A method for DNA molecular signal amplification and nucleic acid sequencing based on solid phase carrier

Abstract

The present invention discloses a method for DNA molecular signal amplification and nucleic acid sequencing based on a solid phase carrier. The method comprises: fixing a carboxylated DNA primer on a solid phase carrier with amino groups through a condensation reaction, adding a circular DNA library and an amplification reagent to the solid phase carrier, and performing rolling circle amplification, wherein the DNA primer is combined with the circular DNA library through base complementation. The present invention fixes the DNA primer on a solid phase carrier, and utilizes rolling circle amplification to amplify the DNA into a linear spiral structure, thereby avoiding the accumulation of errors caused by bridge PCR amplification into clusters, improving sequencing accuracy, and also reducing the high duplicate ratio caused by bridge PCR, saving sequencing costs, and at the same time, performing isothermal DNA amplification, which has low equipment requirements, can reduce the burden on operators, and realize automated preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and relates to a method for DNA molecule signal amplification and nucleic acid sequencing based on a solid phase carrier. Background Art

[0002] High-throughput sequencing technology, also known as "next-generation" sequencing technology, is characterized by its ability to sequence hundreds of thousands to millions of DNA molecules in parallel and generally with shorter read lengths. The steps of high-throughput sequencing include sample preparation, library construction, sequencing reaction and data analysis. In the library construction stage of high-throughput sequencing, the library template can be amplified thousands of times by PCR to reach the library volume on the machine, so as to complete the subsequent determination of the genomic DNA sequence; in the sequencing stage, the signal of a single DNA molecule is difficult to detect, and it is necessary to amplify the single DNA molecule to form a cluster (i.e., cluster) or a ball (DNB) to obtain a signal of sufficient intensity, so that sequencing can detect it. For nucleic acid templates, some have complex secondary structures and some have poor thermal stability. These factors will affect the efficiency of PCR amplification.

[0003] At present, the market has a way to amplify DNA molecular signals in the sequencing stage by amplifying a single DNA molecule to form a cluster. The accumulation of errors in the process of bridge PCR amplification to form clusters will reduce the fidelity of DNA sequence replication, affecting the accuracy and sensitivity of low-depth sequencing variations and low-frequency mutation detection. Due to the PCR amplification of library construction and sequencing, a high duplicate ratio will be brought about, resulting in a waste of DNA data, thereby increasing sequencing costs. When performing RNA sequencing, because it is difficult to distinguish whether it is PCR duplicates or the same template formed by high RNA expression, duplicates cannot be removed, which will affect the accuracy of transcriptome expression, especially the accuracy of transcripts with small and medium expression levels.

[0004] Another way to obtain a signal of sufficient intensity in the sequencing stage is to form a DNA nanoball from a single DNA molecule through amplification. For example, CN113774120A discloses a DNB double-end sequencing method, in which the amplification is first performed in a PCR instrument, the DNA nanoballs are quantified, and finally the DNA nanoballs are loaded into a chip and the DNBs are fixed in the chip in a certain manner. This process is cumbersome and requires the addition of an additional PCR instrument and DNA nanoball quantification equipment, with a long preparation time and a low degree of automation.

[0005] In summary, how to provide a method for efficiently amplifying DNA molecular signals, effectively avoiding error accumulation and reducing costs is one of the urgent problems to be solved in the field of gene sequencing. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides a method for DNA molecule signal amplification and nucleic acid sequencing based on a solid phase carrier, which can amplify the DNA molecule signal in the sequencing process efficiently and at low cost, while improving the sequencing accuracy.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for amplifying a DNA molecule signal based on a solid phase carrier, the method comprising:

[0009] The carboxylated DNA primer is fixed on a solid phase carrier with surface amino through the condensation reaction of carboxyl and amino groups, and a circular DNA library and amplification reagent are added to perform rolling circle amplification. The DNA primer is combined with the circular DNA library through base complementarity.

[0010] In the present invention, a DNA primer containing a carboxyl group is fixed on a solid phase carrier containing an amino group on the surface through a condensation reaction between a carboxyl group and an amino group, so that a solid phase carrier that efficiently loads the DNA primer can be obtained. Subsequently, the circularized library is combined with the DNA primer in a base complementary manner and fixed on the solid phase carrier. The library is subjected to rolling circle amplification under the action of enzymes and reagents to be amplified into a linear spiral structure, thereby avoiding the accumulation of bridge PCR errors. At the same time, the device requirements are low, the device is not overly dependent on PCR instruments, etc., and the device is easy to operate and can realize automated preparation, which is of great significance in the field of high-throughput gene sequencing.

[0011] Preferably, the material of the solid phase carrier includes any one of glass, silica gel, ceramic, plastic or metal.

[0012] Preferably, the carboxylated DNA primer comprises a 5'-end carboxylated DNA primer.

[0013] Preferably, the amplification reagent comprises DNA polymerase, dNTPs and a buffer.

[0014] Preferably, the DNA polymerase comprises phi29 DNA polymerase.

[0015] In the present invention, phi29 DNA polymerase has DNA strand displacement activity and DNA continuous synthesis ability, can synthesize DNA with a length of more than 70 kb, and can perform isothermal DNA amplification in vitro that is independent of thermal cycling.

[0016] Preferably, the condensation agent used in the condensation reaction includes 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and diisopropylethylamine.

[0017] Preferably, the molar concentration ratio of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) and diisopropylethylamine (DIEA) in the condensing agent is 1:(1-4), including but not limited to 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.5, 1:2.8, 1:3, 1:3.5 or 1:3.8, preferably 1:2.

[0018] In the present invention, 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate is used to promote the condensation reaction between the carboxyl group in the DNA primer and the amino group on the solid phase carrier, thereby significantly improving the DNA primer loading rate of the solid phase carrier; diisopropylethylamine can react with the by-product generated by the reaction of the primer-loaded solid phase carrier and TSTU, thereby consuming the by-product and promoting the reaction in the positive direction, thereby further improving the DNA primer loading rate of the solid phase carrier; in addition, by controlling the molar concentration ratio of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and diisopropylethylamine to be 1:2, the DNA primer loading rate of the solid phase carrier can be further improved.

[0019] Preferably, the rolling circle amplification further includes a blocking step.

[0020] Preferably, the blocking comprises adding a blocking reagent to a solid phase carrier for blocking.

[0021] Preferably, the blocking reagent comprises a bovine serum albumin reagent.

[0022] In the present invention, the solid phase carrier is sealed after the amplification reaction is completed, which is conducive to the amplified DNA and phi29 enzyme being fixed on the carrier more stably.

[0023] Preferably, the solid phase carrier-based DNA molecule signal amplification method comprises the following steps:

[0024] (1) performing amination treatment on the solid phase carrier;

[0025] (2) adding a carboxylated DNA primer and a condensing agent to the amino-modified solid phase support to obtain a solid phase support loaded with the DNA primer;

[0026] (3) Adding a circular DNA library and an amplification reagent to the solid phase carrier loaded with the DNA primer to perform rolling circle amplification, and adding a blocking reagent to perform blocking after amplification.

[0027] Preferably, the amination agent comprises 3-aminopropyltriethoxysilane.

[0028] In a second aspect, the present invention provides a method for nucleic acid sequencing based on a solid phase carrier, the method for nucleic acid sequencing based on a solid phase carrier comprising:

[0029] The DNA to be tested is amplified using the solid phase carrier-based DNA molecular signal amplification method described in the first aspect, and nucleic acid sequencing is performed on the amplified product according to the base complementarity principle to obtain nucleic acid sequence information.

[0030] Preferably, the nucleic acid sequencing comprises mixing the amplification product with a sequencing primer to perform a sequencing reaction.

[0031] In the present invention, the solid phase carrier-based DNA molecular signal amplification method described in the first aspect is used to amplify the DNA to be tested, so that the DNA is amplified into a linear spiral structure. All amplification templates are the original inserted fragments, and the errors generated by PCR will not accumulate. At the same time, it does not strictly rely on the PCR instrument to react. The DNA molecular signal is automatically amplified in the chip, which can reduce the burden on operators and realize automated preparation.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) In the present invention, DNA primers are fixed on a solid phase carrier, and the DNA primers are combined with a circular DNA library. Rolling circle amplification (RCA) is used to amplify the DNA into a linear spiral structure, so that all amplified templates are the original inserted fragments, avoiding the accumulation of errors in the process of amplifying into clusters due to bridge PCR, and also reducing the high duplicate rate caused by bridge PCR, thereby reducing the waste of DNA data and saving sequencing costs;

[0034] (2) The preparation method for amplifying DNA molecule signals in a solid phase carrier chip provided by the present invention does not strictly rely on a PCR instrument to perform reactions and DNA nanosphere quality control operations. DNA molecules can be automatically loaded into the chip, and DNA molecule signals can be automatically amplified in the chip, thereby reducing the burden on operators and realizing automated preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the hydroxyl structure diagram on the glass surface;

[0036] Figure 2 Schematic diagram of APTES modified silica surface, a represents physical adsorption, b represents condensation, and c represents the main structure after heating and curing;

[0037] Figure 3 It is a schematic diagram of the condensation reaction between the amino group on the surface of the solid phase carrier and the primer with carboxyl group;

[0038] Figure 4 Schematic diagram of DNA amplification and fixation of samples to be sequenced in a solid phase carrier;

[0039] Figure 5 This is a picture of the DNA amplification product observed under a fluorescence microscope. DETAILED DESCRIPTION

[0040] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0041] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0042] Example 1

[0043] In this embodiment, a glass substrate is used as a solid phase carrier and subjected to an amination treatment.

[0044] The amino groups were connected to the glass substrate by soaking method. The hydroxyl groups on the glass (silicon dioxide) surface were mainly isolated hydroxyl groups, dihydroxyl groups and hydrogen bonded hydroxyl groups ( Figure 1 ), 3-aminopropyltriethoxysilane (APTES) is adsorbed and reacted on the surface of silica under anhydrous conditions, and then heated and cured at 55°C. The reaction diagram is shown in FIG. Figure 2 As shown, a glass solid phase carrier with amino groups modified on the surface was obtained.

[0045] Example 2

[0046] In this example, the 5'-end carboxyl-modified DNA primer is fixed on the glass solid phase carrier prepared in Example 1.

[0047] A 5'-carboxylated DNA primer (sequence: 5'-GCCGTATCATTCAAGCAGAAGACG-3'), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and diisopropylethylamine were added to the glass solid phase carrier prepared in Example 1, the molar concentration ratio of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and diisopropylethylamine was 1:2, and the reaction was carried out at 30° C. for 30 min. The reaction principle diagram is shown in the following figure. Figure 3 As shown, after the reaction is completed, the unreacted reagents are rinsed with anhydrous ethanol to obtain a glass solid phase carrier loaded with DNA primers.

[0048] Example 3

[0049] This example uses the glass solid phase carrier loaded with DNA primers prepared in Example 2 to amplify DNA molecule signals.

[0050] TE buffer was used as the solvent for the circular DNA library. 120 fmol of the library was dissolved in TE buffer in a final volume of 80 μL and injected into the glass solid phase carrier loaded with DNA primers prepared in Example 2. The library and the primers in the carrier formed a double strand through the base complementarity principle. After standing at room temperature for 30 minutes, an amplification reaction reagent was added for rolling circle amplification (the amplification schematic diagram is shown in FIG. 2 ). Figure 4 The rolling circle amplification reaction system is shown in Table 1. The rolling circle amplification reaction conditions are: 30° C., 45 min. After the amplification, 1% BSA reagent is used for blocking.

[0051] Table 1

[0052] Reagent name Initial concentration Final concentration Volume (μL) phi29 nucleotide polymerase 10U / μL 1U / μL 8 10Xphi29 polymerase buffer 10× 1× 8 25mM dNTP Mix 25mM 0.5mM 1.6 Molecular grade water 62.4 Total 80

[0053] Test Example 1

[0054] In this test example, the amplified product of Example 3 was sequenced and analyzed.

[0055] Take the amplified product after blocking in Example 3, add sequencing primers to perform second-generation sequencing, and add the fluorescence image of the sequencing primer with single-color fluorescence as shown in the figure below: Figure 5 shown.

[0056] It can be seen that by the solid phase carrier-based DNA molecular signal amplification method of the present invention, rolling circle amplification is performed on a solid carrier, and sequencing primers are added after amplification. The genes located in the genome can be sequenced by sequencing by synthesis SBS, which can be single-end sequencing (SE) or double-end sequencing (PE).

[0057] In summary, the present invention fixes DNA primers on a solid phase carrier, and the DNA primers are combined with a circular DNA library, and the DNA is amplified into a linear spiral structure by rolling circle amplification, so that all amplification templates are the original inserted fragments, avoiding the error accumulation in the process of amplifying into clusters due to bridge PCR, improving sequencing accuracy, and also reducing the high duplicate ratio caused by bridge PCR, thereby reducing the waste of DNA data volume and saving sequencing costs. At the same time, it does not strictly rely on PCR instruments for reaction and DNA nanoball quality control operations, and can automatically load DNA molecules into the chip, and the DNA molecule signals are automatically amplified in the chip, which can reduce the burden on operators and realize automated preparation.

[0058] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for amplifying a DNA molecule signal based on a solid phase carrier, characterized in that: The method comprises: Through the condensation reaction between carboxyl and amino groups, the carboxylated DNA primer is fixed on the surface of the amino-containing solid phase carrier, and the circular DNA library and amplification reagent are added to perform rolling circle amplification; The DNA primer binds to the circular DNA library in a base complementary manner; The surface amino structure is that the amino group is connected to the Si element of the solid phase carrier through a carbon chain; the condensation agent used in the condensation reaction is composed of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and diisopropylethylamine in a molar concentration ratio of 1:(1-4); The carboxylated DNA primer includes a 5'-end carboxylated DNA primer; The rolling circle amplification further includes a blocking step; The blocking comprises adding a blocking reagent to the solid phase carrier for blocking.

2. The solid phase carrier-based DNA molecular signal amplification method according to claim 1, characterized in that: The material of the solid phase carrier includes any one of glass, silica gel, ceramic, plastic or metal.

3. The method for amplifying DNA molecule signals based on a solid phase carrier according to claim 1, characterized in that: The amplification reagent includes DNA polymerase, dNTPs and buffer.

4. The method for amplifying a DNA molecule signal based on a solid phase carrier according to claim 3, characterized in that: The DNA polymerase comprises phi29 DNA polymerase.

5. The method for amplifying DNA molecule signals based on a solid phase carrier according to claim 1, characterized in that: The blocking reagent includes a bovine serum albumin reagent.

6. The method for amplifying DNA molecule signals based on a solid phase carrier according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) performing amination treatment on the solid phase carrier; (2) adding a carboxylated DNA primer and a condensing agent to the amino-modified solid phase support to obtain a solid phase support loaded with the DNA primer; (3) Adding a circular DNA library and an amplification reagent to the solid phase carrier loaded with the DNA primer to perform rolling circle amplification, and adding a blocking reagent for blocking after the amplification is completed.

7. The solid phase carrier-based DNA molecular signal amplification method according to claim 6, characterized in that: The amination agent includes 3-aminopropyltriethoxysilane.

8. A method for nucleic acid sequencing based on a solid phase carrier, characterized in that: The solid phase carrier-based nucleic acid sequencing method comprises: The DNA to be tested is amplified using the solid phase carrier-based DNA molecular signal amplification method described in any one of claims 1 to 7, and nucleic acid sequencing is performed on the amplified product according to the base complementarity principle to obtain nucleic acid sequence information.

9. The method for nucleic acid sequencing based on a solid phase carrier according to claim 8, characterized in that: The nucleic acid sequencing includes mixing the amplification product with a sequencing primer to perform a sequencing reaction.

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