CN114410764A - Preparation method of long DNA sequence silicon dioxide microbeads - Google Patents
Preparation method of long DNA sequence silicon dioxide microbeads Download PDFInfo
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- CN114410764A CN114410764A CN202210137482.XA CN202210137482A CN114410764A CN 114410764 A CN114410764 A CN 114410764A CN 202210137482 A CN202210137482 A CN 202210137482A CN 114410764 A CN114410764 A CN 114410764A
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B33/00—Silicon; Compounds thereof
- C01B33/113—Silicon oxides; Hydrates thereof
- C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
Abstract
The invention discloses a preparation method of a long DNA sequence silicon dioxide microbead. The method takes silicon dioxide as a carrier, adopts a chemical reagent to activate a surface carboxyl functional group, and can be covalently connected with an amino-modified oligonucleotide sequence. Meanwhile, the subsequent sequence can be introduced (sequence extension is carried out) by using the mode of combining complementary sequence annealing and polymerization reaction. The method can be used for obtaining the silicon dioxide micro-beads with various long sequence codes. Compared with the traditional method, the method has the advantages that the modified oligonucleotide sequences on the silicon dioxide microbeads are more, and the contained biological information is more comprehensive.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of long DNA sequence silicon dioxide microbeads.
Background
Nucleic acids, including deoxyribonucleic acid and ribonucleic acid, are one of the most basic substances of life, and play a vital role in various vital activities such as growth, development, mutation, inflammation, cancer and the like of organisms. The nucleic acid molecules have a close relationship with the occurrence and development of various diseases affecting human health and play an important role. Therefore, the development of an accurate and effective method for sensitively and accurately detecting the nucleic acid has very important significance for deeply exploring the aspects of function regulation and control of the nucleic acid, drug screening, early detection of related diseases, clinical diagnosis and treatment, prognosis evaluation and the like.
The silicon dioxide is a typical inorganic powder material, and has large specific surface area and good chemical stability. The monodisperse silicon dioxide micro-beads have simple preparation process and good biocompatibility, so the silicon dioxide micro-beads with different sizes and different surface modifications can be applied to different fields of information, biology, medicine and the like.
The traditional sequencing method may cause important information omission due to the limitation of action mechanism, however, nucleic acid contains much biological information and has large data volume, so that the method adopts the oligonucleotide chain which is modified on the matrix and is encoded by a long sequence, is further used for DNA sequencing and has important significance for the acquisition of nucleic acid information.
Disclosure of Invention
The invention aims to provide a preparation method of long DNA sequence silicon dioxide microbeads.
In order to achieve the purpose of the invention, the preparation method of the long DNA sequence silicon dioxide microbeads provided by the invention comprises the following steps:
(1) designing four groups of primers which are respectively a primer 1, a primer 2, a primer 3 and a UMI primer: the primers are all oligonucleotide single strands, the sequence lengths of the primer 1, the primer 2 and the primer 3 are consistent, the GC content is 45-55%, and the Tm values of the primers are close to each other;
primer 1 (first round primer) has amino modification at 5 end, and comprises READ1 sequence, barcode 1 sequence and linker 1 sequence of the illiminia sequencing platform from 5 'to 3' (other three rounds of primers synthesize an integral sequence by reverse complementary polymerization); primer 2 (second round primer) comprises a linker 1 reverse complementary sequence, a barcode 2 sequence and a linker2 sequence from 5 'to 3'; primer 3 (third round primer) comprises a linker2 reverse complementary sequence, a barcode 3 sequence and a linker3 sequence from 5 'to 3'; the UMI primer (fourth round primer) comprises a linker3 reverse complementary sequence, a UMI sequence (random sequence) and a polyA sequence from 5 'to 3'; wherein, barcode 1, barcode 2 and barcode 3 are different barcode sequences, and the length of polyA is 20-35 nt;
when designing the primer, on one hand, the principle of reverse complementary synthesis is considered, and on the other hand, the quality inspection difference of various barcode sequences is required to be larger, and the sequences are not the same as the sequences on the genome;
(2) activation of carboxylated silicon dioxide microbeads: placing the carboxylated silicon dioxide microspheres in a mixed solution of EDC and NHS for activation;
(3) and (3) connection reaction: putting the activated microbeads into a primer 1 solution, and carrying out condensation reaction to obtain silicon dioxide microbeads with different sequences;
wherein the amino-modified oligonucleotide strand comprises barcode 1 and linker 1;
(4) synthesis of long DNA sequences: mixing the microbeads obtained in the step (3), the primer 2 and a polymerization reaction reagent, carrying out DNA chain extension reaction, and then removing a reverse complementary sequence with the linker 1; mixing the obtained microbeads with a primer 3 and a polymerization reaction reagent, and further carrying out DNA chain extension reaction; and finally, connecting the microbeads with the UMI sequence to obtain the silicon dioxide microbeads with the long DNA sequences.
The step (2) comprises the following steps: centrifuging 50mg/mL carboxylated silicon dioxide micro-beads, mixing the precipitate with 20-100 mu L of mixed solution of EDC and NHS, and carrying out shaking reaction at the room temperature of 1500-.
The preparation method of the mixed solution of EDC and NHS comprises the following steps: dissolving 1.09mg EDC and 0.65mg NHS in 0.1M MES 100 ul.
The step (3) comprises the following steps: uniformly mixing the activated microbeads with 2.5 mu L of 50 mu M amino modified oligonucleotide chain solution, and carrying out oscillation reaction at room temperature of 2000rpm for overnight; after the reaction is finished, the microbeads are centrifugally collected and are used for subsequent synthesis reaction after being cleaned.
The bead cleaning comprises the following steps: the microbeads were placed in 0.1M PBS containing 0.02% Tween 20, collected by centrifugation, and then washed twice with TE buffer of pH 8.0.
The polymerization reagent of step (4) comprises: dNTPs, Klenow enzyme and Klenow enzyme reaction buffer.
The reaction system used for carrying out DNA chain extension is as follows: mu.L of 50. mu.M primer 21. mu.L, 5. mu.L of 10 XKlenow enzyme reaction buffer, 4. mu.L of 2.5mM dNTPs, 1. mu.L of Klenow enzyme 5U/. mu.L.
The reaction conditions are as follows: the reaction is carried out at 37 ℃ and 2000rpm for 30min-1h with shaking.
The reaction system used for connecting the microbeads and the UMI sequence in the step (4) is as follows: mu.M UMI primer, 5. mu.L of 10 XKlenow enzyme reaction buffer, 4. mu.L of 2.5mM dNTPs and 1. mu.L of Klenow enzyme 5U/. mu.L.
The reaction conditions are as follows: oscillating reaction at 37 deg.C and 2000rpm for 0.5-1h
Preferably, the nucleotide sequence of the primer 1 is shown as SEQ ID NO. 1-5, the nucleotide sequence of the primer 2 is shown as SEQ ID NO. 6-10, the nucleotide sequence of the primer 3 is shown as SEQ ID NO. 11-15, and the nucleotide sequence of the UMI primer is shown as SEQ ID NO. 16.
The carboxylated silicon dioxide microbeads used in the invention are purchased from Shanghai Carbox phenanthrene biomedical science and technology limited, and can also be prepared according to a conventional method.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention provides a method for synthesizing long-sequence coded carboxylated silicon dioxide, which takes silicon dioxide as a carrier, adopts a chemical reagent to activate a carboxyl functional group on the surface, and can be covalently connected with an amino-modified oligonucleotide sequence. Meanwhile, the subsequent sequence can be introduced (sequence extension is carried out) by using the mode of combining complementary sequence annealing and polymerization reaction. The method can be used for obtaining the silicon dioxide micro-beads with various long sequence codes. Compared with the traditional method, the method has the advantages that the modified oligonucleotide sequences on the silicon dioxide microbeads are more, and the contained biological information is more comprehensive.
(II) the activation efficiency is high: EDC and NHS can fully activate the carboxyl structure modified on the surface of the silicon dioxide micro-bead.
(III) the connection efficiency is high: the condensation reaction can effectively connect the carboxyl microbeads with the amino oligonucleotide chains.
(IV) the operation success rate is high: the operation process is simple, the used reagent is less, and the synthesis efficiency is ensured to the maximum extent.
Drawings
FIG. 1 is a schematic diagram of the structure of a long-sequence coded silica microbead synthesized in the preferred embodiment of the present invention.
Detailed Description
The invention provides a synthesis method of long-sequence coded silicon dioxide microbeads. The method adopts a carboxylation reaction to connect the carboxylated silicon dioxide and a sequence with amino, then adds a complementary sequence of a subsequent barcode sequence, and utilizes primer annealing and a polymerization reaction of polymerase to obtain the silicon dioxide micro-beads with long sequence codes, thereby further applying the obtained micro-beads to the decoding of a DNA chip.
The invention adopts the following technical scheme:
1. designing a primer: designing a coding primer sequence.
2. Activation of carboxylated silicon dioxide microbeads: and activating the silicon dioxide micro-beads with carboxyl by using EDC and NHS to obtain the silicon dioxide micro-beads with good dispersity and exposed carboxyl on the surface.
3. And (3) connection reaction: and (3) connecting the carboxylated silicon dioxide microbeads and the oligonucleotide chains with the amino groups modified at the tail ends through a carboxyl reaction to obtain the silicon dioxide microbeads with different sequences.
4. Synthesizing a barcode sequence: and (3) sequentially connecting the later sequences to the silicon dioxide microspheres obtained in the step (3) through polymerization reaction, and finally obtaining the long-sequence coded silicon dioxide microspheres.
The terms referred to in the present invention:
EDC: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
NHS: n-hydroxysuccinimide.
MES: 2-morpholinoethanesulfonic acid.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning Manual of experiments, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or conditions as recommended by the manufacturer's instructions.
Example 1 Synthesis of Long sequence encoded silica Microbeads
The synthesis method of the long-sequence encoded silica microbeads provided by the embodiment comprises the following steps:
1. activated carboxy silica
Accurately weighing 1.09mg of EDC and 0.65mg of NHS, preparing 0.1M MES, and dissolving the weighed EDC and NHS by 100ul MES to obtain mixed solution of EDC and NHS; meanwhile, 100 mu L of carboxylated silicon dioxide micro-beads (50mg/mL) are taken and washed twice by prepared MES solution, and then the mixed solution of EDC and NHS is added into the micro-beads, and the final reaction volume is 100 mu L; the beads were then left at room temperature and the reaction was carried out for 30min with shaking of the metal bath (2000 rpm).
2. Connecting barcode 1 amino modified oligonucleotide chain
After the reaction of the beads, the mixture was divided into 5 tubes, 2.5. mu.L of amino-modified oligonucleotide (dissolved in 0.1M MES at a final concentration of 50. mu.M) was added to each tube, and the mixture was shaken and stirred at room temperature (2000rpm) to react overnight. After completion of the reaction, the beads were collected into 1mL of 0.1M PBS containing 0.02% Tween 20, centrifuged, the supernatant was carefully removed, and then the beads were washed twice in 1mL of TE buffer (pH 8.0).
3. barcode 2 connection
The beads were washed with water and divided equally into 5 tubes, then 1. mu.L of BC2 (dissolved in TE buffer at pH 8.0, final concentration 50. mu.M), 5. mu.L of 10 XKlenow enzyme reaction buffer, final total volume 45. mu.L, were added. The oligonucleotide sequence in each tube was: the following program was run on a metal bath (2000rpm) with linker 1 reverse complement, a unique barcode and linker2 sequence:
after the program run was completed, 4. mu.L of 2.5mM dNTPs, 1. mu.L of 5U/. mu.L Klenow enzyme, 37 ℃ and reaction at 2000rpm were added to each tube for 1 hour.
After the reaction, the microbeads were collected into 0.1M PBS containing 0.02% Tween 20, centrifuged, the supernatant was carefully removed, then the microbeads were washed twice in TE buffer (pH 8.0), 0.1M NaOH was added and left for 2min to remove the sequence complementary to the reverse side of linker2, the supernatant was rapidly removed after centrifugation, and the procedure was repeated twice.
4. barcode 3 and UMI connections
The ligation step for barcode 3 was identical to barcode 2, where the added oligonucleotide sequence was: a sequence complementary to the reverse direction of linker2, a unique barcode and a linker3 sequence; after finishing connection and cleaning of barcode 3, adding 2 muL of UMI for the same treatment, wherein the sequence of the added UMI is as follows: the reverse complement of linker3, a unique barcode, a UMI sequence and a poly-T tail. The barcode 2 ligation step was repeated. The microbeads were stored in TE-TW solution (10mM Tris pH 8.0; 1mM EDTA, 0.01% Tween 20) and stored at 4 ℃.
The experimental results are shown in figure 1, and a plurality of long-sequence coded silica microbeads can be obtained.
The total number of the silica microspheres is 384 multiplied by 384 (the number of the finally synthesized microspheres is more than fifty million), and the silica microspheres can be used for preparing subsequent biochips.
The sequences referred to in this example are shown in Table 1(SEQ ID NOS: 1-16).
TABLE 1
Note: n, B is a degenerate base, N represents A, T, C or G, and B represents G, T or C.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (8)
1. The preparation method of the long DNA sequence silicon dioxide microbeads is characterized by comprising the following steps:
(1) designing four groups of primers which are respectively a primer 1, a primer 2, a primer 3 and a UMI primer: the primers are all oligonucleotide single strands, the sequence lengths of the primer 1, the primer 2 and the primer 3 are consistent, the GC content is 45-55%, and the Tm values of the primers are close to each other;
the 15 ends of the primers are modified by amino groups, and the primers comprise a READ1 sequence, a barcode 1 sequence and a linker 1 sequence of an illumina sequencing platform from 5 'to 3'; the primer 2 comprises a linker 1 reverse complementary sequence, a barcode 2 sequence and a linker2 sequence from 5 'to 3'; primer 3 comprises a linker2 reverse complementary sequence, a barcode 3 sequence and a linker3 sequence from 5 'to 3'; the UMI primer comprises a linker3 reverse complementary sequence, a UMI sequence (random sequence) and a polyA sequence from 5 'to 3'; wherein, barcode 1, barcode 2 and barcode 3 are different barcode sequences, and the length of polyA is 20-35 nt;
(2) activation of carboxylated silicon dioxide microbeads: placing the carboxylated silicon dioxide microspheres in a mixed solution of EDC and NHS for activation;
(3) and (3) connection reaction: putting the activated microbeads into a primer 1 solution, and carrying out condensation reaction to obtain silicon dioxide microbeads with different sequences;
(4) synthesis of long DNA sequences: mixing the microbeads obtained in the step (3), the primer 2 and a polymerization reaction reagent, carrying out DNA chain extension reaction, and then removing a reverse complementary sequence with the linker 1; mixing the obtained microbeads with a primer 3 and a polymerization reaction reagent, and further carrying out DNA chain extension reaction; and finally, connecting the microbeads with the UMI sequence to obtain the silicon dioxide microbeads with the long DNA sequences.
2. The method of claim 1, wherein step (2) comprises: centrifuging 50mg/mL carboxylated silicon dioxide microbeads, mixing the precipitate with 20-100 mu L of mixed solution of EDC and NHS, and carrying out oscillation reaction at the room temperature of 1500-;
the preparation method of the mixed solution of EDC and NHS comprises the following steps: dissolving 1.09mg EDC and 0.65mg NHS in 0.1M MES 100 ul.
3. The method of claim 2, wherein step (3) comprises: uniformly mixing the activated microbeads with 2.5 mu L of 50 mu M amino modified oligonucleotide chain solution, and carrying out oscillation reaction at room temperature of 2000rpm for overnight; after the reaction is finished, the microbeads are centrifugally collected and are used for subsequent synthesis reaction after being cleaned.
4. The method of claim 3, wherein bead washing comprises: the microbeads were placed in 0.1M PBS containing 0.02% Tween 20, collected by centrifugation, and then washed twice with TE buffer of pH 8.0.
5. The method of claim 3, wherein the polymerization reagent of step (4) comprises: dNTPs, Klenow enzyme and Klenow enzyme reaction buffer.
6. The method of claim 5, wherein the DNA chain extension is carried out using a reaction system comprising: mu.L of 50. mu.M primer 21. mu.L, 5. mu.L of 10 XKlenow enzyme reaction buffer, 4. mu.L of 2.5mM dNTPs, 1. mu.L of Klenow enzyme 5U/. mu.L;
the reaction conditions are as follows: the reaction is carried out at 37 ℃ and 2000rpm for 0.5-1h with shaking.
7. The method as claimed in claim 6, wherein the reaction system used for connecting the microbeads and the UMI primers in step (4) is as follows: 50 μ M UMI primer, 5 μ L of 10 XKlenow enzyme reaction buffer, 4 μ L of 2.5mM dNTPs, and 1 μ L of Klenow enzyme 5U/. mu.L;
the reaction conditions are as follows: the reaction is carried out at 37 ℃ and 2000rpm for 0.5-1h with shaking.
8. The method according to any one of claims 1 to 7, wherein the nucleotide sequence of primer 1 is shown as SEQ ID NO. 1 to 5, the nucleotide sequence of primer 2 is shown as SEQ ID NO. 6 to 10, the nucleotide sequence of primer 3 is shown as SEQ ID NO. 11 to 15, and the nucleotide sequence of the UMI primer is shown as SEQ ID NO. 16.
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| PCT/CN2022/140097 WO2023116639A1 (en) | 2021-12-21 | 2022-12-19 | Preparation method for microsphere chip and related application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023116639A1 (en) * | 2021-12-21 | 2023-06-29 | 北京百迈客生物科技有限公司 | Preparation method for microsphere chip and related application |
| CN117089599A (en) * | 2023-10-20 | 2023-11-21 | 青岛百创智能制造技术有限公司 | Long coding sequence microbead and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925572A (en) * | 2016-06-07 | 2016-09-07 | 厦门大学 | DNA encoding microsphere and synthetic method thereof |
| US20190002953A1 (en) * | 2015-10-05 | 2019-01-03 | Helmholtz Zentrum Münchendeutsches Forschungszentrum Für Gesundheit Und Umwelt | A novel method for the preparation of bar-coded primer sets |
| CN112723304A (en) * | 2020-12-14 | 2021-04-30 | 苏州拉索生物芯片科技有限公司 | Microbead chip and preparation method thereof |
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190002953A1 (en) * | 2015-10-05 | 2019-01-03 | Helmholtz Zentrum Münchendeutsches Forschungszentrum Für Gesundheit Und Umwelt | A novel method for the preparation of bar-coded primer sets |
| CN105925572A (en) * | 2016-06-07 | 2016-09-07 | 厦门大学 | DNA encoding microsphere and synthetic method thereof |
| CN112723304A (en) * | 2020-12-14 | 2021-04-30 | 苏州拉索生物芯片科技有限公司 | Microbead chip and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 杨保胜等: "医学遗传学原理与应用 principles and application", 北京：中国人口出版社, pages: 279 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023116639A1 (en) * | 2021-12-21 | 2023-06-29 | 北京百迈客生物科技有限公司 | Preparation method for microsphere chip and related application |
| CN117089599A (en) * | 2023-10-20 | 2023-11-21 | 青岛百创智能制造技术有限公司 | Long coding sequence microbead and preparation method thereof |
| CN117089599B (en) * | 2023-10-20 | 2024-02-13 | 青岛百创智能制造技术有限公司 | Long coding sequence microbead and preparation method thereof |
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