WO2023138206A1 - Dna molecular signal amplification and nucleic acid sequencing method based on solid-phase carrier - Google Patents

Abstract

A DNA molecular signal amplification and nucleic acid sequencing method based on a solid-phase carrier, the method comprising: immobilizing a carboxylated DNA primer on a surface-aminated solid-phase carrier by means of a condensation reaction of carboxyl and amino groups, and adding an annular DNA library and an amplification reagent to the solid-phase carrier for carrying out rolling circle amplification, the DNA primer being bound to the annular DNA library in a base complementary manner. A DNA primer is immobilized to a solid-phase carrier, and DNA is amplified into a linear helix structure by means of rolling circle amplification, so that error accumulation caused by bridge PCR amplification into cluster is avoided, the sequencing accuracy is improved, a relatively high duplicate ratio caused by bridge PCR is also reduced, and the sequencing cost is reduced. In addition, by means of isothermal DNA amplification, demands on equipment is low, the burden on operators can be relieved, and automatic preparation is achieved.

Description

A method for DNA molecule signal amplification and nucleic acid sequencing based on a solid-phase carrier technical field

The application belongs to the technical field of genetic engineering, and relates to a method for DNA molecular signal amplification and nucleic acid sequencing based on a solid-phase carrier.

Background technique

High-throughput sequencing technology (High-throughput sequencing), also known as "next-generation" sequencing technology ("Next-generation" sequencing technology), is marked by the ability to sequence hundreds of thousands to several million DNA molecules in parallel at a time and generally having a short read length. 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, so as to reach the amount of the library 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 ball (DNB) to obtain a signal of sufficient strength before sequencing can be detected. For nucleic acid templates, some secondary structures are complex. Some thermal stability is not good, these factors will affect the efficiency of PCR amplification.

At present, the way to amplify DNA molecular signals in the market during the sequencing stage is to form a cluster of single DNA molecules through amplification, that is, cluster. The accumulation of errors in the process of bridge PCR amplification forming clusters will reduce the fidelity of DNA sequence replication, affecting the accuracy and sensitivity of low-depth sequencing variation and low-frequency mutation detection. Due to the PCR amplification of library construction and sequencing, it will bring a higher duplicate rate, resulting in a waste of DNA data, thereby increasing the cost of sequencing. When performing RNA sequencing, because it is difficult to distinguish between PCR duplicates and the same template formed by high RNA expression, duplicates cannot be removed, which will affect the accuracy of transcriptome expression, especially the accuracy of small and medium expression transcripts.

Another way to obtain a signal of sufficient strength in the sequencing stage is to amplify a single DNA molecule to form DNA nanospheres. For example, CN113774120A discloses a DNB paired-end sequencing method. The process is to first amplify in a PCR machine, quantify the DNA nanospheres, and finally load the DNA nanospheres into the chip and fix the DNB in the chip in a certain way.

To sum up, 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.

Contents of the invention

The present application provides a method for DNA molecular signal amplification and nucleic acid sequencing based on a solid-phase carrier, which can efficiently and cost-effectively amplify DNA molecular signals during the sequencing process while improving sequencing accuracy.

In the first aspect, the present application provides a method for amplifying DNA molecular signals based on a solid-phase carrier, the method comprising:

Through the condensation reaction of carboxyl and amino groups, the carboxylated DNA primers are immobilized on the surface aminated solid phase carrier, and the circular DNA library and amplification reagents are added to perform rolling circle amplification;

Wherein, the DNA primers are combined with the circular DNA library through base complementation.

In this application, DNA primers containing carboxyl groups are immobilized on the solid-phase carrier containing amino groups on the surface through the condensation reaction of carboxyl groups and amino groups, and a solid-phase carrier for efficiently loading DNA primers can be obtained. Then, the circularized library and DNA primers are combined and fixed on the solid-phase carrier through base complementarity. The field of high-throughput sequencing is of great significance.

Preferably, the material of the solid phase carrier includes any one of glass, silica gel, ceramics, plastic or metal.

Preferably, the carboxylated DNA primer comprises a 5' end carboxylated DNA primer.

Preferably, said amplification reagents include DNA polymerase, dNTPs and buffer.

Preferably, the DNA polymerase comprises phi29 DNA polymerase.

In this application, phi29 DNA polymerase has DNA strand displacement activity and continuous DNA synthesis ability, can synthesize DNA with a length of more than 70kb, and can perform isothermal DNA amplification in vitro that does not depend on thermal cycles.

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

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 .

In this application, 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, which significantly increases the loading rate of the DNA primer on the solid-phase carrier. Diisopropylethylamine can react with the by-product generated by the reaction between the solid-phase carrier and TSTU loaded with the primer, thereby consuming the by-product and promoting the reaction to proceed in the positive direction, further improving the DNA primer loading rate of the solid-phase carrier. In addition, by controlling the 2-succinimide The molar concentration ratio of imido-1,1,3,3-tetramethyluronium tetrafluoroborate and diisopropylethylamine is 1:2, which can further increase the DNA primer loading rate of the solid phase carrier.

Preferably, a blocking step is also included after the rolling circle amplification.

Preferably, the blocking includes adding a blocking reagent to a solid phase carrier for blocking.

Preferably, the blocking reagent comprises bovine serum albumin reagent.

In this application, the solid-phase carrier is blocked after the amplification reaction is completed, which is beneficial for the amplified DNA and phi29 enzyme to be more firmly immobilized on the carrier.

Preferably, the DNA molecular signal amplification method based on a solid phase carrier comprises the following steps:

(1) Amination treatment is carried out to the solid phase carrier;

(2) adding carboxylated DNA primers and condensing agents to the aminated solid phase carrier to obtain a solid phase carrier loaded with DNA primers; and

(3) adding a circular DNA library and amplification reagents to the solid phase carrier loaded with DNA primers for rolling circle amplification, and adding a blocking reagent for blocking after amplification.

Preferably, the reagent for amination treatment includes 3-aminopropyltriethoxysilane.

In a second aspect, the present application provides a method for nucleic acid sequencing based on a solid-phase carrier, and the method for nucleic acid sequencing based on a solid-phase carrier includes:

Using the DNA molecular signal amplification method based on the solid-phase carrier described in the first aspect to amplify the DNA to be tested, and perform nucleic acid sequencing on the amplified product according to the principle of base complementarity to obtain nucleic acid sequence information.

Preferably, the nucleic acid sequencing includes mixing the amplification product with sequencing primers to perform a sequencing reaction.

In this application, 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 helical structure. All amplified templates are the original insert fragments, and the errors generated by PCR will not accumulate. At the same time, the reaction is not strictly dependent on the PCR instrument. The DNA molecular signal is automatically amplified in the chip, which can reduce the burden on operators and realize automatic preparation.

Compared with the prior art, the present application has the following beneficial effects:

(1) In this application, the DNA primers are immobilized on the solid-phase carrier, the DNA primers are combined with the circular DNA library, and the DNA is amplified into a linear helical structure by rolling circle amplification (Rolling circle replication, RCA), so that all the amplified templates are the original insert fragments, avoiding the accumulation of errors in the process of amplifying into clusters due to bridge PCR, and also reducing the high duplicate ratio caused by bridge PCR, thereby reducing the waste of DNA data and saving sequencing costs.

(2) The preparation method for DNA molecular signal amplification in a solid-phase carrier chip provided by this application does not strictly rely on PCR equipment for reaction and DNA nanosphere quality control operations, and can automatically load DNA molecules into the chip, and DNA molecular signals are automatically amplified in the chip, which can reduce the burden on operators and realize automatic preparation.

Description of drawings

Figure 1 is a diagram of the structure of hydroxyl groups on the glass surface.

Figure 2 is a schematic diagram of the surface of 3-aminopropyltriethoxysilane (APTES) modified silica, a represents physical adsorption, b represents condensation, and c represents the main structure after heating and curing.

Fig. 3 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.

Fig. 4 is a schematic diagram of DNA amplification and immobilization of a sample to be sequenced in a solid phase carrier.

Fig. 5 is a picture of DNA amplification products observed under a fluorescence microscope.

Detailed ways

In order to further illustrate the technical means and effects adopted by the present application, the present application will be further described below in conjunction with the embodiments and accompanying drawings. It can be understood that the specific implementation manners described here are only used to explain the present application, but not to limit the present application.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products commercially available through formal channels.

Example 1

In this embodiment, a glass substrate is used as a solid phase carrier and amination treatment is performed on it.

Amino groups were connected to the glass substrate by soaking method. The hydroxyl groups on the surface of glass (silica) were mainly isolated hydroxyl groups, dihydroxyl groups and hydrogen-bonded hydroxyl groups (Figure 1). 3-Aminopropyltriethoxysilane (APTES) was used to absorb and react on the surface of silica under anhydrous conditions, and heat and solidify at 55°C.

Example 2

In this example, the 5' end carboxylated DNA primer was immobilized on the glass solid phase carrier prepared in Example 1.

Add the 5' end carboxylated DNA primer (SEQ ID NO: 1: 5'-GCCGTATCATTCAAGCAGAAGACG-3') and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and diisopropylethylamine to the glass solid phase carrier prepared in Example 1. The ratio is 1:2, and react at 30°C for 30 minutes. The schematic diagram of the reaction is shown in Figure 3. After the reaction is completed, rinse with absolute ethanol to wash away the unreacted reagents to obtain a glass solid-phase carrier loaded with DNA primers.

Example 3

In this example, the glass solid-phase carrier loaded with DNA primers prepared in Example 2 was used to amplify DNA molecular signals.

Using TE buffer as the solvent for the circular DNA library, take 120 fmol of the circular DNA library and dissolve it in the TE buffer, so that the final volume is 80 μL, and inject it into the glass solid-phase carrier loaded with DNA primers prepared in Example 2. The primers in the library and the carrier form a double strand through the principle of base complementarity. After standing at room temperature for 30 minutes, add the amplification reaction reagents for rolling circle amplification (the schematic diagram of the amplification is shown in Figure 4). The rolling circle amplification reaction system is shown in Table 1. The rolling circle amplification reaction conditions are: 30 ℃, 45min, after the end of the amplification, use 1% BSA reagent to block.

Table 1

Reagent name	initial concentration	Final concentration	Volume (μL)
phi29 nucleotide polymerase	10U/μL	1U/μL	8
10 x phi29 polymerase buffer	10×	1×	8
25mM dNTP mix	25mM	0.5mM	1.6
molecular grade water	the	the	62.4
Total	the	the	80

Test example 1

In this test example, the amplification product of Example 3 was sequenced and analyzed.

Take the amplified product after sealing in Example 3, add sequencing primers to carry out next-generation sequencing, and the fluorescence picture of adding sequencing primers with monochromatic fluorescence is shown in Figure 5.

It can be seen that the rolling circle amplification method based on the solid-phase carrier of the present application is performed on a solid carrier, and sequencing primers are added after amplification, and the genes located in the genome can be sequenced by sequencing while synthesizing SBS, that is, single-end sequencing (SE testing) or double-end sequencing (PE testing).

In summary, this application immobilizes DNA primers on a solid-phase carrier, combines the DNA primers with a circular DNA library, and uses rolling circle amplification to amplify the DNA into a linear helical structure, so that all amplified templates are the original insert fragments, avoiding the accumulation of errors in the process of amplifying bridge PCR into a cluster, improving the accuracy of sequencing, and reducing the high duplicate ratio caused by bridge PCR, thereby reducing the waste of DNA data and saving sequencing costs. Molecules are automatically loaded into the chip, and DNA molecular signals are automatically amplified in the chip, which can reduce the burden on operators and realize automatic preparation.

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

Claims (11)

1. A method for amplifying DNA molecular signals based on a solid phase carrier, comprising:

   Through the condensation reaction of carboxyl and amino groups, the carboxylated DNA primers are immobilized on the surface aminated solid phase carrier, and the circular DNA library and amplification reagents are added to perform rolling circle amplification;

   Wherein, the DNA primers are combined with the circular DNA library through base complementation.
2. The method for amplifying DNA molecular signals based on a solid-phase carrier according to claim 1, wherein the material of the solid-phase carrier comprises any one of glass, silica gel, ceramics, plastic or metal.
3. The DNA molecular signal amplification method based on a solid-phase carrier according to claim 1 or 2, wherein the carboxylated DNA primer comprises a 5' end carboxylated DNA primer.
4. The DNA molecular signal amplification method based on a solid phase carrier according to any one of claims 1-3, wherein the amplification reagent comprises DNA polymerase, dNTPs and a buffer;

   Preferably, said DNA polymerase comprises phi29 DNA polymerase.
5. The DNA molecular signal amplification method based on a solid phase carrier according to any one of claims 1-4, wherein the condensing agent used in the condensation reaction comprises 2-succinimidyl-1,1,3,3-tetramethylurea tetrafluoroborate and diisopropylethylamine;

   Preferably, the molar concentration ratio of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and diisopropylethylamine in the condensing agent is 1:(1-4).
6. The solid-phase carrier-based DNA molecular signal amplification method according to any one of claims 1-5, wherein a sealing step is further included after the rolling circle amplification.
7. The DNA molecular signal amplification method based on a solid phase carrier according to claim 6, wherein said blocking comprises adding a blocking reagent to a solid phase carrier for blocking;

   Preferably, the blocking reagent comprises bovine serum albumin reagent.
8. The DNA molecular signal amplification method based on a solid phase carrier according to any one of claims 1-7, wherein the method comprises the following steps:

   (1) Amination treatment is carried out to the solid phase carrier;

   (2) adding carboxylated DNA primers and condensing agents to the aminated solid phase carrier to obtain a solid phase carrier loaded with DNA primers; and

   (3) Adding a circular DNA library and amplification reagents to the solid phase carrier loaded with DNA primers for rolling circle amplification, adding a blocking reagent for blocking after the amplification is completed.
9. The method for amplifying DNA molecular signals based on a solid-phase carrier according to claim 8, wherein the reagent for amination treatment includes 3-aminopropyltriethoxysilane.
10. A method for nucleic acid sequencing based on a solid phase carrier, comprising:

    Using the DNA molecular signal amplification method based on a solid-phase carrier according to any one of claims 1-9 to amplify the DNA to be tested, and perform nucleic acid sequencing on the amplified product according to the principle of base complementarity to obtain nucleic acid sequence information.
11. The method for nucleic acid sequencing based on a solid phase carrier according to claim 10, wherein the nucleic acid sequencing comprises mixing the amplification product with sequencing primers to perform a sequencing reaction.

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