleic acid fragment to be detected is added in the step (2); when the microsphere is coated with a single-stranded nucleotide fragment containing a complementary sequence of the nucleic acid fragment to be detected, a modified unidirectional primer for guiding the synthesis of the nucleic acid fragment to be detected is added in the step (2).

Furthermore, the invention takes a single-stranded nucleotide fragment containing a complementary sequence of a nucleic acid fragment to be detected as a template, under the catalysis of nucleic acid polymerase, one part of microsphere extends a single non-fluorescence labeled base from the end of the unidirectional primer by adding ribose 3'-OH protected dNTP, the other part of microsphere extends a single fluorescence labeled base from the end of the unidirectional primer by adding fluorescence labeled ddNTP or/and ribose 3' -OH protected fluorescence labeled dNTP, and the part of microsphere is reserved for base sequencing. After single non-fluorescence labeled base is extended at the tail end of the one-way primer, ribose 3'-OH of the nucleic acid at the tail end of the one-way primer extension chain is deprotected by adding ribose 3' -OH deprotecting agent, then the microsphere is divided into two parts, one part is continuously added with ribose 3'-OH protected dNTP, the other part is added with fluorescence labeled ddNTP or/and ribose 3' -OH protected fluorescence labeled dNTP, after ribose 3'-OH protected dNTP is added, the tail end of the one-way primer is extended with single non-fluorescence labeled base again, after fluorescence labeled ddNTP or/and ribose 3' -OH protected fluorescence labeled dNTP is added, the tail end of the one-way primer is extended with single fluorescence labeled base, and the obtained microsphere is reserved for sequencing. Adding ribose 3'-OH protected dNTP into microspheres incubated by adding ribose 3' -OH protected dNTP for deprotection, then dividing into two parts again, adding ribose 3'-OH protected dNTP and fluorescence labeled ddNTP or/and ribose 3' -OH protected fluorescence labeled dNTP according to the same steps, and repeating the operation until all sequencing microspheres needed for sequencing n bases are obtained. The invention selects microspheres obtained by adding fluorescence-labeled ddNTP or/and ribose 3' -OH protected fluorescence-labeled dNTP for incubation as sequencing microspheres, and the sequencing microspheres can be detected by a flow cytometer and used for identifying the base sequence of a nucleic acid fragment to be detected.

Further, in the above preparation method, the microspheres are microspheres used in flow cytometry, such as polystyrene microspheres, and the microspheres can be obtained commercially. The microspheres used in the present invention may be either uniform size or size-encoded microspheres, or non-fluorescent or fluorescent-encoded microspheres. The diameter of the microspheres may be chosen in the range of 0.5-50 μm, preferably 1-10 μm.

Further, the microsphere of the present invention is a microsphere modified by one or more of the following means: modified with at least one of carboxyl, amino, hydroxyl, hydrazide, aldehyde, chloromethyl, oxirane, antibody, aptamer, streptavidin, poly thymidylate (poly T), poly adenylate (poly a), poly guanylate (poly G), polycytidylic acid (poly C), polyuridylate (poly U), and methylated CpG binding domain (MBD) protein, dimethylamine, thiol. The purpose of microsphere modification is to introduce a group capable of combining with a modified unidirectional primer or a single-stranded nucleotide fragment containing a complementary sequence of a nucleic acid fragment to be detected on the surface of the microsphere so as to coat the modified unidirectional primer or the single-stranded nucleotide fragment containing the complementary sequence of the nucleic acid fragment to be detected on the surface of the microsphere. In general, the most widely used microspheres are those modified with carboxyl groups, i.e., carboxylated microspheres. The modification of the microspheres can be carried out by methods reported in the prior art, or the modified microspheres can be purchased directly.

Furthermore, in the above preparation method, the nucleic acid fragment to be detected is a single-stranded deoxyribonucleic acid (DNA) fragment, the nucleic acid fragment to be detected in the present invention refers to a certain fragment specifically required, for example, a human whole genome sequence has been disclosed, a whole genome sequence of other animals or microorganisms has been reported, and if a certain sequence specific to a human, an animal or a microorganism of a known whole genome sequence is to be detected, the sequence to be detected is the nucleic acid fragment to be detected. A one-way primer is designed for the complementary sequence of the nucleic acid fragment to be detected.

Further, in the above preparation method, the one-way primer is a primer capable of extending a base at the end by nucleic acid polymerase catalysis using a single-stranded nucleotide fragment containing a sequence complementary to a nucleic acid fragment to be detected as a template. If the one-way primer is coated on the surface of the microsphere, the one-way primer needs to be modified so as to form a group capable of being combined with the modification group on the microsphere. Therefore, the modified unidirectional primer coated on the surface of the microsphere used in the invention is a unidirectional primer modified by at least one of amino, carboxyl, digoxin, biotin, poly A, poly G, poly C, poly T, poly U and methyl. The design and modification of the one-way primer can be carried out by a corresponding company, such as Shanghai Biotech.

Furthermore, in the above preparation method, the modified unidirectional primer that guides the synthesis of the nucleic acid fragment to be detected or the single-stranded nucleotide fragment containing the complementary sequence of the nucleic acid fragment to be detected is coated on the surface of the microsphere, and the coating means that the modified unidirectional primer or the single-stranded nucleotide fragment is immobilized on the surface of the microsphere through a covalent bond or a non-covalent bond. The modified unidirectional primer or the single-stranded nucleotide fragment containing the complementary sequence of the nucleic acid fragment to be detected can be coated on the surface of the microsphere by the method disclosed in the prior art, for example, by electrostatic adsorption, chemical bonding, etc.

Further, in the above preparation method, the nucleic acid polymerase is a DNA-dependent DNA polymerase, and includes Taq DNA polymerase, Klenow fragment, and sequencing enzyme (Sequenase).

Further, in the above preparation method, the dNTP is one of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxyuridine triphosphate (dUTP), deoxythymidine triphosphate (dTTP), or a combination of two or more thereof, and the deoxyuridine triphosphate and the deoxythymidine triphosphate do not occur simultaneously.

Further, in the above preparation method, the ddNTP is one of dideoxy adenosine triphosphate (ddATP), dideoxyguanosine triphosphate (ddGTP), dideoxycytidine triphosphate (ddCTP), dideoxyuridine triphosphate (ddUTP), dideoxythymidine triphosphate (ddTTP), or a combination of two or more of them, and the dideoxynucleotide triphosphate and the dideoxythymidine triphosphate do not occur simultaneously.

Further, in the above preparation method, among the fluorescently labeled ddNTP and ribose 3' -OH protected fluorescently labeled dntps, ddNTP and ribose 3' -OH protected dntps are fluorescently labeled for use in a subsequent sequencing process, and in the present invention, the ddNTP and ribose 3' -OH protected dntps may be labeled with any one or more of the following fluorescent substances: fluorescein Isothiocyanate (FITC), Alexa Fluor 610, Alexa Fluor 488, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 700, cyanine dye Cy5(cyanine 5), Texas Red (Texas Red), cyanine dye Cy3(cyanine 3), cyanine dye Cy7(cyanine 7), hydroxyfluorescein (FAM), lucifer yellow (luciferase yellow), cyanine dye Cy5.5(cyanine 5.5), rhodamine 110 (rhodomine 110, R110), ROX, rhodamine 6G (rhodomine 6G, R6G), TAMRA.

Further, in the above-mentioned production method, the ribose 3'-OH protected dNTP, the fluorescently labeled ddNTP, and the ribose 3' -OH protected fluorescently labeled dNTP may be commercially available, or may be produced by a method described in protecting group chemical mobile (wu kinje, li bengilene, chemical industry press, beijing, 2007), Bioconjugate technologies (second classification, Greg t. hermanson, Elsevier press, 2008).

Further, in the above preparation method, the buffer solution comprises Tris-HCl (pH7-7.5) and MgCl as basic components₂、NaCl。

Further, in the steps (2), (3) and (5), different components are added to prepare an incubation system, and then incubation is performed under appropriate conditions. The incubation can be performed in containers such as 24-well plate wells, 24-well filter plate wells, 96-well filter plate wells, 384-well filter plate wells, centrifuge tubes, EP tubes, and the like, and the containers used for each incubation can be the same or different. The total volume of the incubation system is 10. mu.l-10 ml, preferably 10. mu.l-1000. mu.l, for each incubation. The incubation system refers to a mixture formed by mixing various components as required for each incubation. The final concentration of microspheres in the incubation system was 5X 10 for each incubation²Per ml to 2.5X 10⁸Nucleic acid polymerase at final concentration of 2-480 units/ml, ribose 3' -OH protected dNTP at final concentration of 0.1-50 μmol/L, and fluorescently labeled ddNTP at final concentration0.1-50. mu. mol/L, and the final concentration of each ribose 3' -OH protected fluorescently labeled dNTP is 0.1-50. mu. mol/L. Ribose 3' -OH protected dntps are: ribose 3 '-OH-protected dATP, ribose 3' -OH-protected dTTP/dUTP (dTTP/dUTP means dTTP or dUTP, the same applies hereinafter), a mixture of ribose 3 '-OH-protected dGTP and ribose 3' -OH-protected dCTP, and the final concentration of any one of ribose 3 '-OH-protected dATP, ribose 3' -OH-protected dTTP/dUTP, ribose 3 '-OH-protected dGTP and ribose 3' -OH-protected dCTP is 0.1 to 50. mu. mol/L. The fluorescence labeling ddNTP is any one or more of fluorescence labeling ddATP, fluorescence labeling ddTTP/ddUTP, fluorescence labeling ddGTP and fluorescence labeling ddCTP, and the final concentration of any one (if added) of the fluorescence labeling ddATP, the fluorescence labeling ddTTP/ddUTP, the fluorescence labeling ddGTP and the fluorescence labeling ddCTP is 0.1-50 mu mol/L. The ribose 3' -OH protected fluorescently labeled dNTP is any one or more of ribose 3' -OH protected fluorescently labeled dATP, ribose 3' -OH protected fluorescently labeled dTTP/dUTP, ribose 3' -OH protected fluorescently labeled dGTP and ribose 3' -OH protected fluorescently labeled dCTP, and the final concentration of any one of ribose 3' -OH protected fluorescently labeled dATP, ribose 3' -OH protected fluorescently labeled dTTP/dUTP, ribose 3' -OH protected fluorescently labeled dGTP and ribose 3' -OH protected fluorescently labeled dCTP (if added) is 0.1-50 mu mol/L.

Further, in the above steps (2), (3) and (5), the temperature for each incubation is 20 to 95 ℃, preferably 32 to 70 ℃. The time of each incubation can be selected according to actual needs.

Further, in the step (4), a ribose 3'-OH deprotecting agent is added to the incubated microspheres to deprotect ribose 3' -OH. The ribose 3' -OH deprotecting agent can be purchased from the market. The final concentration of the ribose 3' -OH protective agent is 0.1-10 mol/L.

Further, in the above steps (3) and (5), the microspheres are divided into two parts, wherein one part of the microspheres is added with ribose 3'-OH protected dNTP, and the other part of the microspheres is added with fluorescently labeled ddNTP or/and ribose 3' -OH protected fluorescently labeled dNTP. According to the method, the sequencing microspheres with the same group number as the base number of the nucleic acid fragment to be detected can be obtained by continuously repeating the steps (4) and (5) according to the base number of the nucleic acid fragment to be detected, for example, when the base number of the nucleic acid fragment to be detected is n, the steps (4) and (5) are repeated to obtain n groups of sequencing microspheres, the n groups of sequencing microspheres are detected by a flow cytometer, and the sequence of the microsphere groups corresponds to the base arrangement sequence, so that the base sequence of the nucleic acid fragment to be detected can be obtained. When 4 kinds of fluorescence respectively labeled ddATP, ddTTP/ddUTP, ddGTP, ddCTP or/and ribose 3' -OH protected dATP, dTTP/dUTP, dGTP and dCTP are added in the steps (3) and (5), only n groups of sequencing microspheres are prepared and sequenced to obtain the base sequence of the fragment to be detected, for example, when 4 kinds of fluorescence respectively labeled ddATP, ddTTP/ddUTP, ddGTP and ddCTP are simultaneously added, n groups of microspheres are prepared and detected to obtain the base sequence of the nucleic acid fragment. And when 1-2 kinds of fluorescence respectively labeled ddATP, ddTTP/ddUTP, ddGTP, ddCTP or/and ribose 3'-OH protected dATP, dTTP/dUTP, dGTP and dCTP are added in the steps (3) and (5), the base sequence of the whole nucleic acid fragment to be detected cannot be obtained by only the n groups of sequencing microspheres, and the step (7) is required to replace the fluorescence labeled ddNTP or/and the ribose 3' -OH protected fluorescence labeled dNTP, replace the container and repeat the steps (2) - (6) to obtain 4 Xn or 2 Xn groups of sequencing microspheres so as to ensure that all possible bases of all sites can be detected. In a specific embodiment of the present invention, when only 2 kinds of ddntps labeled with fluorescence respectively or 2 kinds of dntps labeled with fluorescence respectively and protected with ribose 3'-OH are added, the obtained n groups of sequencing microspheres can only detect the two specific bases, so in order to obtain the whole base sequence, the fluorescence-labeled ddntps or the ribose 3' -OH-protected dntps need to be replaced, the experiment is repeated to obtain n groups of sequencing microspheres for detecting another two bases, and the microspheres are combined to obtain the whole base sequence.

Further, in the step (7), the number of sequencing repetitions is determined by the number of fluorescently labeled ddNTP and/or ribose 3' -OH protected fluorescently labeled dNTP species added per time, the number of sequencing repetitions is small for a large number of species added per time, and the number of sequencing repetitions is large for a small number of species added per time. When only n groups of sequencing microspheres are prepared and all possible bases are detected at one time, the configuration requirement on the flow cytometer is high, and in order to reduce the cost, the method is suitable for the existing flow cytometer, and 2n groups of sequencing microspheres are preferably prepared. When the 2 n-group microspheres are prepared by adopting the method, any two kinds of fluorescently-labeled ddNTP or ribose 3' -OH protected fluorescently-labeled dNTPs are added in the steps (3) and (5) to obtain n-group microspheres, then the two kinds of fluorescently-labeled ddNTP or ribose 3' -OH protected fluorescently-labeled dNTPs in the steps (3) and (5) are replaced by the other two kinds of fluorescently-labeled ddNTPs or ribose 3' -OH protected fluorescently-labeled dNTPs, a container is replaced, the steps (2) to (6) are repeated to obtain n-group microspheres, and the 2 n-group microspheres are obtained. Compared with the 4n group microspheres, the 2n group microspheres are simple and convenient to operate, and compared with the n group microspheres, the method has low configuration requirements on a flow cytometer and low sequencing cost. The two fluorescently-labeled ddNTPs or the fluorescently-labeled dNTPs protected by ribose 3' -OH added each time can be combined randomly, for example, the fluorescently-labeled ddTTP/ddUTP and ddATP can be added to obtain n groups of microspheres, and then the n groups of microspheres are replaced by the fluorescently-labeled ddGTP and ddCTP to obtain n groups of microspheres; or adding fluorescence labeled ddTTP/ddUTP and ddGTP to obtain n-group microspheres, and replacing the microspheres with fluorescence labeled ddATP and ddCTP to obtain n-group microspheres; or adding fluorescence labeled ddTTP/ddUTP and ddCTP to obtain n groups of microspheres, and replacing the microspheres with fluorescence labeled ddATP and ddGTP to obtain n groups of microspheres, and the like.

Further, the invention provides a specific method for single-base continuous extension flow-type targeted sequencing, which comprises the following steps:

(2) adding a coating microsphere, a single-stranded nucleotide fragment containing a complementary sequence of the nucleic acid fragment to be detected or a modified unidirectional primer and a buffer solution for guiding the synthesis of the nucleic acid fragment to be detected into a container I, uniformly mixing, and carrying out incubation hybridization;

(3) washing and suspending the microspheres by using a buffer solution, keeping part of the microspheres in a container I, transferring the other part of the microspheres from the container I to a container II, and adding dNTP protected by ribose 3' -OH, nucleic acid polymerase and the buffer solution into the container I for incubation; adding one or a combination of the fluorescence labeled ddNTP and the ribose 3' -OH protected fluorescence labeled dNTP, nucleic acid polymerase and buffer solution into a container II for incubation (preferably, the final volume of liquid in each container is 10 mu l to 10ml, mixing uniformly, and incubating at 20 ℃ to 95 ℃);

(4) washing microspheres in a container I, adding a ribose 3' -OH deprotecting agent into the container I, and uniformly mixing;

(5) washing and suspending the microspheres in the container I by using a buffer solution, wherein part of the microspheres are remained in the container I, the rest microspheres are moved into a container III from the container I, and dNTP protected by ribose 3' -OH, nucleic acid polymerase and the buffer solution are added into the container I for incubation; adding one or a combination of the fluorescence labeled ddNTP and the ribose 3' -OH protected fluorescence labeled dNTP, nucleic acid polymerase and buffer solution into a container III for incubation (preferably, the final volume of liquid in each container is 10 mu l to 10ml, mixing uniformly, and incubating at 20 ℃ to 95 ℃);

(6) repeating the operations from (4) to (5) continuously, wherein n bases of the sequenced nucleic acid fragment are required to be continuously prepared into microspheres of n containers; (the microspheres are microspheres incubated with fluorescently labeled ddNTPs or/and ribose 3' -OH protected fluorescently labeled dNTPs added);

(7) if all bases cannot be detected by using the n containers of microspheres, replacing the fluorescently-labeled ddNTP or/and ribose 3' -OH protected fluorescently-labeled dNTP in the steps (3) and (5), replacing the containers, and repeating the steps (2) to (6) to obtain 4 xn or 2 xn containers of microspheres, wherein the microspheres in all the containers are sequencing microspheres;

(8) and (3) washing and suspending the 4 xn or 2 xn or n groups of microspheres obtained in the steps (6) and (7), and further detecting by a flow cytometer to obtain the base sequence of the nucleic acid fragment to be detected.

Further, the invention also provides a preferred single-base continuous extension flow-type target sequencing method, which comprises the following steps:

(2) adding the coated microspheres obtained in the step (1), a single-stranded nucleotide fragment containing a complementary sequence of a nucleic acid fragment to be detected or a modified unidirectional primer and a buffer solution for guiding the synthesis of the nucleic acid fragment to be detected into a container I, uniformly mixing, and carrying out incubation hybridization;

(3) washing and suspending the microspheres by using a buffer solution, keeping part of the microspheres in a container I, transferring the other part of the microspheres from the container I to a container II, and adding nucleic acid polymerase, the buffer solution and ribose 3' -OH protected dNTP into the container I for incubation; adding nucleic acid polymerase, buffer solution, two fluorescence-labeled ddNTPs or two fluorescence-labeled dNTPs with protected ribose 3' -OH into a container II, and incubating, wherein the sequencing microsphere obtained in the container II is marked as A1; the ribose 3' -OH protected dNTP is ribose 3' -OH protected dATP, ribose 3' -OH protected dTTP/dUTP, ribose 3' -OH protected dGTP and ribose 3' -OH protected dCTP; preferably, the final volume of the reaction in each container is 10 mu l to 10ml, the mixture is mixed evenly and incubated at the temperature of 20 ℃ to 95 ℃;

(5) washing and suspending the microspheres in the container I by using a buffer solution, wherein part of the microspheres are remained in the container I, the other part of the microspheres are moved into a container III from the container I, and nucleic acid polymerase, the buffer solution and ribose 3' -OH protected dNTP are added into the container I for incubation; adding nucleic acid polymerase, buffer solution, two fluorescence-labeled ddNTPs or two fluorescence-labeled dNTPs with protected ribose 3' -OH into a container III, and incubating, wherein the sequencing microsphere obtained in the container III is marked as A2; the ribose 3' -OH protected dNTP is ribose 3' -OH protected dATP, ribose 3' -OH protected dTTP/dUTP, ribose 3' -OH protected dGTP and ribose 3' -OH protected dCTP; preferably, the final volume of the reaction in each container is 10 mu l to 10ml, the mixture is mixed evenly and incubated at the temperature of 20 ℃ to 95 ℃;

(6) continuously repeating the steps similar to (4) and (5) to obtain n groups of sequencing microspheres added with fluorescence-labeled ddNTP or ribose 3' -OH protected fluorescence-labeled dNTP for incubation, wherein the last group of sequencing microspheres is marked as An, and n is the number of basic groups of the nucleic acid fragment to be detected;

(7) adding the coated microspheres obtained in the step (1), a single-stranded nucleotide fragment containing a complementary sequence of the nucleic acid fragment to be detected or a modified unidirectional primer and a buffer solution for guiding the synthesis of the nucleic acid fragment to be detected into containers ①, uniformly mixing, and carrying out incubation hybridization;

(8) washing and suspending the microspheres with a buffer solution, leaving a portion of the microspheres in container ①, transferring another portion of the microspheres from container ① to container ②, adding a nucleic acid polymerase, a buffer solution, and ribose 3' -OH-protected dNTPs to container ①, incubating, adding a nucleic acid polymerase, a buffer solution, two additional fluorescently labeled ddNTPs different from step (3) or two additional ribose 3' -OH-protected fluorescently labeled dNTPs different from step (3) to container ②, incubating, and incubating, wherein the sequencing microspheres obtained in container ② are labeled B1, wherein the ribose 3' -OH-protected dNTPs are ribose 3' -OH-protected dATP, ribose 3' -OH-protected dTTP/dUTP, ribose 3' -OH-protected dGTP and ribose 3' -OH-protected dCTP, preferably, the reaction volume in each container is 10. mu.l to 10ml, mixing, and incubating at 20 ℃ to 95 ℃;

(9) after the microspheres in the container ① are washed, a ribose 3' -OH deprotecting agent is added into the container ① and mixed evenly;

(10) buffer washing and suspending the microspheres in container ①, leaving a portion of the microspheres in container ① and another portion of the microspheres in container ①, transferring the remaining portion of the microspheres in container ③, adding nucleic acid polymerase, buffer, and ribose 3' -OH protected dNTPs to container ①, incubating, adding nucleic acid polymerase, buffer, another two fluorescently labeled ddNTPs different from step (5) or another two fluorescently labeled dNTPs protected from ribose 3' -OH different from step (5) to container ③, incubating, and incubating, wherein the sequencing microspheres obtained in container ③ are labeled B2, wherein the ribose 3' -OH protected dNTPs are ribose 3' -OH protected dATP, ribose 3' -OH dTTP/dUTP, ribose 3' -OH protected dGTP and ribose 3' -OH protected dCTP, preferably, the final reaction volume in each container is 10. mu.l to 10ml, mixing, and incubating at 20 ℃ to 95 ℃;

(11) continuously repeating the steps (9) and (10) to obtain n groups of sequencing microspheres added with fluorescence-labeled ddNTP or ribose 3' -OH protected fluorescence-labeled dNTP for incubation, wherein the last group of sequencing microspheres is marked as Bn, and n is the number of basic groups of the nucleic acid fragment to be detected;

(12) washing and suspending the microspheres A1-An and B1-Bn, and further detecting by a flow cytometer to obtain the base sequence of the nucleic acid fragment to be detected.

Further, the invention also provides a more preferable single-base continuous extension flow-type target sequencing method, which comprises the following steps:

(1) coating amino modified unidirectional primers for guiding the synthesis of the nucleic acid fragment to be detected or a single-stranded nucleotide fragment containing the complementary sequence of the nucleic acid fragment to be detected on the surface of a carboxylated polystyrene microsphere with the diameter of 1-10 mu m to obtain a coated microsphere;

(2) based on a full-automatic pipetting workstation, adding coating microspheres, single-stranded nucleotide fragments containing complementary sequences of nucleic acid fragments to be detected or modified unidirectional primers and buffer solution for guiding the synthesis of the nucleic acid fragments to be detected into holes I of a 96-hole filter plate A, uniformly mixing, and carrying out incubation hybridization;

(3) washing and suspending the microspheres by using a buffer solution, wherein the retained microspheres are partially placed in a well I of a 96-well filter plate A, the other microspheres are moved to a well I of a 96-well filter plate B from the well I of the 96-well filter plate A, nucleic acid polymerase, buffer solution, dATP protected by ribose 3'-OH, dUTP/dTTP protected by ribose 3' -OH, dGTP protected by ribose 3'-OH and dCTP protected by ribose 3' -OH are continuously added into the well I of the 96-well filter plate A, the nucleic acid polymerase, the buffer solution, ddATP marked by fluorescence and ddUTP/ddTTP are continuously added into the well I of the 96-well filter plate B, the total reaction volume in the well I of the 96-well filter plate A, B is 10-1000 mul, the two wells are mixed and incubated at 37 ℃ for 60 seconds, and the sequencing microspheres obtained after incubation in the well I of the 96-well filter plate B are marked as A1;

(4) carrying out suction filtration and washing on microspheres in the holes I of the 96-hole filter plate A, adding a ribose 3' -OH deprotecting agent into the holes I of the 96-hole filter plate A, and uniformly mixing;

(5) washing and suspending microspheres in a hole I of a 96-hole filter plate A by using a buffer solution, wherein a part of microspheres are remained in the hole I of the 96-hole filter plate A, and the other part of microspheres are moved from the hole I of the 96-hole filter plate A to a hole II of a 96-hole filter plate B;

(6) adding nucleic acid polymerase and buffer solution into the first hole of the 96-hole filter plate A and the second hole of the 96-hole filter plate B, continuously adding dATP with protected ribose 3'-OH, dUTP/ddTTP with protected ribose 3' -OH, dGTP with protected ribose 3'-OH and dCTP with protected ribose 3' -OH into the first hole of the 96-hole filter plate A, continuously adding ddATP with fluorescent label and ddUTP/ddTTP with fluorescent label into the second hole of the 96-hole filter plate B, wherein the total reaction volumes of the first hole of the 96-hole filter plate A and the second hole of the 96-hole filter plate B are 10-1000 mul, uniformly mixing the two holes, and incubating at 37 ℃ for 60 seconds, wherein the sequencing microsphere obtained after incubation in the second hole of the 96-hole filter plate B is marked as A2;

(7) repeating the operations from (4) to (6) continuously to obtain n groups of sequencing microspheres added with fluorescence labeled ddATP and fluorescence labeled ddUTP/ddTTP for incubation, and marking the last group of sequencing microspheres as An, wherein n is the number of bases of the nucleic acid fragment to be detected;

(8) replacing the fluorescence-labeled ddATP and the fluorescence-labeled ddUTP/ddTTP in the steps (3) and (6) with fluorescence-labeled ddGTP and fluorescence-labeled ddCTP, repeating the operations from (2) to (7) in a 96-hole filter plate C to obtain n groups of sequencing microspheres added with the fluorescence-labeled ddGTP and the fluorescence-labeled ddCTP for incubation, wherein the first group of sequencing microspheres is labeled as B1, the last group of sequencing microspheres is labeled as Bn, and n is the base number of the nucleic acid fragment to be detected; the A1-An group microspheres prepared in the step (7) are used for identifying thymine (T) and adenine (A) in the nucleic acid fragment to be detected, and the B1-Bn group microspheres prepared in the step (8) are used for identifying cytosine (C) or guanine (G) in the nucleic acid fragment to be detected;

(9) washing and suspending the microspheres A1-An and B1-Bn, and further detecting by a flow cytometer to obtain the base sequence of the nucleic acid fragment to be detected.

Furthermore, in the sequencing method, the obtained sequencing microspheres are n groups, or 2n groups, or 4n groups, so that correct bases of each site of the nucleic acid fragment can be detected, the preparation sequence of the sequencing microspheres corresponds to the base arrangement sequence during detection, and the detection results are integrated to obtain the whole base sequence of the nucleic acid fragment.

Further, in the above sequencing method, the nucleic acid fragment is a single-stranded DNA fragment. The nucleic acid fragment may be any biological nucleic acid fragment, and may be human, animal or microbial. The method is simple, accurate in result and easy to read, and has good application prospects in the fields of gene detection, disease screening, gene-guided targeted drug delivery, single nucleotide polymorphism and the like.

Furthermore, in the sequencing method, a plurality of nucleic acid fragment sequences can be detected in parallel by selecting the coding microspheres and regulating the preparation process of the sequencing microspheres.

The invention establishes a single-base continuous extension flow-type target sequencing method through single-base continuous extension of a primer, can obtain sequencing microspheres by applying the method, and can obtain a base sequence of a nucleic acid fragment to be detected by detecting the sequencing microspheres by a flow cytometer. Compared with the existing sequencing method, the single-base continuous extension flow type target sequencing method for carrying out nucleic acid target sequencing has the obvious advantages of simplicity, accuracy, easiness in reading and the like, is suitable for detecting a nucleic acid base sequence by a flow cytometer, can be widely applied to the nucleic acid sequencing fields of gene detection, microbial detection, genetics, exons, Single Nucleotide Polymorphism (SNP), genomics, proteomics and the like, has good application prospects in disease screening, gene-guided target drug delivery and particularly in the tumor companion diagnosis field.

Drawings

FIG. 1 is a flow chart of the single-base continuous extension flow-type target sequencing method of example 1. A. Coating microspheres with one-way primers; B. hybridizing a single-stranded nucleotide fragment containing a complementary sequence of the nucleic acid fragment to be detected with a one-way primer; C. taking a single-stranded nucleotide fragment containing a complementary sequence of a nucleic acid fragment to be detected as a template, and continuously extending a single fluorescence labeling base from the tail end of a microsphere coating primer under the catalysis of nucleic acid polymerase; D. the microspheres were detected by flow cytometry to identify base sequences.

FIG. 2 is a Sanger sequencing map of a T790M nucleic acid fragment of the EGFR gene, wherein FIG. 2A is a Sanger sequencing map of a wild-type T790M nucleic acid fragment; FIG. 2B is a Sanger sequencing chart of a mutant T790M nucleic acid fragment.

FIG. 3 is a flow chart of parallel target sequencing of mixed samples of an exon 21L 858R nucleic acid fragment (wild-type plasmid DNA fragment), an exon 18G719X nucleic acid fragment (mixture of wild-type and mutant plasmid fragments) of the EGFR gene starting with the sequencing of the 1 st base, wherein FIG. 3(i) is the detection of thymine (T), FIG. 3(ii) is the detection of adenine (A), FIG. 3(iii) is the detection of cytosine (C), and FIG. 3(iv) is the detection of guanine (G); as a result, thymine (T) was used as the base at the 1 st position from exon 21L 858R, and adenine (A) and guanine (G) were used as the allelic bases at the 1 st position from exon 18G 719X.

Detailed Description

The following examples are given to further illustrate the present invention, but not to limit the scope of the invention.

The functionalized polystyrene microspheres used in the examples were obtained from Spherotech, Inc., USA, Cyanine dye Cy 3-labeled ddATP, Cyanine dye Cy 3-labeled ddGTP, Cyanine dye Cy 5-labeled ddGTP, FITC-labeled ddUTP, FITC-labeled ddCTP, ROX-labeled ddCTP from PE, Ribose 3 '-OH-protected dNTP, Ribose 3' -OH-deprotecting agent from Hippon-Cretaceae Biotech, Ltd, DNA polymerase Sequenase, DNA polymerase Klenow fragment from Thermoher Fis Scientific.

Unless otherwise stated, the following asymmetric PCR products and modified one-way primers were obtained according to the PCR amplification method reported in the prior art or were obtained by the corresponding company.

Example 1 Single-base continuous extension flow-directed sequencing of EGFR Gene T790M nucleic acid fragment [ FIG. 1]

(1) The base sequence of the T790M fragment of the EGFR gene is searched from NCBI Genebank, a one-way primer is designed according to the sequence and is subjected to amino modification, and the amino-modified one-way primer is as follows: 5 'GGAAGCCTACGTGATGG CCA 3', coating the amino modified one-way primer on the surface of a carboxylated polystyrene microsphere with the diameter of 7 mu m;

(2) based on a full-automatic pipetting workstation, adding 4 multiplied by 10 into a hole I of a 96-hole filter plate A⁵Carrying out incubation hybridization on microspheres coated by each primer, T790M fragment (wild type/mutant plasmid mixture) asymmetric PCR products and buffer solution for 2min at 65 ℃ and naturally cooling for 45 min; washing and suspending the microspheres with a buffer solution, sucking 4000 microspheres in a well I of a 96-well filter plate A into a well I of a 96-well filter plate B, continuously adding 10 units of DNA polymerase Sequenase, the buffer solution, 10 mu M of dATP protected by ribose 3'-OH, 10 mu M of dUTP protected by ribose 3' -OH, 10 mu M of dGTP protected by ribose 3'-OH and 10 mu M of dCTP protected by ribose 3' -OH into the well I of the 96-well filter plate A, continuously adding 10 units of DNA polymerase Sequenase, the buffer solution, 0.5 mu M of ddATP marked by cyanine dye Cy3 and 0.5 mu M of ddUTP marked by FITC into the well I of the 96-well filter plate B, uniformly mixing, and incubating for 60 seconds at 37 ℃;

(3) carrying out suction filtration and washing on microspheres in the holes I of the 96-hole filter plate A, adding a ribose 3' -OH deprotecting agent into the holes I of the 96-hole filter plate A, and uniformly mixing;

(4) washing and suspending microspheres in a hole I of a 96-hole filter plate A by buffer solution suction filtration, and sucking 4000 microspheres in the hole I of the 96-hole filter plate A to a hole II of a 96-hole filter plate B;

(5) adding 10 units of DNA polymerase Sequenase and buffer solution into the first hole of the 96-hole filter plate A and the second hole of the 96-hole filter plate B, continuously adding 10 mu M of ribose 3'-OH protected dATP, 10 mu M of ribose 3' -OH protected dUTP, 10 mu M of ribose 3'-OH protected dGTP and 10 mu M of ribose 3' -OH protected dCTP into the first hole of the 96-hole filter plate A, continuously adding 0.5 mu M of cyanine dye Cy3 labeled ddATP and 0.5 mu M of FITC labeled ddUTP into the second hole of the 96-hole filter plate B, wherein the final volume of each hole is 10 mu l-1000 mu l, uniformly mixing, and incubating for 60 seconds at 37 ℃;

(6) washing the microspheres in the I wells of the 96-well filter plate A, adding a ribose 3' -OH deprotecting agent, dividing the washed microspheres into two wells, adding 10 units of DNA polymerase Sequenase, a buffer solution, 10. mu.M of dATP protected by ribose 3' -OH, 10. mu.M of dUTP protected by ribose 3' -OH, 10. mu.M of dGTP protected by ribose 3' -OH, 10. mu.M of dCTP protected by ribose 3' -OH, 10 units of DNA polymerase Sequenase added to the III wells of the 96-well filter plate B, a buffer solution, 0.5. mu.M of cyanine dye Cy3 labeled ddATP, 0.5. mu.M of FITC labeled ddUTP, repeating the steps (3) to (5), and continuously preparing 76-well microspheres in the 96-well filter plate B;

(7) the "0.5. mu.M cyanine dye Cy 3-labeled ddATP and 0.5. mu.M FITC-labeled ddUTP" in the above-mentioned (2), (5) and (6) were replaced with "0.5. mu.M cyanine dye Cy 3-labeled ddGTP and 0.5. mu.M FITC-labeled ddCTP", and similar operations were repeated in 96-well filter plates C according to the procedures of (2) to (6), thereby preparing 76-well microspheres in succession.

(8) And (4) carrying out suction filtration and washing on the 76-hole microspheres prepared in the step (6) and the 76-hole microspheres prepared in the step (7), and respectively loading the microspheres and the 76-hole microspheres to a flow cytometer for detection.

Since the DNA fragment does not contain uracil (U), when the detection result shows that uracil is uracil, the base is thymine (T). The 76-well microspheres in the 96-well filter plate B prepared in (6) above were used for sequencing thymine (T) and adenine (A) in the T790M fragment, and the 76-well microspheres in the 96-well filter plate C prepared in (7) above were used for sequencing cytosine (C) and guanine (G) in the T790M fragment.

All the results of the examination were integrated to obtain the sequencing result of the T790M nucleic acid fragment: GCGTG GACAACCCCC ACGTG TGCCG CCTGC TGGGC ATCTG CCTCA CCTCC ACCGT GCAGC TCATC AC (C → T, T790M), GCA GCTCA T, the result that C and T occur simultaneously at the 67 th base, indicates that wild type and mutant type exist simultaneously in the nucleic acid fragment. The detection result is consistent with the sample Sanger sequencing method, and as can be seen from FIG. 2A, the target base of the wild type T790M plasmid DNA fragment is C (bar-shaped frame); as can be seen in FIG. 2B, the target base of the mutant T790M plasmid DNA fragment is T (bar box).

Example 2 Single base continuous extension flow Targeted sequencing of the EGFR Gene T790M nucleic acid fragment

(1) Finding a base sequence of an EGFR gene T790M from NCBI Genebank, artificially synthesizing wild type/mutant plasmids according to the sequence, taking plasmids as a template, synthesizing a single-stranded DNA fragment containing a complementary sequence of a T790M target fragment by asymmetric PCR, designing a one-way primer according to the single-stranded DNA fragment, and coating the single-stranded DNA fragment on the surface of a dimethylamine modified polystyrene microsphere with the diameter of 5 mu m to obtain a coated microsphere;

(2) based on a full-automatic pipetting workstation, adding 4 multiplied by 10 into a hole I of a 96-hole filter plate A⁵Carrying out incubation hybridization on the coated microspheres, the one-way primers and the buffer solution for 2min at 65 ℃ and naturally cooling for 45 min; washing and suspending the microspheres with a buffer solution, sucking 4000 microspheres in a well I of a 96-well filter plate A into a well I of a 96-well filter plate B, continuously adding 100units of DNA polymerase Sequenase, the buffer solution, 20 mu M of dATP protected by ribose 3'-OH, 20 mu M of dUTP protected by ribose 3' -OH, 20 mu M of dGTP protected by ribose 3'-OH, 20 mu M of dCTP protected by ribose 3' -OH in the well I of the 96-well filter plate A, continuously adding 100units of DNA polymerase Sequenase, the buffer solution, 2 mu M of ddATP marked by cyanine dye Cy3, 2 mu M of ddUTP marked by FITC, 2 mu M of GTP ddmarked by cyanine dye Cy5 and 2 mu M of ddCTP marked by ROX in the well I of the 96-well filter plate B, incubating the mixture to obtain a final CTP volume of 10 mu l-1000 mu l per well, and mixing the final CTP for 60 seconds at 37 ℃;

(5) adding 100units of DNA polymerase Sequenase and buffer solution into the first hole of the 96-hole filter plate A and the second hole of the 96-hole filter plate B, continuously adding 20 mu M of dATP protected by ribose 3'-OH, 20 mu M of dUTP protected by ribose 3' -OH, 20 mu M of dGTP protected by ribose 3'-OH and 20 mu M of dCTP protected by ribose 3' -OH into the first hole of the 96-hole filter plate A, continuously adding 2 mu M of ddATP 3 labeled by cyanine dye, 2 mu M of ddUTP labeled by FITC, 2 mu M of ddGTP labeled by cyanine dye Cy5 and 2 mu M of ROX labeled ddCTP into the second hole of the 96-hole filter plate B, wherein the final volume of each hole is 10 mu l-1000 mu l, uniformly mixing, and incubating for 60 seconds at 37 ℃;

(6) washing microspheres in a well I of a 96-well filter plate A, adding a ribose 3' -OH deprotecting agent, dividing the washed microspheres into two wells after washing, adding 100units of DNA polymerase Sequenase to the well I of the 96-well filter plate A, a buffer solution, 20. mu.M of dATP protected by ribose 3' -OH, 20. mu.M of dUTP protected by ribose 3' -OH, 20. mu.M of dGTP protected by ribose 3' -OH, 20. mu.M of dCTP protected by ribose 3' -OH, adding 100units of DNA polymerase Sequenase to a well III of the 96-well filter plate B, a buffer solution, 2. mu.M of cyanine dye Cy 3-labeled ddATP, 2. mu.M of FITC-labeled ddUTP, 2. mu.M of cyanine dye Cy 5-labeled ddGTP, and 2. mu.M of ROX-labeled ddCTP, repeating the steps (3) - (5), and preparing 76-well microspheres in the 96-well filter plate B successively;

(7) and (4) washing the 76-hole microspheres in the 96-hole filter plate B prepared in the step (6), and detecting by using a sample flow cytometer to obtain a detection result at one time.

The data analysis shows that the sequencing result of the T790M nucleic acid fragment is as follows: GCGTG GACAA CCCCC ACGTGTGCCG CCTGC TGGGC ATCTG CCTCA CCTCC ACCGT GCAGC TCATC AC (C → T, T790M), GCA GCTCAT, the result that C and T occur simultaneously at the 67 th base, indicates that wild type and mutant type exist simultaneously in the nucleic acid fragment.

The sequencing result of the EGFR gene T790M nucleic acid fragment sample by the Sanger method is consistent with the sequencing result of the invention.

Example 3 EGFR Gene exon 21L 858R, exon 18G719X Point mutated Single-base continuous extension flow-Targeted sequencing

(1) Finding out base sequences of EGFR genes containing exon 21L 858R and exon 18G719X targets from NCBI Genebank, respectively designing one-way primers according to the two sequences, and performing biotin modification at the tail ends of the one-way primers, wherein the biotin-modified exon 21L 858R one-way primer is as follows: 5 'TGTCAAGATCACAGATTTTGGGC 3'; the biotin-modified exon 18G719X one-way primer is: 5 'CTGAATTCAAAAAGATCAAAGTGCTG 3', coating two biotin-modified one-way primers on the surface of two groups of streptavidin-modified polystyrene microspheres with the diameter of about 3 mu m and coded by PE-Cy5 respectively;

(2) based on a full-automatic pipetting workstation, 20000 exon 21L 858R primer coated microspheres, 20000 exon 18G719X primer coated microspheres, an exon 21L 858R fragment (wild type plasmid fragment) and an exon 18G719X fragment (mixture of wild type/mutant type plasmid fragments) mixed sample double asymmetric PCR products and buffer are added into a well I of a 96-well filter plate A, and incubation hybridization is carried out for 2min 65 ℃ and natural cooling for 45 min; washing and suspending the microspheres with a buffer solution, sucking 2000 microspheres in a well I of a 96-well filter plate A into a well I of a 96-well filter plate B, continuously adding 50units of DNA polymerase klenow fragment, the buffer solution, 5 mu M of dATP protected by ribose 3'-OH, 5 mu M of dUTP protected by ribose 3' -OH, 5 mu M of dGTP protected by ribose 3'-OH and 5 mu M of dCTP protected by ribose 3' -OH in the well I of the 96-well filter plate A, continuously adding 50units of DNA polymerase klenow fragment, the buffer solution, 5 mu M of ddATP marked by cyanine dye Cy3 and 5 mu M of ddUTP marked by FITC in the well I of the 96-well filter plate B, uniformly mixing, and incubating for 15 minutes at 37 ℃;

(4) washing and suspending microspheres in a hole I of a 96-hole filter plate A by buffer solution suction filtration, and sucking 2000 microspheres in the hole I of the 96-hole filter plate A to a hole II of a 96-hole filter plate B;

(5) adding 50units of DNA polymerase klenow fragment and buffer solution into the first hole of the 96-hole filter plate A and the second hole of the 96-hole filter plate B, continuously adding 5 mu M of dATP protected by ribose 3'-OH, 5 mu M of dUTP protected by ribose 3' -OH, 5 mu M of dGTP protected by ribose 3'-OH and 5 mu M of dCTP protected by ribose 3' -OH into the first hole of the 96-hole filter plate A, continuously adding 5 mu M of cyanine dye Cy 3-labeled ddUTP and 5 mu M of FITC-labeled ddUTP into the second hole of the 96-hole filter plate B, wherein the final volume of each hole is 10 mu l-1000 mu l, uniformly mixing, and incubating for 15 minutes at 37 ℃;

(6) washing microspheres in a well I of a 96-well filter plate A, adding a ribose 3' -OH deprotecting agent, dividing the microspheres into two wells after washing according to the operations of the steps (3) to (5), adding 50units of DNA polymerase klenow fragment, a buffer solution, 5 mu M of dATP protected by ribose 3' -OH, 5 mu M of dUTP protected by ribose 3' -OH, 5 mu M of dGTP protected by ribose 3' -OH, 5 mu M of dCTP protected by ribose 3' -OH into the well I of the 96-well filter plate A, adding 50units of DNA polymerase klenow fragment, a buffer solution, 5 mu M of ddATP marked by cyanine dye Cy3 and 5 mu M of ddUTP marked by FITC into a well III of the 96-well filter plate B, and repeating the operations according to the steps (3) to (5) to prepare 10-well microspheres in the 96-well filter plate B continuously;

(7) the "5. mu.M cyanine dye Cy 3-labeled ddATP, 5. mu.M FITC-labeled ddUTP" in the above (2), (5), (6) was replaced with "5. mu.M cyanine dye Cy 3-labeled ddGTP, 5. mu.M FITC-labeled ddCTP", and similar operations were repeated in 96-well filter plates C according to the procedures of (2) to (6), and 10-well microspheres were prepared continuously.

The 10-well microspheres in the 96-well filter plate B prepared in (6) above were used for parallel sequencing of thymine (T) and adenine (A) in the exon 21L 858R and exon 18G719X fragments, and the 10-well microspheres in the 96-well filter plate C prepared in (7) above were used for parallel sequencing of cytosine (C) and guanine (G) in the exon 21L 858R and exon 18G719X mutant fragments.

All flow cytometry results were integrated to give the sequencing results of the exon 21L 858R nucleic acid fragment as: TGGCCAAACT, respectively; the sequencing result of the exon 18G719X nucleic acid fragment was: g (G → A, G719X) GCTC CGGTG. The detection results of the first bases of these two nucleic acid fragments are shown in FIG. 3, the first base at the beginning of the exon 21L 858R nucleic acid fragment is T, and no mutation exists; and the first base of the exon 18G719X fragment begins to simultaneously generate G and A, which indicates that the wild type and the mutant type exist in the nucleic acid fragment at the same time.

The result of the Sanger sequencing method of the EGFR gene nucleic acid fragment sample is consistent with the sequencing result of the invention.

Example 4

The EGFR gene T790M nucleic acid fragment was sequenced according to the method of example 1, except that: the final concentration of the nucleic acid polymerase was 200 units/ml, the final concentration of each fluorescently labeled ddNTP was 10. mu. mol/L, and the final concentration of each ribose 3' -OH protected dNTP was 10. mu. mol/L in each incubation system. The results were the same as in example 1.

Example 5

The EGFR gene T790M nucleic acid fragment was sequenced according to the method of example 1, except that: in each incubation system, the final concentration of the nucleic acid polymerase was 2 units/ml, the final concentration of each fluorescently labeled ddNTP was 50. mu. mol/L, and the final concentration of each ribose 3' -OH protected dNTP was 50. mu. mol/L, and the incubation time was extended. The results were the same as in example 1.

Claims

1. The single-base continuous extension flow type target sequencing method is characterized by comprising the following steps:

(2) mixing the coated microspheres, the single-stranded nucleotide fragment containing the complementary sequence of the nucleic acid fragment to be detected or the modified unidirectional primer for guiding the synthesis of the nucleic acid fragment to be detected and a buffer solution, and carrying out incubation hybridization on the obtained mixture;

(3) washing and suspending the microspheres by using a buffer solution, dividing the microspheres into two parts, mixing one part of microspheres with nucleic acid polymerase, the buffer solution and the dNTP with protected ribose 3' -OH, and incubating the obtained mixture; mixing the other microsphere with nucleic acid polymerase, buffer solution, fluorescence-labeled ddNTP or/and ribose 3' -OH protected fluorescence-labeled dNTP, and incubating the obtained mixture;

2. The method of claim 1, further comprising: the fluorescence-labeled ddNTP is at least one of fluorescence-labeled ddATP, ddGTP, ddCTP and ddUTP/ddTTP;

preferably, the ribose 3 '-OH-protected fluorescently labeled dNTP is at least one of ribose 3' -OH-protected fluorescently labeled dATP, dGTP, dCTP and dUTP/dTTP;

preferably, the ribose 3 '-OH-protected dNTP is a mixture of ribose 3' -OH-protected dATP, dTTP/dUTP, dGTP and dCTP;

preferably, when the microspheres are coated with modified unidirectional primers for guiding the synthesis of the nucleic acid fragment to be detected, the single-stranded nucleotide fragment containing the complementary sequence of the nucleic acid fragment to be detected is added in the step (2); when the microsphere is coated with a single-stranded nucleotide fragment containing a complementary sequence of the nucleic acid fragment to be detected, a modified unidirectional primer for guiding the synthesis of the nucleic acid fragment to be detected is added in the step (2).

3. A method according to claim 1 or 2, characterized by: the fluorescently labeled ddNTP or ribose 3' -OH protected fluorescently labeled dNTP is labeled with any one or more of the following fluorescent substances: fluorescein isothiocyanate, AlexaFluor 610, Alexa Fluor 488, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 700, cyanine dye Cy5, Texas Red, cyanine dye Cy3, cyanine dye Cy7, hydroxyfluorescein, lucifer yellow, cyanine dye Cy5.5, rhodamine 110, ROX, rhodamine 6G, TAMRA.

4. A method according to claim 1 or 2, characterized by: adding any two fluorescence-labeled ddNTPs or ribose 3' -OH protected fluorescence-labeled dNTPs in steps (3) and (5) to obtain n groups of microspheres, then replacing the two fluorescence-labeled ddNTPs or ribose 3' -OH protected fluorescence-labeled dNTPs in steps (3) and (5) with the other two fluorescence-labeled ddNTPs or ribose 3' -OH protected fluorescence-labeled dNTPs, replacing containers, and adopting steps (2) - (6) to obtain n groups of microspheres to obtain 2n groups of microspheres.

5. The method according to claim 1 or 4, characterized by comprising the steps of:

(3) washing and suspending the microspheres by using a buffer solution, keeping part of the microspheres in a container I, transferring the other part of the microspheres from the container I to a container II, and adding nucleic acid polymerase, the buffer solution and ribose 3' -OH protected dNTP into the container I for incubation; adding nucleic acid polymerase, buffer solution, two fluorescence-labeled ddNTPs or two fluorescence-labeled dNTPs with protected ribose 3' -OH into a container II, and incubating, wherein the sequencing microsphere obtained in the container II is marked as A1; the ribose 3' -OH protected dNTP is ribose 3' -OH protected dATP, ribose 3' -OH protected dTTP/dUTP, ribose 3' -OH protected dGTP and ribose 3' -OH protected dCTP;

(4) after the microspheres in the container I are washed, adding a ribose 3' -OH deprotecting agent into the container I, and uniformly mixing;

(5) washing and suspending the microspheres in the container I by using a buffer solution, wherein part of the microspheres are remained in the container I, the other part of the microspheres are moved into a container III from the container I, and nucleic acid polymerase, the buffer solution and ribose 3' -OH protected dNTP are added into the container I for incubation; adding nucleic acid polymerase, buffer solution, two fluorescence-labeled ddNTPs or two fluorescence-labeled dNTPs with protected ribose 3' -OH into a container III, and incubating, wherein the sequencing microsphere obtained in the container III is marked as A2; the ribose 3' -OH protected dNTP is ribose 3' -OH protected dATP, ribose 3' -OH protected dTTP/dUTP, ribose 3' -OH protected dGTP and ribose 3' -OH protected dCTP;

(7) adding the coated microspheres obtained in the step (1), a single-stranded nucleotide fragment containing a complementary sequence of the nucleic acid fragment to be detected or a modified unidirectional primer and a buffer solution for guiding the synthesis of the nucleic acid fragment to be detected into a container ①, uniformly mixing, and carrying out incubation hybridization;

(8) washing and suspending the microspheres with a buffer solution, wherein a part of the microspheres are in a container ①, another part of the microspheres are moved from a container ① to a container ②, a nucleic acid polymerase, a buffer solution and ribose 3'-OH protected dNTP are added into a container ① for incubation, a nucleic acid polymerase, a buffer solution, two other fluorescently labeled ddNTPs different from step (3) or two other ribose 3' -OH protected fluorescently labeled dNTPs different from step (3) are added into a container ② for incubation, and the obtained sequencing microspheres are marked as B1 in a container ②;

(10) washing and suspending the microspheres in container ① with buffer solution, wherein the remaining part of the microspheres is in container ①, the other part of the microspheres is transferred from container ① to a new container ③, adding nucleic acid polymerase, buffer solution and ribose 3' -OH protected dNTP to container ①, incubating, adding nucleic acid polymerase, buffer solution, two other fluorescently labeled ddNTPs different from step (5) or two other ribose 3' -OH protected fluorescently labeled dNTPs different from step (5) to container ③, incubating, and incubating, wherein the sequencing microspheres obtained in container ③ are labeled B2, and the ribose 3' -OH protected dNTPs are ribose 3' -OH protected dATP, ribose 3' -OH protected dTTP/dUTP, ribose 3' -OH protected dGTP and ribose 3' -OH protected dCTP;

(11) continuously repeating the steps (9) and (10) to obtain n groups of microspheres added with the fluorescence-labeled ddNTP or the fluorescence-labeled dNTP with protected ribose 3' -OH for incubation, wherein the last group of microspheres is marked as Bn, and n is the number of basic groups of the nucleic acid segment to be detected;

6. The method of claim 5, wherein: in steps (3) and (5), the 2 fluorescently labeled ddNTPs are fluorescently labeled ddATP and fluorescently labeled ddTTP/ddUTP, and the 2 ribose 3' -OH protected fluorescently labeled dNTPs are ribose 3' -OH protected fluorescently labeled dATP and ribose 3' -OH protected fluorescently labeled dTTP/dUTP; in steps (8) and (10), the fluorescently labeled ddNTP is fluorescently labeled ddGTP and fluorescently labeled ddCTP, and the ribose 3' -OH-protected fluorescently labeled dNTP is ribose 3' -OH-protected fluorescently labeled dGTP and ribose 3' -OH-protected fluorescently labeled dCTP.

7. The method according to any of claims 1-5, characterized by: the microspheres are microspheres with uniform size, size-coded microspheres, microspheres without fluorescence or fluorescence-coded microspheres;

preferably, the microspheres have a diameter of 0.5 to 50 μm, more preferably 1 to 10 μm;

preferably, the microspheres are modified with at least one of carboxyl, amino, hydroxyl, hydrazide, aldehyde, chloromethyl, oxirane, antibody, aptamer, streptavidin, poly T, poly a, poly G, poly C, poly U, and methylated CpG binding domain protein, dimethylamine, thiol, more preferably carboxylated microspheres.

8. The method according to any of claims 1-5, characterized by: the nucleic acid fragment to be detected is a single-stranded DNA fragment;

preferably, the nucleic acid polymerase is a DNA-dependent DNA polymerase;

preferably, the modified one-way primer is a one-way primer modified with at least one of amino group, carboxyl group, digoxin, biotin, poly a, poly G, poly C, poly T, poly U and methyl group, more preferably an amino group-modified one-way primer.

9. The method of claim 1, 2 or 5, wherein: the final concentration of microspheres in the incubation system was 5X 10 for each incubation²2.5X 10 pieces/ml⁸Nucleic acid polymerase at a final concentration of 2-480 units/ml, each fluorescently labeled ddNTP at a final concentration of 0.1-50. mu. mol/L, each ribose 3'-OH protected fluorescently labeled dNTP at a final concentration of 0.1-50. mu. mol/L, and each ribose 3' -OH protected dNTP at a final concentration of 0.1-50. mu. mol/L.

10. The method of claim 1 or 9, wherein: the incubation was carried out in the following containers: the wells of 24-well plate, 24-well filter plate, 96-well filter plate, 384-well filter plate, centrifuge tube or EP tube are the same or different in container used for each incubation;

preferably, the temperature of each incubation is 20-95 ℃, more preferably 32-70 ℃;

preferably, the total volume of the incubation system is 10. mu.l-10 ml, more preferably 10. mu.l-1000. mu.l, per incubation.