CN116536399A - Gene chip coding and decoding method based on microbead sequencing and gene chip - Google Patents

Abstract

The invention discloses a gene chip coding and decoding method based on microbead sequencing and a gene chip, wherein the method comprises the following steps: a) Providing a gene chip; the gene chip comprises: a microbead and an oligonucleotide chain covalently coupled to said microbead; each oligonucleotide strand consists of a tag sequence and a probe sequence from the 5 'end to the 3' end; the tag sequence is composed of a coding sequence and a primer binding sequence from a 5 'end to a 3' end; incubating the oligonucleotide chain modified microbeads in the gene chip with a reversible fluorescent terminator, a primer and a polymerase; and selecting a fluorescence channel for fluorescence photographing based on the reversible fluorescence terminator. The coding and decoding method has the advantages of strong specificity, low cost, less damage to the oligonucleotide chain and higher decoding efficiency.

Description

A gene chip encoding and decoding method and gene chip based on microbead sequencing

technical field

The invention relates to the technical field of gene chips, in particular to a microbead sequencing-based gene chip encoding and decoding method and the gene chip.

Background technique

The most mature structural form of gene chip technology is the microbead chip, which includes microbeads and probe molecules connected to the microbeads. The part of the probe molecule close to the microbeads is the tag sequence. The tag sequence is specific. The design of the tag sequence It is the code; after the microbeads connected with the probe molecules are loaded on the chip, due to the randomness of the loading process, it is necessary to adopt a certain method to obtain the information of the probe type corresponding to each microbead on the chip, that is, to obtain the information of each microbead on the chip. The tag sequence information of the beads, this process is called decoding.

The traditional gene chip encoding and decoding method uses the principle of complementary hybridization. Specifically, using the principle of complementary base pairing, the label sequence of the probe on the microbeads is hybridized with its complementary sequence (decoding sequence). Since the decoding sequence has a fluorescent group, the microbeads are equipped with corresponding fluorescence. The signal is obtained by fluorescent scanning, and then converted into the corresponding label sequence, so that the type of probe carried on the microbead can be known. Due to the limited color types of fluorophores, for high-density gene chips, multiple rounds of color development are required to form color codes for each microbead, and the label sequence on the microbeads is interpreted according to the color codes. Each microbead requires multiple bands. There are complementary hybridization probes with different fluorophores to decode, which is costly. At the same time, multiple rounds of hybridization and color development involve multiple DNA denaturation and renaturation processes, which will damage the oligonucleotide chains on the microbeads, which is not conducive to subsequent Riddle. In addition, when there are many types of encoded microbeads, a large number of tag sequences need to be generated, and repetitive sequences are prone to appear, resulting in poor DNA hybridization specificity and reduced accuracy.

In the prior art, some studies have proposed a hybridization method in which multiple tag sequences are sequentially hybridized, that is, a probe has multiple tag sequences, the first decoding experiment hybridizes with the first tag sequence, and the second decoding experiment hybridizes with the second tag sequence. Tag sequence hybridization, and so on, but this method still does not break away from the principle of complementary hybridization, it can only reduce the number of DNA denaturation and renaturation, but cannot fundamentally solve the problem.

Contents of the invention

The invention provides a gene chip encoding and decoding method to solve the shortcomings of high cost and large damage to oligonucleotide chains in the existing gene chip encoding and decoding method.

The gene chip encoding and decoding method provided by the present invention includes:

a) Provide a gene chip; the gene chip includes:

Microbeads modified with k kinds of oligonucleotide chains;

Each of said oligonucleotide strand-modified beads comprises a bead and an oligonucleotide strand covalently coupled to said bead;

The microbeads modified with k kinds of oligonucleotide chains include k kinds of oligonucleotide chains;

Each oligonucleotide chain is composed of a tag sequence and a probe sequence from the 5' end to the 3' end;

The tag sequence consists of a coding sequence and a primer binding sequence from the 5' end to the 3' end;

The probe sequences in the k oligonucleotide chains are different from each other;

The coding sequences in the tag sequences in the k oligonucleotide chains are different from each other, where k≥1;

The tag sequence meets the following conditions:

The number of consecutive identical bases contained does not exceed 3, the GC content is 30%-60%, the length of the hairpin structure does not exceed 4 bases, and the self-complementary fragments between the tag sequences do not exceed 6 bases, and the similarity between the tag sequence and the target genome is less than 0.05;

b) incubating microbeads modified with k types of oligonucleotide chains in the gene chip together with m types of reversible fluorescent terminators, primers and polymerases, wherein m is an integer between 2 and 4;

c) based on the reversible fluorescent terminator, select a fluorescent channel to take a fluorescent photo, and then determine whether the coding sequence in the k oligonucleotide chains can be determined;

d) If not, incubate the reducing agent with the microbeads modified with the k kinds of oligonucleotide chains after fluorescent photography, and then repeat steps b) and c) until the k kinds of oligonucleotide chains can be determined The coding sequence in .

In one embodiment, the length of the primer binding sequence is 16-23 mer.

In one embodiment, the coding sequence is 1-n in length, where m ⁿ >K.

In one embodiment, the reducing agent is any of tris(2-formylethyl)phosphine hydrochloride, dithiothreitol, dithioerythritol, glutathione, and β-mercaptoethanol. A sort of.

In one embodiment, the oligonucleotide chains are amino-terminal modified oligonucleotide chains; the microbeads are carboxyl-modified polystyrene microbeads or silica microbeads.

The present invention also provides a gene chip, the gene chip comprises microbeads modified by k kinds of oligonucleotide chains; each of the microbeads modified by oligonucleotide chains comprises microbeads and the oligonucleotide chains that are valence-coupled; the microbeads modified by the k oligonucleotide chains include k oligonucleotide chains; each oligonucleotide chain is labeled from the 5' end to the 3' end sequence and probe sequence; the tag sequence is composed of coding sequence and primer binding sequence from the 5' end to the 3' end; the probe sequences in the k oligonucleotide chains are different from each other; the k oligonucleotides The coding sequences in the tag sequence in the nucleotide chain are different from each other; wherein, k≥1; the tag sequence meets the following conditions: the number of consecutive identical bases contained is not more than 3, and the GC content 30%-60%, the length of the hairpin structure does not exceed 4 bases, the self-complementary segment between the tag sequences does not exceed 6 bases, and the similarity between the tag sequence and the target genome is low at 0.05.

The encoding and decoding method of the gene chip of the present invention uses a reversible fluorescent terminator to read, and compared with traditional fluorescent probe decoding, it has stronger specificity and lower cost; decoding does not involve the denaturation operation of oligonucleotide chains, The oligonucleotide chain is less damaged, and the decoding efficiency is higher, which is more conducive to subsequent analysis and detection experiments.

Description of drawings

Fig. 1 shows a schematic diagram of the principle of the encoding and decoding method of the gene chip of the present invention.

FIG. 2 shows the fluorescent image of the decoded oligonucleotide chain-modified microbeads according to Example 1 of the present invention.

FIG. 3 shows the fluorescent image of the decoded gene chip according to Example 2 of the present invention.

Fig. 4 shows the fluorescent image of the decoded gene chip according to embodiment 3 of the present invention.

Detailed ways

The following examples are intended to illustrate the present invention without further limiting the invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of the present application, and are not intended to limit the present application.

The first aspect of the present invention provides a codec method for a gene chip, the codec method comprising:

a) A gene chip is provided, the gene chip includes microbeads modified with k kinds of oligonucleotide chains; each of the microbeads modified with oligonucleotide chains includes microbeads and is covalently coupled to the microbeads The oligonucleotide chains; the microbeads modified by the k oligonucleotide chains include k oligonucleotide chains; each oligonucleotide chain is composed of a tag sequence and a tag sequence from the 5' end to the 3' end The probe sequence is composed; the tag sequence is composed of a coding sequence and a primer binding sequence from the 5' end to the 3' end; the probe sequences in the k oligonucleotide chains are different from each other; the k oligonucleotides The coding sequences in the tag sequence in the acid chain are different from each other; wherein, k≥1; the tag sequence meets the following conditions: the number of consecutive identical bases contained is not more than 3, and the GC content is 30 %-60%, the length of the hairpin structure does not exceed 4 bases, the self-complementary segment between the tag sequences does not exceed 6 bases, and the similarity between the tag sequence and the target genome is less than 0.05 (compared with BLAST);

b) incubating microbeads modified with k types of oligonucleotide chains in the gene chip together with m types of reversible fluorescent terminators, primers and polymerases, wherein m is an integer between 2 and 4;

c) based on the reversible fluorescent terminator, select a fluorescent channel to take a fluorescent photo, and then determine whether the coding sequence in the k oligonucleotide chains can be determined;

d) If not, incubate the reducing agent with the microbeads modified with the k kinds of oligonucleotide chains after fluorescent photography, and then repeat steps b) and c) until the k kinds of oligonucleotide chains can be determined The coding sequence in .

Fig. 1 shows a schematic diagram of the principle of the encoding and decoding method of the gene chip of the present invention.

The encoding and decoding method of the gene chip provided by the first aspect of the present invention uses a reversible fluorescent terminator to read, and compared with traditional fluorescent probe decoding, it has stronger specificity and lower cost; decoding does not involve oligonucleotide chains The denaturation operation has less damage to the oligonucleotide chain, higher decoding efficiency, and is more conducive to subsequent analysis and detection experiments.

In one embodiment, the length of the primer binding sequence is 16-23 mer. If the primer binding sequence is too short, the primer binding efficiency will be low, and if it is too long, it will easily cause non-specific binding.

In one embodiment, the coding sequence is 1-n in length, where m ⁿ >K. The coding sequence uses a single nucleotide as a coding unit, and compared with the traditional tag sequence coding, the amount of information that can be encoded is larger.

In one embodiment, the reducing agent is tris(2-formylethyl)phosphine hydrochloride, dithiothreitol, dithioerythritol, glutathione, β-mercaptoethanol Any of them is preferably tris(2-formylethyl)phosphine hydrochloride.

In one embodiment, the concentration of the reducing agent is 50-250 mM. In one embodiment, the reducing agent is incubated with the microbeads modified with the k kinds of oligonucleotide chains after fluorescence photography for 15-30 min. If the reducing agent concentration is too low or the incubation time is too short, the reduction effect will not be good; if the reducing agent concentration is too high or the incubation time is too long, other side reactions may occur and unnecessary waste will be caused.

In one embodiment, the reversible fluorescent terminator is a nucleotide with a chemically cleavable azido group at the 3' hydroxyl end and a fluorescent group that can be cleaved by a reducing agent on the base. There are 2 to 4 types of reversible fluorescent terminators according to the different fluorescent groups.

In one embodiment, the oligonucleotide chain is an amino-terminally modified oligonucleotide chain. In one embodiment, the microbeads are carboxyl-modified polystyrene microbeads or silica microbeads. In one embodiment, the covalent coupling is by reacting an amino group with a carboxyl group to form an amide bond.

The second aspect of the present invention provides a kind of gene chip, and this gene chip comprises the microbead of k kinds of oligonucleotide chain modification; The microbead of each described oligonucleotide chain comprises microbead and described microbead Bead covalently coupled oligonucleotide chains; said k oligonucleotide chain modified microbeads comprise k oligonucleotide chains; each oligonucleotide chain is from said 5' end to 3' end It consists of a tag sequence and a probe sequence; the tag sequence is composed of a coding sequence and a primer binding sequence from the 5' end to the 3' end; the probe sequences in the k oligonucleotide chains are different from each other; the k The coding sequences in the tag sequences in the oligonucleotide chains are different from each other; wherein, k≥1; the tag sequences meet the following conditions: the number of consecutive identical bases contained is not more than 3, The GC content is 30%-60%, the length of the hairpin structure does not exceed 4 bases, the self-complementary fragment between the tag sequences does not exceed 6 bases, and the similarity between the tag sequence and the target genome The sex is lower than 0.05 (compared with BLAST).

Example 1.

Preparation of Polystyrene Microspheres Modified by Oligonucleotide Chains

Carboxyl-modified polystyrene microspheres (5.5 μm, Jiangsu Xianfeng Nano Material Technology Co., Ltd., number: XFB13, product number: 104300) were prepared with 4-morpholineethanesulfonic acid buffer (MES, pH = 6) into 5 mg/mL suspension, use MES to prepare 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution, make the system The concentration of EDC and NHS was 25 mg/mL, and the reaction was shaken at room temperature for 1 h.

Dissolve 10 nmol of the dry powder of the oligonucleotide chain to be connected with MES buffer, and then mix it with the obtained suspension of activated polystyrene microspheres, in which the content of polystyrene microspheres is 5 mg. After shaking at room temperature for 4 hours , centrifuged (5000rpm, 5 min), washed three times with PBST, and then resuspended with ultrapure water.

The sequence (5'-3') of the oligonucleotide chain is:

_NH2 -GTCACTACGCTATCAGTGCCACGCCTTGTTCCTTTCCTATGTTGTTGTCTTGC;

Wherein, the primer binding sequence is CTACGCTATCAGTGCCAC; the coding sequence is GTCA.

decoding

a. Configure decoding solution: Sequenase version 2.0 DNA polymerase working solution [200 mM Tris-HCl (pH7.5), 100 mM MgCl ₂ , 250 mM NaCl], 0.5 μM reversible fluorescent terminator (AF532-dATP, Cy5-dGTP, Cy5 .5-dCTP, ROX-dTTP), 0.1 µM primer (GTGGCACTGATAGCGTAG).

b. Add Sequenase version 2.0 DNA polymerase (0.05 U/μL) to the decoding solution, incubate with microbeads, incubate at 37°C for 1 minute, centrifuge (10000 rpm, 3 min), wash 3 times with PBST, and fluorescent microscope ROX, Cy5, AF532, and Cy5.5 channels were photographed and recorded sequentially, as shown in the first round of Figure 2.

c. Co-incubate with tris(2-formylethyl)phosphine hydrochloride (TCEP, 100 mM) and microbeads after fluorescence photography, centrifuge (10000 rpm, 3 min), and wash with PBST for 3 to 5 times.

d. Repeat steps b and c three times. The resulting fluorescence images are shown in the second, third and fourth rounds of Figure 2.

As shown in Figure 2, ROX-dTTP, Cy5-dGTP, AF532-dATP, and Cy5.5-dCTP were successfully displayed in the four rounds of color development, and the remaining three channels in each round of color development did not show fluorescence, so it can be proved that The encoding and decoding method of the gene chip of the present invention is feasible and has good specificity.

Example 2.

Preparation of gene chips

Referring to Example 1, four kinds of oligonucleotide chain-modified microbeads were prepared, and the oligonucleotide sequences (5′-3′) coupled to the microspheres were as follows:

Oligonucleotide strand 1:

ACCTACGCTATCAGTGCCACGCCTTGTTCCTTTCCTATGTTGTTGTCTTGC

Oligonucleotide strand 2:

CCCTACGCTATCAGTGCCACACTTGTTTGTCTGGAGGCCTATGCTTAAAT

Oligonucleotide strand 3:

CACTACGCTATCAGTGCCACCGGCGTTCGCGGAGGATCCGCAGGGCGCG

Oligonucleotide strand 4:

AACTACGCTATCAGTGCCACAATTCTCAACAGGGCACTTAAAAATCTTCC

Wherein, the primer binding sequence is CTACGCTATCAGTGCCAC; the coding sequence: probe 1 is AC, probe 2 is CC, probe 3 is CA, probe 4 is AA.

Forming a groove array area on the first surface of the silicon substrate (10mm×10mm×1mm) by photolithography;

Inject the microbeads of polystyrene covalently bound to oligonucleotide chains into ultrapure water, and prepare a monodisperse microbead solution with a concentration of 1 mg/mL through ultrasonic dispersion; put the etched silicon substrate into the assembly In the mounting groove (10mm×10mm×2mm) of the mold (75mm×25mm×3mm), 150 μL of monodisperse microbead solution was evenly dropped on the first surface of the silicon substrate, and the glass slide was covered on the assembled mold , and make sure that there is no air in the liquid under the glass slide, wrap it with parafilm and seal it, put it face up into the ultrasonic cleaner, use 120 W ultrasonic assembly for 10 min, and clean the silicon with deionized water Twice on the surface of the substrate to complete the assembly of microbeads;

Place the silicon substrate assembled with microbeads face up, put it into a microplate centrifuge, centrifuge at the speed of the centrifuge at 2000rpm for 5 minutes, wash the surface of the silicon substrate with deionized water three times, and complete the sedimentation of the microbeads, and prepare Get a gene chip.

decoding

a. Configure decoding solution: Sequenase version 2.0 DNA polymerase working solution [200 mM Tris-HCl (pH 7.5), 100 mM MgCl ₂ , 250 mM NaCl], 0.5 μM reversible fluorescent terminator (Cy5-dGTP, ROX-dTTP ), 0.1 µM primer (GTGGCACTGATAGCGTAG).

b. Add Sequenase version 2.0 DNA polymerase (0.05 U/μL) to the decoding solution, drop it on the bead chip, incubate at 37°C for 1 minute, wash with PBST for 3 times, and take pictures in turn on the ROX and Cy5 channels of the fluorescence microscope , as shown in the first round of Figure 3.

c. Add tris(2-formylethyl)phosphine hydrochloride (TCEP, 250 mM) dropwise on the gene chip after fluorescence photography, incubate at room temperature, and wash with PBST for 3 to 5 times.

d. Repeat step b. The resulting fluorescence images are shown in the second round of Figure 3.

In this experiment, two kinds of reversible fluorescent terminators encode four kinds of probe microspheres, that is, two colors determine the four probe sequences, and each base sequence is divided into two parts according to the color of the microbeads. 2 ² =4, the type of microbead can be confirmed by two experiments, that is, the type of probe connected to the microbead can be known. As shown in Figure 3, each round of color development can successfully divide the microbead into two parts without overlapping. Good color rendering effect and high specificity.

Example 3.

Preparation of gene chips

Referring to Example 1, nine kinds of microbeads modified with oligonucleotide chains were prepared, and the oligonucleotide sequences (5′-3′) coupled to the microspheres were as follows:

Oligonucleotide strand 1:

ACCTACGCTATCAGTGCCACGCCTTGTTCCTTTCCTATGTTGTTGTCTTGC

Oligonucleotide strand 2:

CCCTACGCTATCAGTGCCACACTTGTTTGTCTGGAGGCCTATGCTTAAAT

Oligonucleotide strand 3:

TCCTACGCTATCAGTGCCACCGGCGTTCGCGGAGGATCCGCAGGGCGCG

Oligonucleotide strand 4:

CACTACGCTATCAGTGCCACAATTCTCAACAGGGCACTTAAAAATCTTCC

Oligonucleotide strand 5:

TACTACGCTATCAGTGCCACCAATTTTTGTATTTTCCTCATCAAGGTTGCG

Oligonucleotide strand 6:

AACTACGCTATCAGTGCCACGTGTTCGGGGAAGCTTGATCTTAAAAGGAG

Oligonucleotide strand 7:

CTCTACGCTATCAGTGCCACGTTAAGTGAATTTGGAGAGGATCATCAGTA

Oligonucleotide strand 8:

ATCTACGCTATCAGTGCCACAATGATAAAGCGCATAGCAGTTCATCCCGA

Oligonucleotide strand 9:

TTCTACGCTATCAGTGCCACGTTTGAAGAATTTTGTAGAAGCGAAGGCAT

Among them, the primer binding sequence is CTACGCTATCAGTGCCAC, and the coding sequence: probe 1 is AC, probe 2 is CC, probe 3 is TC, probe 4 is CA, probe 5 is TA, probe 6 is AA, probe 7 is CT, probe 8 is AT, and probe 9 is TT.

Forming a groove array area on the first surface of the silicon substrate (10mm×10mm×1mm) by photolithography;

Inject the microbeads of polystyrene covalently bound to oligonucleotide chains into ultrapure water, and prepare a monodisperse microbead solution with a concentration of 1 mg/mL through ultrasonic dispersion; put the etched silicon substrate into the assembly In the mounting groove (10mm×10mm×2mm) of the mold (75mm×25mm×3mm), 150 μL of monodisperse microbead solution was evenly dropped on the first surface of the silicon substrate, and the assembled mold was covered with a glass slide After making sure that there is no air in the liquid under the slide glass, wrap it with a parafilm and seal it, put it face up into an ultrasonic cleaner, use 120 W ultrasonic assembly for 10 min, and clean it with deionized water Silicon substrate surface twice to complete the assembly of microbeads;

Place the silicon substrate assembled with microbeads face up, put it into a microplate centrifuge, centrifuge at the speed of the centrifuge at 2000rpm for 5 minutes, wash the surface of the silicon substrate with deionized water three times, and complete the sedimentation of the microbeads, and prepare Get a gene chip.

decoding

a. Configure decoding solution: Sequenase version 2.0 DNA polymerase working solution [200 mM Tris-HCl (pH 7.5), 100 mM MgCl ₂ , 250 mM NaCl], 0.5 μM reversible fluorescent terminator (Cy5-dGTP, ROX-dTTP, AF532 -dATP), 0.1 µM primer (GTGGCACTGATAGCGTAG).

b. Add Sequenase version 2.0 DNA polymerase (0.05 U/μL) to the decoding solution, drop it on the microbead chip, incubate at 37°C for 1 minute, wash with PBST three times, and use the AF532, ROX, and Cy5 channels of the fluorescence microscope in sequence Take pictures and record, as shown in the first round of Figure 4.

c. Add tris(2-formylethyl)phosphine hydrochloride (TCEP, 250 mM) dropwise on the gene chip after fluorescence photography, incubate at room temperature, and wash with PBST for 3 to 5 times.

d. Repeat step b. The resulting fluorescence image is shown in the second round of Figure 4.

In this embodiment, three kinds of reversible fluorescent terminators encode nine kinds of probe microspheres, that is, nine kinds of probe sequences are determined by three colors, and the microbeads are divided into three parts according to the color of the microbeads for each base sequence. According to 3 ² =9, the type of microbead can be confirmed by two experiments, that is, the type of probe connected to the microbead can be known. As shown in Figure 4, each round of color development can successfully divide the microbead into three parts without overlapping , good color rendering effect and high specificity.

Claims (10)

1. a codec method of gene chip, is characterized in that, described codec method comprises: a) Provide a gene chip; the gene chip includes: Microbeads modified with k kinds of oligonucleotide chains; Each of said oligonucleotide strand-modified beads comprises a bead and an oligonucleotide strand covalently coupled to said bead; The microbeads modified with k kinds of oligonucleotide chains include k kinds of oligonucleotide chains; Each oligonucleotide chain is composed of a tag sequence and a probe sequence from the 5' end to the 3' end; The tag sequence consists of a coding sequence and a primer binding sequence from the 5' end to the 3' end; The probe sequences in the k oligonucleotide chains are different from each other; The coding sequences in the tag sequences in the k oligonucleotide chains are different from each other, Among them, k≥1; The tag sequence meets the following conditions: The number of consecutive identical bases contained does not exceed 3, the GC content is 30%-60%, the length of the hairpin structure does not exceed 4 bases, and the self-complementary fragment between the tag sequences does not exceed 6 bases base, and the similarity between the tag sequence and the target genome is less than 0.05; b) incubating microbeads modified with k types of oligonucleotide chains in the gene chip together with m types of reversible fluorescent terminators, primers and polymerases, wherein m is an integer between 2 and 4; c) based on the reversible fluorescent terminator, select a fluorescent channel to take a fluorescent photo, and then determine whether the coding sequence in the k oligonucleotide chains can be determined; d) If not, incubate the reducing agent with the microbeads modified with the k kinds of oligonucleotide chains after fluorescent photography, and then repeat steps b) and c) until the k kinds of oligonucleotide chains are determined The coding sequence of . 2. The encoding and decoding method according to claim 1, wherein the length of the primer binding sequence is 16-23mer. 3. The encoding and decoding method according to claim 1, wherein the encoding sequence has a length of 1-n, wherein m ⁿ ≥ K. 4. The encoding and decoding method according to claim 1, wherein the reducing agent is tris(2-formylethyl)phosphine hydrochloride, dithiothreitol, dithioerythritol, gluten Either of glutathione and β-mercaptoethanol. 5. The encoding and decoding method according to claim 1, wherein the oligonucleotide chain is an oligonucleotide chain modified by terminal amino groups; the microbead is a carboxyl-modified polystyrene microbead or two Silica microbeads. 6. A gene chip, comprising microbeads modified by k kinds of oligonucleotide chains; Each of said oligonucleotide strand-modified beads comprises a bead and an oligonucleotide strand covalently coupled to said bead; The microbeads modified with k kinds of oligonucleotide chains include k kinds of oligonucleotide chains; Each oligonucleotide chain is composed of a tag sequence and a probe sequence from the 5' end to the 3' end; The tag sequence is composed of a coding sequence and a primer binding sequence from the 5' end to the 3' end; The probe sequences in the k oligonucleotide chains are different from each other; The coding sequences in the tag sequences in the k oligonucleotide chains are different from each other; Among them, k≥1; The tag sequence meets the following conditions: the number of consecutive identical bases contained does not exceed 3, the GC content is 30%-60%, the length of the hairpin structure does not exceed 4 bases, and the number of consecutive identical bases between the tag sequences The self-complementary fragment does not exceed 6 bases, and the similarity between the tag sequence and the target genome is lower than 0.05. 7. The gene chip according to claim 6, wherein the length of the primer binding sequence is 16-23mer. 8 . The gene chip according to claim 6 , wherein the length of the coding sequence is 1-n, wherein, m ⁿ ≥ K. 9 . 9. The gene chip according to claim 6, characterized in that, the oligonucleotide chain is an oligonucleotide chain modified with terminal amino groups. 10. The gene chip according to claim 9, characterized in that, the microbeads are carboxy-modified polystyrene microbeads or silica microbeads.