Double-stranded oligonucleotide melting temperature analysis method

Abstract

The invention provides a method for analyzing the melting temperature of double-stranded oligonucleotide, which comprises the following steps: dissolving the double-stranded oligonucleotide with water to prepare a 0.2 mg / ml detection solution, and analyzing the absorbance of the detection solution at different temperatures by using an ultraviolet-visible spectrophotometer to obtain S-shaped curves of the absorbance at different temperatures; and collecting the absorbance increase initial temperature value T1 and the absorbance increase final temperature value T2 in the S-shaped curve to obtain the melting temperature (Tm), Tm = (T1 + T2) / 2. The absorbance increasing initial temperature value T1 is the minimum temperature value with the slope greater than 0 in the S-shaped curve, and the absorbance increasing final temperature value T2 is the maximum temperature value with the slope greater than 0 in the S-shaped curve. The method has the advantages of being convenient to operate, high in accuracy, capable of rapidly obtaining a result and the like, achieves the purposes of thermodynamic stability, property and identification of the oligonucleotide double-strand, and analyzes the structure of the double-strand oligonucleotide.

Description

Technical Field

[0001] The invention relates to a method for analyzing the melting temperature of double-stranded oligonucleotide using an ultraviolet-visible spectrophotometer. Background Art

[0002] Double-stranded DNA (dsDNA) or double-stranded RNA (dsRNA) is a DNA or RNA with two strands, and the two strands are complementary. The melting temperature (Tm) of an oligonucleotide is the temperature at which 50% of the oligonucleotide reacts with its perfect complement and 50% is free in the solution, and the temperature at which the color enhancement effect shown in ultraviolet absorption reaches half. The size of the Tm value is related to factors such as the composition, length, salinity, and pH of the DNA or RNA sequence. The melting temperature of DNA is higher than that of RNA because the double-stranded structure of DNA is more stable than that of RNA. The hydrogen bonds in the double-stranded DNA mainly come from the pairing between AT and GC base pairs, while in RNA, since only CG and AU base pairs exist, the number of hydrogen bonds is less, so the melting temperature is relatively low.

[0003] In the oligonucleotide technology path, there are two major types based on molecular structure: single-stranded oligonucleotides (generally antisense oligonucleotide, referred to as ASO) and double-stranded oligonucleotides (generally small interfering RNA, referred to as siRNA); they are roughly composed of 16-30 bases, and the Tm value is only applicable to the study of the properties of double-stranded oligonucleotides.

[0004] Currently, there are many softwares that have introduced various prediction methods for melting temperatures (Tm) of dsDNA or dsRNA, but there are disadvantages that the errors of some sequences are relatively large, and they are only theoretical values. Therefore, the prediction method through software is unreliable. For double-stranded oligonucleotides with different sequences and modifications (including base modification, phosphate bond modification and ribose modification), it is particularly important and necessary to measure their Tm values ​​with precision instruments. Summary of the invention

[0005] The present invention aims to provide a method for analyzing the melting temperature (Tm) of double-stranded oligonucleotides, which is single and scarce and has low accuracy.

[0006] The present invention adopts the following technical scheme to achieve the purpose of the present invention: a method for analyzing the melting temperature of double-stranded oligonucleotides: dissolving the double-stranded oligonucleotides in water to prepare a 0.2 mg / ml detection solution, using a UV-visible spectrophotometer to analyze the absorbance of the detection solution at different temperatures, and obtaining an absorbance S-shaped curve at different temperatures; collecting the initial temperature value T1 of the absorbance increase and the end temperature value T2 of the absorbance increase in the S-shaped curve, thereby obtaining the melting temperature (Tm), Tm = (T1+T2) / 2. The initial temperature value T1 of the absorbance increase is the minimum temperature value in the S-shaped curve where the slope is greater than 0, and the end temperature value T2 of the absorbance increase is the maximum temperature value in the S-shaped curve where the slope is greater than 0.

[0007] Furthermore, the test temperature range is 0-100°C, preferably 30-85°C.

[0008] Furthermore, within the temperature test range, the absorbance test is performed at intervals of 5°C.

[0009] Further, the detection wavelength is 260 nm;

[0010] Furthermore, using Thermo Scientific TM Evolution 201 UV-Vis spectrophotometer with Peltier single cell holder and quartz cuvette with 1 cm pathlength.

[0011] The above technical solution has the following beneficial effects:

[0012] (1) Wide temperature range, suitable for Tm value detection of double-stranded oligonucleotides of various sequences and lengths;

[0013] (2) Rapid analysis of sample Tm values, simple data processing, and the ability to obtain raw data;

[0014] (3) High analytical precision;

[0015] (4) The operation is simple and the analysis is fully automated. The detection is completed by the instrument. Regardless of whether you have experimental knowledge or not, you can operate it as long as you have received operation training. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Example 1 Analyze the results of sample detection.

[0017] Figure 2 : Example 2 Analyze the sample detection results. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0019] The present invention intends to establish a rapid, economical and accurate double-stranded oligonucleotide melting temperature (Tm) analysis method. It uses a UV-visible spectrophotometer equipped with a Peltier single sample cell holder for analysis, and the heating module can monitor the sample at 0°C-100°C. It has the advantages of convenient operation, high accuracy, and rapid results, so as to achieve the purpose of thermodynamic stability, properties and identification of oligonucleotide double chains, and analyze the structure of double-stranded oligonucleotides.

[0020] Example 1, Tm analysis of ASO nucleic acid samples,

[0021] 1. Instruments and materials used

[0022] (1) Thermo Scientific TM Evolution 201 UV-Vis spectrophotometer with Peltier single cell holder and xenon flash lamp;

[0023] (2) Quartz cuvette with an optical path of 1 cm;

[0024] (3) The ASO nucleic acid sample came from Notaino and, based on the sequence, its theoretical Tm value was 61.3°C.

[0025] 2. Test conditions

[0026] (1) Diluent: Water

[0027] (2) Detection wavelength: 260nm

[0028] (3) Target temperature: 85°C

[0029] (4) Temperature change rate: 5℃ / min

[0030] 3. Detection method:

[0031] (1) Accurately weigh 2 mg of sample and place it in a 10 ml volumetric flask. Add water to dissolve and make up to volume.

[0032] (2) Place the test solution on a heating device for heating and monitor the temperature in real time.

[0033] (3) The absorbance of the solution at different temperatures was analyzed using a UV-visible spectrophotometer.

[0034] (4) Draw the absorbance curve at different temperatures, such as Figure 1 .

[0035] (5) Collect the initial temperature value T1 of the absorbance increase and the end temperature value T2 of the absorbance increase in the S-shaped curve to obtain the melting temperature (Tm), Tm = (T1 + T2) / 2≈61.4°C.

[0036] It can be seen that the analysis method provided by the present invention has high accuracy, and the heating process only takes 10 minutes, and the entire analysis process is completed within 15 minutes. It has the advantages of easy operation, high accuracy, and rapid results.

[0037] Example 2: Tm analysis of RNA nucleic acid samples.

[0038] 4. Instruments and materials used

[0039] (4) Thermo Scientific TM Evolution 201 UV-Vis spectrophotometer with Peltier single cell holder and xenon flash lamp;

[0040] (5) Quartz cuvette with an optical path of 1 cm;

[0041] (6) The RNA nucleic acid sample was from Notaino and, based on the sequence, its theoretical Tm value was 67.7°C.

[0042] 5. Test conditions

[0043] (5) Diluent: 0.05M NaCl

[0044] (6) Detection wavelength: 260nm

[0045] (7) Target temperature: 80°C

[0046] (8) Temperature change rate: 5℃ / min

[0047] 6. Detection method:

[0048] (6) Accurately weigh 2 mg of sample and place it in a 10 ml volumetric flask. Add 0.05 M NaCl to dissolve and make up to volume.

[0049] (7) Place the test solution on a heating device for heating, and monitor the temperature in real time.

[0050] (8) The absorbance of the solution at different temperatures was analyzed using a UV-visible spectrophotometer.

[0051] (9) Draw the absorbance curve at different temperatures, such as Figure 2 .

[0052] (10) The initial temperature value T1 of the absorbance increase and the end temperature value T2 of the absorbance increase in the S-shaped curve are collected to obtain the melting temperature (Tm), Tm = (T1 + T2) / 2≈67.9°C.

[0053] It can be seen that the analysis method provided by the present invention has high accuracy, and the heating process only takes 10 minutes, and the entire analysis process is completed within 15 minutes. It has the advantages of easy operation, high accuracy, and rapid results.

Claims

1. A method for analyzing the melting temperature of a double-stranded oligonucleotide, characterized in that: The double-stranded oligonucleotide is dissolved in water to prepare a 0.2 mg / ml detection solution, and the absorbance of the detection solution at different temperatures is analyzed by a UV-visible spectrophotometer to obtain an absorbance S-shaped curve at different temperatures; the initial temperature value T1 of the absorbance increase and the end temperature value T2 of the absorbance increase in the S-shaped curve are collected to obtain the melting temperature (Tm), Tm=(T1+T2) / 2; the initial temperature value T1 of the absorbance increase is the minimum temperature value in the S-shaped curve where the slope is greater than 0, and the end temperature value T2 of the absorbance increase is the maximum temperature value in the S-shaped curve where the slope is greater than 0.

2. The method for analyzing the melting temperature of double-stranded oligonucleotide according to claim 1, characterized in that: The test temperature range is 0~100℃.

3. The method for analyzing the melting temperature of double-stranded oligonucleotide according to claim 2, characterized in that: Preferably, the temperature is 30 to 85°C.

4. The method for analyzing the melting temperature of double-stranded oligonucleotide according to claim 1, characterized in that: Within the temperature test range, the absorbance test was performed at intervals of 5°C.

5. The method for analyzing the melting temperature of double-stranded oligonucleotide according to claim 1, characterized in that: Detection wavelength: 260nm.

6. The method for analyzing the melting temperature of double-stranded oligonucleotide according to claim 1, characterized in that: Using ThermoScientific TM Evolution 201 UV-Vis spectrophotometer with Peltier single cell holder and quartz cuvette with 1 cm pathlength.

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