A method for the preservation and release of DNA in reverse micelles

Abstract

The present invention provides a method for the preservation and release of reverse micelle DNA, belonging to the technical field of DNA preservation. The method for preserving reverse micelle DNA provided by the present invention comprises the following steps: mixing isooctane and 1-hexanol to obtain an organic solvent, and mixing cetyltrimethylammonium bromide with the organic solvent to obtain an organic phase; dissolving DNA in water to obtain an aqueous phase; mixing the organic phase and the aqueous phase to obtain a reverse micelle system for preservation. The method for preserving reverse micelle DNA provided by the present invention can significantly extend the preservation time of DNA at room temperature, specifically up to more than half a year. Moreover, the present invention uses reverse micelles for the room temperature preservation of DNA, which has the advantages of simple operation and low material cost.

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 DNA from reverse micelles, which includes the following steps: mixing a sodium chloride solution, 1-butanol, and the reverse micelle system obtained by the above method, and centrifuging to collect the lower-layer DNA aqueous solution; 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 centrifuging at 4°C and 10,000 rpm for 5 min.

[0014] Advantages of the present invention:

[0015] The method for preserving DNA in reverse micelles provided by the present invention can significantly extend the preservation time of DNA at room temperature, specifically up to more than half a year. Moreover, the present invention uses reverse micelles for room-temperature preservation of DNA, which has the advantages of simple operation and low material cost. Description of the drawings

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

[0017] Figure 2 is the DNA aqueous solution obtained by the method for releasing reverse micelle DNA described in Example 2;

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

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

[0020] The present invention has no special limitation on the specific sources of isooctane, 1-hexanol, cetyltrimethylammonium bromide (CTAB), and water, and conventional commercially available products in the art can be used. In the present invention, CTAB is used as the surfactant constituting the reverse micelles, isooctane is used as the solvent, and 1-hexanol is used as the co-surfactant. The three are mixed to form the 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 cetyltrimethylammonium bromide to the organic solvent is preferably 0.1458 g:10 mL, and the molar concentration ratio of water to cetyltrimethylammonium bromide in the reverse micelle system is preferably 10:1. By controlling the water content in the reverse micelle system in the present invention, most of the water in the reverse micelle can exist 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, it is preferably stirred for 1 h, and an optically transparent reverse micelle system can be observed to form. The DNA is dispersed in isooctane in the form of reverse micelle loading. In the present invention, the reverse micelle acts as a shell to protect the DNA from contacting the outer organic solvent, and the outer organic solvent isolates the DNA from the external 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 a sodium chloride solution, 1-butanol and the reverse micelle system obtained by the above method, and centrifuging to collect the lower-layer DNA aqueous solution; the concentration of the sodium chloride solution is 1 mol / L.

[0025] In the present invention, the volume ratio of the 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 the art can be used. In the present invention, the sodium chloride solution serves as an aqueous receiving phase, and the mixing is preferably stirred for 1 h. In the present invention, the centrifugation conditions are preferably centrifugation at 4°C and 10,000 rpm for 5 min. Through the method of back extraction in the present invention, the DNA stored in the reverse micelle can be released into the aqueous phase and can be applied to experimental work with requirements for DNA solvents.

[0026] The technical solutions provided by the present invention will be 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 are conventional methods.

[0028] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0029] Example 1

[0030] A method for preserving reverse micelle DNA consists of 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 cetyltrimethylammonium bromide was weighed and added to the organic solvent, and the mixture was stirred at room temperature for 0.5 h to form an organic phase of reverse micelles.

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

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

[0034] Example 2

[0035] A method for releasing reverse micelle DNA consists of 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, and stir and mix at 1000 rpm for 1 h. After that, centrifuge at 4 °C and 10,000 rpm for 5 min, and collect the lower aqueous phase solution, which is the DNA aqueous solution. The results are as Figure 2 shown.

[0037] Comparative Example 1

[0038] The same DNA as in Example 1 was dissolved in phosphate buffer (pH 7.4) with the same volume as 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-phase DNA obtained in Comparative Example 1 were stored at room temperature simultaneously. After being stored 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 a control group. The results are as Figure 3 shown. Using TIANGEN's DNA Marker Ⅲ as the molecular weight standard, it can be observed that compared with the fresh DNA solution ( Figure 3 with 2 bands in Figure 3 ), the DNA stored in the reverse micelle system of Example 1 (Figure 3 In the 4 bands) among them, the DNA with a molecular weight greater than 800 bp has undergone obvious degradation. It can be seen that the cationic reverse micelle system provided by the present invention can significantly improve the stability of DNA preservation at room temperature.

[0041] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope 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; mixing the organic phase with the aqueous phase to obtain a reverse micelle system for storage; The volume ratio of isooctane to 1-hexanol is 9.4:0.6; The mass volume ratio of hexadecyltrimethylammonium bromide to the organic solvent is 0.1458 g:10 mL; The molar concentration ratio of water to hexadecyltrimethylammonium bromide in the reverse micelle system is 10:

1.

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

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