A DNA purification reagent, preparation method thereof and DNA purification method

Abstract

The present invention provides a DNA purification reagent, a preparation method thereof and a DNA purification method. The raw materials for preparing the DNA purification reagent include component A and component B; the component A includes carboxyl magnetic beads; the component B includes: 33% to 38% by volume of ethanol, 3% to 5% by volume of tetraethylene glycol and 2.0 mol / L to 3.0 mol / L of NaCl; the ratio of the component A to the component B is: (1.5 to 2.5) g:1L. Compared with the prior art, the present invention adopts ethanol and tetraethylene glycol to absorb water molecules in the system together, and then cooperates with NaCl to precipitate DNA fragments, thereby increasing the precipitation amount, thereby increasing the subsequent adsorption amount of carboxyl magnetic beads, and improving the subsequent magnetic bead recovery rate.

Description

Technical Field

[0001] The present invention relates to the field of DNA purification, and in particular to a DNA purification reagent, a preparation method thereof and a DNA purification method. Background Art

[0002] Sanger sequencing is a method in which a nucleotide starts at a fixed point, terminates randomly at a specific base, and performs fluorescent labeling after each base to produce a series of nucleotides of four different lengths ending with A, T, C, and G. The sequence is then detected by electrophoresis on a urea-denatured PAGE gel to obtain a visible DNA base sequence. This method is widely used in sequencing bacterial plasmids and amplified products.

[0003] In the entire Sanger sequencing process, DNA needs to be purified and precipitated. The commonly used purification and precipitation method is the alcohol sodium acetate method. The magnetic bead method uses a solid phase carrier to adsorb DNA in a polyethylene glycol with a molecular weight of several thousand and a high salt environment, and finally washes and desorbs it with ethanol for recovery. The existing magnetic bead system includes: magnetic beads, polyethylene glycol, and salt ions. The system can precipitate and adsorb DNA. The magnetic bead reagent system can adsorb DNA fragments with a narrow molecular weight range, but cannot adsorb all fragments generated by PCR amplification. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a DNA purification reagent, a preparation method thereof and a DNA purification method.

[0005] The present invention adopts the following technical solution:

[0006] A DNA purification reagent, which is different in that its preparation raw materials include component A and component B; the component A includes carboxyl magnetic beads; the component B includes: 33% to 38% by volume of ethanol, 3% to 5% by volume of tetraethylene glycol and 2.0 mol / L to 3.0 mol / L of NaCl; the ratio of the component A to the component B is: (1.5 to 2.5) g:1L.

[0007] Compared with the prior art, the present invention uses ethanol and tetraethylene glycol to absorb water molecules in the system, and then cooperates with NaCl to precipitate DNA fragments, which can specifically bind to all fragments in the Sanger sequencing reaction, but not to dNTPs, ddNTPs, enzymes and other raw materials.

[0008] Furthermore, the B component also includes 6.0 mmol / L to 6.5 mmol / L EDTA.

[0009] The beneficial effect of adopting the above further technical solution is that the addition of EDTA removes salt ions in the purification system through complex reaction, thereby improving its purity.

[0010] The method for preparing the above DNA purification reagent is different in that it comprises:

[0011] Step S1: taking a carboxyl magnetic bead solution, removing the supernatant, and obtaining carboxyl magnetic beads;

[0012] Step S2: mixing ethanol, tetraethylene glycol and NaCl to obtain a premixed solution;

[0013] Step S3: mixing the carboxyl magnetic beads and the premixed solution into the DNA purification reagent.

[0014] Furthermore, in step S2, ethanol, tetraethylene glycol, NaCl and EDTA are mixed to obtain a premixed solution.

[0015] A method for DNA purification comprising:

[0016] Step A1: adding the above DNA purification reagent and ethanol to the sample to be purified, mixing well, and obtaining a mixed solution to be processed;

[0017] Step A2: placing the mixed solution to be treated on a magnetic rack for adsorption, and discarding the supernatant;

[0018] Step A3: then adding ethanol and mixing evenly to obtain a solution to be purified, placing the solution to be purified on a magnetic rack again for adsorption, and discarding the supernatant;

[0019] Step A4: Repeat step A3 1 to 3 times to recover the magnetic beads.

[0020] Compared with the prior art, the invention has the following beneficial effects: by adding the DNA purification reagent of the invention and ethanol to the DNA sample to be purified, all fragments generated by unidirectional PCR amplification can be adsorbed, and then all raw materials remaining in the reaction are removed by washing with 80% ethanol, thereby achieving the purpose of purification. The purified product can be directly subjected to electrophoresis sequencing on a genetic analyzer such as 3730xl; the signal is strong and uniform, and the background noise is extremely low.

[0021] Furthermore, the volume ratio of the sample to be purified to the DNA purification reagent is 1:(0.8-1.2).

[0022] Furthermore, the volume percentage of ethanol in the mixed liquid to be treated and the liquid to be purified is 40% to 50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a magnetic bead recovery test diagram of Example 5;

[0024] Figure 2 This is the magnetic bead recovery test diagram of Company A in Comparative Example 1;

[0025] Figure 3 This is the magnetic bead recovery test chart of the comparative example 1B company. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0027] The following embodiments are only used to illustrate the present invention, but are not limited to the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0028] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0029] In the examples of the present invention, the raw materials used are all conventional commercially available products.

[0030] In the embodiments of the present invention, the experimental raw materials involved are described as follows:

[0031] Carboxyl magnetic bead solution: The carboxyl magnetic bead solution of the present invention was purchased from Biomag Biotechnology, with a specification of 300 nm, 50 mg / ml, and a product number of 191116;

[0032] Ethanol solution: 80% v / v ethanol solution was purchased from (Sinopharm Group) 500 ml of anhydrous ethanol;

[0033] Tetraethylene glycol: sigma lot:04818AHV.

[0034] Embodiment 1 to Embodiment 4

[0035] Examples 1 to 4 provide DNA purification reagents, the specific components and contents of which are shown in Table 1:

[0036] Table 1 DNA purification reagent components and contents in Examples 1 to 4

[0037]

[0038] The preparation methods of Examples 1 to 4 are all prepared by the following steps:

[0039] Step S1: taking a carboxyl magnetic bead solution, adsorbing and removing the supernatant to obtain component A;

[0040] Step S2: mixing ethanol solution, tetraethylene glycol, NaCl and EDTA to obtain component B;

[0041] Step S3: Mix component A and component B to obtain a DNA purification reagent.

[0042] Embodiment 5 to Embodiment 8

[0043] Examples 5 to 8 provide a method for purifying DNA, and the specific operation method is as follows:

[0044] Step A1: Add 5ul of sequencing product to a 96-well plate, add 5ul of carboxyl magnetic bead mixture and ethanol, shake and mix to obtain a mixture to be processed;

[0045] Step A2: Place the 96-well plate on a magnetic stand, adsorb for 30 seconds, and remove the supernatant;

[0046] Step A3: Add ethanol and shake to mix for 1 min to obtain the solution to be treated. Place the 96-well plate on a magnetic rack, adsorb for 30 seconds, and remove the supernatant.

[0047] Repeat step A3 once and let stand at room temperature for 5 min.

[0048] After adding ethanol in step A1 and step A3, the final concentrations are the same, as shown in Table 2.

[0049] Table 2 Final concentration of ethanol in step A1 and step A3 of Example 5 to Example 8

[0050] Example DNA Purification Agents Ethanol (% v / v) Example 5 Example 1 45 Example 6 Example 2 50 Example 7 Example 3 40 Example 8 Example 4 40

[0051] Comparative Example 1

[0052] The commercially available MCLAB lot: L580A59M (referred to as Company A) magnetic bead system and Thrmo lot: 1706084 magnetic bead system (referred to as Company B) were used to purify the DNA samples according to the instructions.

[0053] 40 μL of sterilized water was added to the wells of the DNA magnetic beads purified by Example 5, Comparative Example 1 and Comparative Example 2, and the magnetic beads recovery efficiency was detected by centrifugation after mixing. The detection method was: 20 ul of each purified sample was taken for capillary electrophoresis detection. The higher the recovery efficiency, the stronger the fluorescence signal value.

[0054] It can be seen that the fluorescence signal values ​​of each figure are distributed from strong to weak, but compared with Company A and Company B, the embodiment has stronger y-axis signals corresponding to the x-axis as a whole, proving that the amount of DNA molecules recovered in each regional fragment using the magnetic bead system of Example 5 is greater than that of the commercially available magnetic bead system.

[0055] The results of Example 5, Comparative Example 1 and Comparative Example 2 are as follows Figures 1 to 3 The signal strength of the magnetic beads in each embodiment and comparative example is shown in Table 3.

[0056] Table 3 The strongest signal detected by magnetic beads

[0057] Detection object Magnetic bead signal Example 5 18000 Comparative Example 1A Company 6000 Comparative Example 1 Company B 12000

[0058] As can be seen from Table 3, the magnetic bead signal using the DNA purification reagent and extraction method of the present invention is much greater than that of commercially available companies.

[0059] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A DNA purification reagent, characterized in that The preparation raw materials include component A and component B; the component A includes carboxyl magnetic beads; the component B includes: 33% to 38% by volume of ethanol, 3% to 5% by volume of tetraethylene glycol and 2.0 mol / L to 3.0 mol / L of NaCl; the ratio of the component A to the component B is: (1.5 to 2.5) g:1L; The specification of the carboxyl magnetic beads is 300nm; The B component also includes 6.0 mmol / L to 6.5 mmol / L EDTA.

2. The method for preparing a DNA purification reagent according to claim 1, wherein include: Step S1: taking a carboxyl magnetic bead solution, removing the supernatant, and obtaining carboxyl magnetic beads; Step S2: mixing ethanol, tetraethylene glycol, NaCl and EDTA to obtain a premixed solution; Step S3: mixing the carboxyl magnetic beads and the premixed solution into the DNA purification reagent.

3. A DNA purification method, characterized in that: include: Step A1: adding the DNA purification reagent of claim 1 and ethanol to the sample to be purified, and mixing well to obtain a mixed solution to be treated; Step A2: placing the mixed solution to be treated on a magnetic rack for adsorption, and discarding the supernatant; Step A3: then adding ethanol and mixing evenly to obtain a solution to be purified, placing the solution to be purified on a magnetic rack again for adsorption, and discarding the supernatant; Step A4: Repeat step A31 to 3 times to recover the magnetic beads.

4. The DNA purification method according to claim 3, characterized in that The volume ratio of the sample to be purified to the DNA purification reagent is 1:(0.8-1.2).

5. The DNA purification method according to claim 3, characterized in that The volume percentage of ethanol in the mixed liquid to be treated and the liquid to be purified is 40% to 50%.

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