Reverse micelle DNA preservation and release method

Abstract

The invention provides a reverse micelle DNA preservation and release method, and belongs to the technical field of DNA preservation. The reverse micelle DNA preservation method provided by the invention comprises the following steps: mixing isooctane and 1-hexanol to obtain an organic solvent, and mixing hexadecyl trimethyl ammonium bromide and the organic solvent to obtain an organic phase; dissolving DNA in water to obtain a water phase; and mixing the organic phase with the water phase to obtain a reverse micelle system for storage. According to the reverse micelle DNA preservation method provided by the invention, the preservation time of the DNA under the room temperature condition can be obviously prolonged, and specifically, the preservation time can reach more than half a year. And the reverse micelle is adopted to preserve the DNA at room temperature, so that the method has the advantages of simplicity and convenience in operation and low cost of required materials.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA preservation, and in particular relates to a method for preserving and releasing reverse micelle DNA. Background Art

[0002] As the information carrier of life, DNA has a very high information density, so it can be used in electronic data storage, molecular computing and other fields. However, in addition to these core technologies, how to store DNA conveniently and at low cost is also crucial.

[0003] Most DNA operations are performed in the form of solutions. Although DNA is relatively stable in aqueous solutions, conditions such as temperature, pH, ionic strength, and ROS may damage the structure of DNA and cause loss of its function. It is generally believed that DNA molecules can be stored at room temperature for no more than one month, so when these solutions need to be used multiple times over a period of time, they are usually stored in a refrigerator. However, this type of storage method based on lowering the temperature needs to prevent unexpected situations such as power outages. Therefore, finding a method that can stably store DNA for a long time at room temperature can further promote the application of DNA in various fields. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for preserving reverse micelle DNA, which can achieve the purpose of preserving DNA for a long time at room temperature.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a method for preserving reverse micelle DNA, comprising the following steps: mixing isooctane and 1-hexanol to obtain an organic solvent, mixing hexadecyltrimethylammonium bromide and the organic solvent to obtain an organic phase; dissolving DNA in water to obtain an aqueous phase; and mixing the organic phase with the aqueous phase to obtain a reverse micelle system for preservation.

[0007] Preferably, the volume ratio of isooctane to 1-hexanol is 9.4:0.6.

[0008] Preferably, the mass volume ratio of hexadecyltrimethylammonium bromide to the organic solvent is 0.1458 g:10 mL.

[0009] Preferably, the molar concentration ratio of water to hexadecyltrimethylammonium bromide in the reverse micelle system is 10:1.

[0010] Preferably, the storage temperature is room temperature.

[0011] The present invention also provides a method for releasing reverse micelle DNA, comprising the following steps: mixing sodium chloride solution, 1-butanol and the reverse micelle system obtained by the above method, and collecting the DNA aqueous solution at the lower layer by centrifugation; the concentration of the sodium chloride solution is 1 mol / L.

[0012] Preferably, the volume ratio of the sodium chloride solution, 1-butanol and the reverse micelle system is 1:0.4:1.

[0013] Preferably, the centrifugation conditions are 4°C and 10,000 rpm for 5 min.

[0014] Beneficial effects of the present invention:

[0015] The reverse micelle DNA storage method provided by the present invention can significantly prolong the storage time of DNA at room temperature, specifically more than half a year. In addition, the reverse micelle is used to store DNA at room temperature, which has the advantages of simple operation and low material cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The reverse micelle system prepared according to the method of Example 1 at different times;

[0017] Figure 2 The DNA aqueous solution obtained by the reverse micelle DNA release method described in Example 2;

[0018] Figure 3 1 is the result of agarose gel electrophoresis identification, wherein 1 is DNA Marker, 2 is fresh DNA solution, 3 is the reverse micelle system preservation result described in Example 1, and 4 is the phosphate buffer storage result of Comparative Example 1. DETAILED DESCRIPTION

[0019] The invention provides a method for preserving reverse micelle DNA, comprising the following steps: mixing isooctane and 1-hexanol to obtain an organic solvent, mixing hexadecyltrimethylammonium bromide and the organic solvent to obtain an organic phase; dissolving DNA in water to obtain an aqueous phase; and mixing the organic phase with the aqueous phase to obtain a reverse micelle system for preservation.

[0020] The present invention has no particular limitation on the specific sources of isooctane, 1-hexanol, cetyltrimethylammonium bromide (CTAB) and water, and conventional commercial products in the art can be used. In the present invention, CTAB is used as a surfactant constituting reverse micelles, isooctane is used as a solvent, and 1-hexanol is used as a co-surfactant, and the three are mixed to form an organic phase of the reverse micelles.

[0021] In the present invention, the volume ratio of isooctane to 1-hexanol is preferably 9.4:0.6, the mass volume ratio of hexadecyltrimethylammonium bromide to the organic solvent is preferably 0.1458g:10mL, and the molar concentration of water in the reverse micelle system to the molar concentration of hexadecyltrimethylammonium bromide is preferably 10:1. The present invention controls the water content in the reverse micelle system, so that most of the water in the reverse micelle exists in the form of bound water rather than solvent water, thereby reducing the possibility of DNA hydrolysis. In the present invention, the water is preferably double distilled water.

[0022] In the present invention, when the organic phase and the aqueous phase are mixed, preferably stirred for 1 hour, an optically transparent reverse micelle system can be observed to be formed, and the DNA is dispersed in isooctane in the form of reverse micelle loading. In the present invention, the reverse micelle acts as an outer shell to protect the DNA from contacting with the outer organic solvent, while the outer organic solvent isolates the DNA from the outside air, thereby reducing the possibility of DNA oxidation.

[0023] In the present invention, the storage temperature is preferably room temperature.

[0024] The present invention also provides a method for releasing reverse micelle DNA, comprising the following steps: mixing sodium chloride solution, 1-butanol and the reverse micelle system obtained by the above method, and collecting the DNA aqueous solution at the lower layer by centrifugation; the concentration of the sodium chloride solution is 1 mol / L.

[0025] In the present invention, the volume ratio of sodium chloride solution, 1-butanol and the reverse micelle system is preferably 1:0.4:1. The present invention has no special limitation on the specific sources of sodium chloride and 1-butanol, and conventional commercially available products in this field can be used. In the present invention, the sodium chloride solution is used as the aqueous receiving phase, and the mixing is preferably stirred for 1 hour. In the present invention, the centrifugation conditions are preferably 4°C and 10,000 rpm for 5 minutes. The present invention can release the DNA stored in the reverse micelle into the aqueous phase through the back extraction method, and can be applied to experimental work that requires DNA solvents.

[0026] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0027] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Example 1

[0030] A method for preserving reverse micelle DNA, comprising the following steps:

[0031] 9.4 mL of isooctane and 0.6 mL of 1-hexanol were mixed to obtain an organic solvent; 0.1458 g (40 mM) of hexadecyltrimethylammonium bromide was weighed and added to the organic solvent, and the mixture was stirred for 0.5 h at room temperature to form an organic phase of reverse micelles.

[0032] The DNA was dissolved in double distilled water to form the aqueous phase of reverse micelles.

[0033] The organic phase and the aqueous phase were stirred for 1 h to form an optically transparent reverse micelle system, such as Figure 1 As shown, DNA is dispersed in isooctane in the form of reverse micelle loading; the molar concentration ratio of water to hexadecyltrimethylammonium bromide in the reverse micelle system is 10:1.

[0034] Example 2

[0035] A method for reverse micelle DNA release, comprising the following steps:

[0036] Take 1 mL of the reverse micelle system obtained in Example 1, add 1 mL of 1 mol / L sodium chloride solution (the preparation method of 1 mol / L sodium chloride solution is: 1.1688 g of sodium chloride is dissolved in 20 mL of double distilled water), then add 0.4 mL of 1-butanol, stir at 1000 rpm for 1 h, and then centrifuge at 4°C and 10000 rpm for 5 min. Collect the lower aqueous phase solution, which is the DNA aqueous solution. The results are as follows Figure 2 shown.

[0037] Comparative Example 1

[0038] The same DNA as in Example 1 was dissolved in a phosphate buffer (pH 7.4) having the same volume as that of the reverse micelle system described in Example 1 to obtain liquid phase DNA.

[0039] Example 3

[0040] The reverse micelle system obtained in Example 1 and the liquid DNA obtained in Comparative Example 1 were placed at room temperature for storage at the same time. After being placed at room temperature for half a year, the reverse micelle system obtained in Example 1 was released by the method of Example 2, and the DNA aqueous solution was collected. The quality of the DNA stored in Example 1 and Comparative Example 1 was identified by agarose gel electrophoresis, and fresh DNA that had not been stored for a long time was used as the control group. The results are as follows Figure 3 As shown, using TIANGEN's DNAMarkerⅢ as the molecular weight standard, it can be observed that compared with the fresh DNA solution ( Figure 3 Compared with the 2 bands in the reverse micelle system of Example 1, the DNA ( Figure 3 The 3 bands in the sample were not significantly degraded, and the gel electrophoresis results of the two samples were similar. Figure 3 In the 4 bands in the sample, DNA with a molecular weight greater than 800 bp was significantly degraded. It can be seen that the cationic reverse micelle system provided by the present invention can significantly improve the stability of DNA stored at room temperature.

[0041] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preserving reverse micelle DNA, characterized in that: The method comprises the following steps: mixing isooctane and 1-hexanol to obtain an organic solvent, mixing hexadecyltrimethylammonium bromide with the organic solvent to obtain an organic phase; dissolving DNA in water to obtain an aqueous phase; and mixing the organic phase with the aqueous phase to obtain a reverse micelle system for storage.

2. The method according to claim 1, characterized in that The volume ratio of isooctane to 1-hexanol is 9.4:0.

6.

3. The method according to claim 1, characterized in that The mass volume ratio of hexadecyltrimethylammonium bromide to the organic solvent is 0.1458 g:10 mL.

4. The method according to claim 1, characterized in that The molar concentration ratio of water to hexadecyltrimethylammonium bromide in the reverse micelle system is 10:

1.

5. The method according to claim 1, characterized in that The storage temperature is room temperature.

6. A method for releasing reverse micelle DNA, characterized in that: The method comprises the following steps: mixing sodium chloride solution, 1-butanol and the reverse micelle system obtained by the method according to any one of claims 1 to 5, and collecting the DNA aqueous solution at the lower layer by centrifugation; the concentration of the sodium chloride solution is 1 mol / L.

7. The method according to claim 6, characterized in that The volume ratio of the sodium chloride solution, 1-butanol and the reverse micelle system is 1:0.4:

1.

8. The method according to claim 6, characterized in that The centrifugation conditions are 4° C. and 10,000 rpm for 5 min.

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