Method for carrying out nucleic acid amplification by fixing probe on inner wall of capillary tube

Abstract

A method for nucleic acid amplification by fixing a probe on the inner wall of a capillary relates to the field of nucleic acid amplification, and comprises the following steps: 1) pretreatment and modification of the inner wall of the capillary: heating in an alkaline solution, immersing in a mixed solution of ethanol, water and APTES, reacting in a dark place, immersing in a glutaraldehyde solution, and treating in a dark place at room temperature; 2) fixing a probe on the inner wall of the capillary tube: alternately extracting an organic phase and a probe solution into the capillary tube to form a segmented probe array; and 3) carrying out PCR nucleic acid amplification by using the capillary tube fixed with the probe in the step 2). According to the invention, multiple sections of different probes can be fixed on the inner wall of the capillary tube, multiple DNA products can be simultaneously amplified in one capillary tube, and multi-index amplification on the inner wall of the capillary tube can be realized.

Description

Technical Field

[0001] The invention relates to the field of nucleic acid amplification, and in particular to a method for nucleic acid amplification by fixing a probe on the inner wall of a capillary. Background Art

[0002] In the past few decades, the field of molecular biology has made great progress, among which the development of nucleic acid amplification technology is particularly noteworthy. Nucleic acid amplification is an important experimental technique used to amplify DNA or RNA molecules in the laboratory. The most commonly used method is the polymerase chain reaction (PCR), which is widely used in genomic research, medical diagnosis, forensics and other fields. However, traditional PCR technology usually requires a large reaction volume, a long amplification time, and has certain limitations when processing trace samples. In recent years, with the development of microfluidics technology, people have begun to explore the possibility of nucleic acid amplification on a microscale in order to achieve more efficient and faster amplification effects. Summary of the invention

[0003] The purpose of the present invention is to solve the above problems in the prior art and provide a method for nucleic acid amplification by fixing probes on the inner wall of a capillary, fixing multiple different probes on the inner wall of the capillary, and amplifying multiple DNA products in one capillary at the same time, thereby realizing multi-index amplification on the inner wall of the capillary.

[0004] To achieve the above object, the technical solution of the present invention is as follows:

[0005] A method for nucleic acid amplification by fixing a probe on the inner wall of a capillary comprises the following steps:

[0006] 1) Pretreatment and modification of the inner wall of the capillary: first, immerse the capillary in an alkaline solution and heat it, then wash it with water and blow it dry with nitrogen; immerse the treated capillary in a solution of ethanol, water and APTES, react in the dark, then wash it with water, blow it dry with nitrogen, and bake it; immerse the treated capillary in a glutaraldehyde solution, treat it in the dark at room temperature, then wash it with acetone and water in turn, and blow it dry with nitrogen;

[0007] 2) Fixing the probe on the inner wall of the capillary: First, use a syringe pump to control the syringe to alternately draw the organic phase and the probe solution into the capillary to form a segmented probe array, in which the probe array is separated by the organic phase; then, incubate the capillary at room temperature to fix the probe on the inner wall of the capillary; finally, wash the capillary with a fixing buffer and water in turn, blow dry with nitrogen, chemically reduce, and then wash with water and dry with nitrogen;

[0008] 3) Perform PCR nucleic acid amplification using the capillary tube fixed with the probe in step 2).

[0009] In step 3), the capillary with the fixed probe is first filled with the amplification solution, and then the capillary is mounted on a platform with a temperature control function, and a semiconductor refrigeration chip is used to perform a temperature cycle of 60 to 95°C on the capillary.

[0010] A heat-conducting pad is arranged between the semiconductor refrigeration sheet and the capillary tube.

[0011] The thermal pad is provided with a groove for the capillary to be placed.

[0012] In step 1), the alkaline solution is a sodium hydroxide solution.

[0013] In step 1), the ethanol, water and APTES are mixed in a volume ratio of 95:3:2.

[0014] In step 2), multiple probes targeting different target sequences are fixed to form a probe array for multi-index amplification.

[0015] Now with respect to the prior art, the present invention has the following outstanding beneficial effects:

[0016] 1. The present invention performs surface modification on the inner wall of the capillary by multi-step treatment of the capillary, thereby fixing probes on the inner wall of the capillary, and forming a probe array by using an organic phase spacer on the inner wall of the capillary.

[0017] 2. The method of the present invention can design multiple probes for different target sequences to form a probe array for multi-index amplification, thereby achieving high efficiency of nucleic acid amplification, thereby simultaneously detecting multiple nucleic acid targets in one experiment, thereby improving the throughput and efficiency of detection.

[0018] 3. The present invention uses capillary nucleic acid amplification to greatly increase the contact opportunities between the target nucleic acid and the probe, thereby improving the sensitivity of detection, and can effectively capture and amplify the target nucleic acid even at low concentrations. The small volume of the capillary and the design of high-density probes make the contact between the reactants closer and the reaction kinetics are optimized, which helps to speed up the reaction, shorten the time, and improve the experimental efficiency.

[0019] 4. Due to the small size of the capillary and the high density of the probe, the consumption of reagents is significantly reduced. This is of great significance for reducing experimental costs, reducing environmental pollution and achieving sustainable development. And the capillary as a reaction container reduces contact with the external environment, reduces the risk of contamination, and ensures the accuracy and reliability of the experiment.

[0020] 5. The present invention uses a temperature control platform to control the temperature of the capillary and ensure temperature cycling during PCR nucleic acid amplification. Since the capillary is small and easy to handle, the present invention can be easily integrated with other microfluidic technologies or portable devices to achieve portability and on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A schematic diagram of a device for extracting a solution into a capillary;

[0022] Figure 2 Schematic diagram of the inner wall of the capillary filled with an organic phase and a fixed probe array;

[0023] Figure 3 Schematic diagram of capillary fixed probe array;

[0024] Figure 4 Schematic diagram of PCR amplification after the inner wall of the capillary forming the probe array is filled with the amplification solution and then mounted on a constructed temperature-controlled platform. DETAILED DESCRIPTION

[0025] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0026] Example 1

[0027] 1. Before fixing the DNA probe on the inner wall of the capillary to form a probe array, the inner wall of the capillary needs to be surface modified, including the following steps:

[0028] 1. First, immerse the capillary to be treated in a 10% mass fraction NaOH solution, ensure that the NaOH solution completely fills the capillary, heat it to 100℃ for 10 min, take it out and wash it with deionized water, and blow it dry with nitrogen;

[0029] 2. Prepare a solution by mixing ethanol, deionized water, and APTES in a volume ratio of 95:3:2. Immerse the capillary treated in step 1) in the solution and react for 1-2 h in the dark (cover the glass dish with tin foil to avoid light). Take it out and wash it with deionized water, blow dry it with nitrogen, and then put the capillary in a 120 °C oven for 1 h.

[0030] 3. Use 0.1 M PBS buffer to dilute the 50% glutaraldehyde solution ten times to obtain a 5% glutaraldehyde solution. Immerse the capillary treated in step 2) in the glutaraldehyde solution and treat it overnight at room temperature away from light. After the treatment, take out the capillary, wash it with acetone and deionized water in turn, blow dry with nitrogen, and store it in a dry place at 4°C.

[0031] 2. Fix the probe on the inner wall of the capillary:

[0032] Figure 1Schematic diagram of the device for extracting solution into the capillary. The syringe pump 4 is used to control the syringe 5 to extract the liquid in the centrifuge tube 7 into the capillary 6. The organic phase is first extracted into the capillary, and then the probe solution is extracted. This process is repeated continuously to form a segmented probe array of appropriate length in the capillary, and the arrays are separated by the organic phase. The capillary is incubated at room temperature for 1 hour, and the probes in the droplets are fixed on the inner wall of the capillary. Figure 2 The capillary includes a capillary segment 2 filled with an organic phase and a capillary segment 3 with a fixed probe array. After fluorescence detection, the fluorescence value of the capillary segment 3 with the fixed probe array is much higher than the fluorescence value of the capillary segment 2 filled with an organic phase.

[0033] After the probe is fixed, the syringe pump 4 is used to extract the fixation buffer and deionized water to wash the capillary. After washing, it is blown dry with nitrogen. Then, NaBH4 solution (100 mg NaBH4 dissolved in 30 mL 1 PBS buffer and 10 mL 95% anhydrous ethanol) is used for chemical reduction for 45 minutes. Then, the capillary is washed with deionized water and dried with nitrogen to obtain a capillary with a probe array formed on the inner wall of the capillary, which is ready for PCR nucleic acid amplification. Figure 3 , is a schematic diagram of the capillary-immobilized DNA probe array 1.

[0034] 3. PCR nucleic acid amplification:

[0035] Figure 4 After the probe is fixed on the inner wall of the capillary, the capillary is filled with amplification solution and installed on a temperature control platform for PCR nucleic acid amplification. The capillary is subjected to 40 temperature cycles from 60 to 95 °C using a semiconductor refrigeration chip 9. A thermal pad 11 is placed on the semiconductor refrigeration chip to transfer heat from the surface of the semiconductor refrigeration chip to the capillary. Two grooves 12 and 13 are designed on the thermal pad to embed the capillary, so that the capillary is in close contact with the semiconductor refrigeration chip, reducing heat loss during nucleic acid amplification. The semiconductor refrigeration chip dissipates heat through the heat sink 10 and the fan 8.

[0036] The probe fixing method of the present invention is used to form a probe array on the inner wall of the capillary, and then the capillary is filled with an amplification solution, and the capillary to be amplified is mounted on the temperature control platform designed by the present invention, and amplified by PCR nucleic acid amplification technology. Referring to the above-mentioned inventive method, a variety of probes can be fixed on the inner wall of the capillary to form a probe array to achieve multi-index amplification, and probes for different target sequences can be designed, and these probes have high specificity to ensure binding to the target sequence.

Claims

1. A method for nucleic acid amplification by fixing a probe on the inner wall of a capillary, characterized in that: The following steps are involved: 1) Pretreatment and modification of the inner wall of the capillary: First, immerse the capillary in an alkaline solution and heat it, then wash it with water and blow it dry with nitrogen; The treated capillary was immersed in a solution of ethanol, water and APTES, and reacted in the dark, then washed with water, dried with nitrogen, and baked; the treated capillary was immersed in glutaraldehyde solution, treated in the dark at room temperature, then washed with acetone and water in turn, and dried with nitrogen; 2) Fixing the probe on the inner wall of the capillary: First, use a syringe pump to control the syringe to alternately draw the organic phase and the probe solution into the capillary to form a segmented probe array, in which the probe array is separated by the organic phase; then, incubate the capillary at room temperature to fix the probe on the inner wall of the capillary; finally, wash the capillary with a fixing buffer and water in turn, blow dry with nitrogen, chemically reduce, and then wash with water and dry with nitrogen; 3) Perform PCR nucleic acid amplification using the capillary tube fixed with the probe in step 2).

2. A method for nucleic acid amplification by fixing a probe on the inner wall of a capillary as claimed in claim 1, characterized in that: In step 3), the capillary with the fixed probe is first filled with the amplification solution, and then the capillary is mounted on a platform with a temperature control function, and a semiconductor refrigeration chip is used to perform a temperature cycle of 60 to 95°C on the capillary.

3. A method for nucleic acid amplification by fixing a probe on the inner wall of a capillary as claimed in claim 2, characterized in that: A heat-conducting pad is arranged between the semiconductor refrigeration sheet and the capillary tube.

4. A method for nucleic acid amplification by fixing a probe on the inner wall of a capillary as claimed in claim 3, characterized in that: The thermal pad is provided with a groove for the capillary to be placed.

5. A method for nucleic acid amplification by fixing a probe on the inner wall of a capillary as claimed in claim 1, characterized in that: In step 1), the alkaline solution is a sodium hydroxide solution.

6. A method for nucleic acid amplification by fixing a probe on the inner wall of a capillary as claimed in claim 1, characterized in that: In step 1), the ethanol, water and APTES are mixed in a volume ratio of 95:3:

2.

7. A method for nucleic acid amplification by fixing a probe on the inner wall of a capillary as claimed in claim 1, characterized in that: In step 2), multiple probes targeting different target sequences are fixed to form a probe array for multi-index amplification.

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