NUCLEIC ACID HOMOGENIZATION METHOD, AND KIT AND USE THEREOF

Abstract

The present disclosure provides a nucleic acid homogenization method, and a kit and use thereof. The method including at least the following steps: respectively adding nucleic acid adsorption materials having the same nucleic acid saturation adsorption amount into a plurality of nucleic acid solutions, and the nucleic acid adsorption materials added into each nucleic acid solution can all achieve a nucleic acid adsorption saturation state; separating the nucleic acid adsorption materials of the saturation absorbed nucleic acids; eluting the nucleic acids from the separated nucleic acid adsorption materials. The nucleic acid homogenization method disclosed in the present disclosure is easy to operate, and allows for rapid and stable equal proportional dilution of a nucleic acid, a PCR product or a high throughput sequencing library concentration.

Claims

1. A nucleic acid homogenization method, comprising at least the following steps: (1) respectively adding nucleic acid adsorption materials having a same nucleic acid saturation adsorption amount into a plurality of nucleic acid solutions, wherein the nucleic acid adsorption materials added into each nucleic acid solution achieves a nucleic acid adsorption saturation state; (2) separating the nucleic acid adsorption materials of the saturation absorbed nucleic acids; (3) eluting the nucleic acids from the separated nucleic acid adsorption materials.

2. The nucleic acid homogenization method according to claim 1, wherein the nucleic acid adsorption materials are coated with any one or more of a carboxyl group, an amino group, a hydroxyl group, and a silicon group.

3. The nucleic acid homogenization method according to claim 1, wherein the nucleic acid adsorption materials added into each of the nucleic acid solutions is the same and has the same amount.

4. The nucleic acid homogenization method according to claim 3, wherein the nucleic acid adsorption materials are nano-microspheres or glass particles.

5. The nucleic acid homogenization method according to claim 4, wherein the nucleic acid adsorption materials are monodisperse nano-microspheres or monodisperse glass particles.

6. The nucleic acid homogenization method according to claim 3, wherein the nano-microspheres are capable of being magnetically adsorbed.

7. The nucleic acid homogenization method according to claim 3, wherein the nano-microspheres are formed by coating Fe.sub.3O.sub.4 with oleic acid.

8. The nucleic acid homogenization method according to claim 3, wherein an average particle diameter of the nano-microspheres is 0.5 to 2 m.

9. The nucleic acid homogenization method according to claim 1, further comprising step (4): adding a solvent to the nucleic acid.

10. A kit for nucleic acid homogenization, wherein the kit comprises a nucleic acid adsorption material, and the nucleic acid adsorption material is coated with any one or more of a carboxyl group, an amino group, a hydroxyl group, and a silicon group.

11. The kit according to claim 10, wherein the nucleic acid adsorption material is nano-microspheres or glass particles.

12. The kit according to claim 10, wherein the kit further comprises at least one of ethanol with a volume fraction of 70-85%, ddH.sub.2O or Tris-HCL buffer.

13. Use of the kit according to claims 10 to 12 for nucleic acid homogenization.

14. Use of the kit according to claim 11 for nucleic acid homogenization.

15. Use of the kit according to claim 12 for nucleic acid homogenization.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The implementation mode of the present disclosure will be described below through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present disclosure according to contents disclosed by the specification. The present disclosure can also be implemented or applied through other different specific implementation modes. Various modifications or changes can also be made to all details in the specification based on different points of view and applications without departing from the spirit of the present disclosure. It should be noted that processing equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the field. In addition, it should be understood that one or more method steps mentioned in the present disclosure are not exclusive of other method steps that may exist before or after the combined steps or that other method steps may be inserted between these explicitly mentioned steps, unless otherwise stated; it should also be understood that the combined connection relationship between one or more equipment/devices mentioned in the present disclosure does not exclude that there may be other equipment/devices before or after the combined equipment/devices or that other equipment/devices may be inserted between these explicitly mentioned equipment/devices, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of each method step or to limit the scope of the present disclosure. The change or adjustment of the relative relationship shall also be regarded as the scope in which the present disclosure may be implemented without substantially changing the technical content.

[0031] In the present specification and claims, the singular forms a, an and the include the plural forms, unless specifically stated otherwise.

[0032] When the numerical values are given by the embodiments, it is to be understood that the two endpoints of each numerical range and any value between the two endpoints may be selected unless otherwise stated. Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one skilled in the art. In addition to the specific method, equipment and material used in the embodiments, any method, equipment and material in the existing technology similar or equivalent to the method, equipment and material mentioned in the embodiments of the present disclosure may be used to realize the invention according to the grasp of the existing technology and the record of the invention by those skilled in the art.

[0033] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all employ conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology in the technical field and related fields. These techniques are well described in the prior literature. For details, please see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third Edition, Academic Press, San Diego, 1998; METHOD IN ENZYMOLOGY, Vol. 304, Chromatin (PMWassarman and APWolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, etc.

[0034] The reagents in the following embodiments are all commercially available.

Embodiment 1

Preparation of Nano-Microspheres

[0035] 1.1 Under an atmosphere of nitrogen, adding excess aqueous ammonia to the Fe2+/Fe3+ salt solution, and reacting for 0.5 hours at 80 C. to obtain Fe.sub.3O.sub.4. Adding 20% oleic acid, incubating for 1.5 hours at room temperature, and then washing with ddH.sub.2O until pH reaches 7.0. After the supernatant is removed by magnetic separation, washing with 100% ethanol for 3 times, and vacuum-drying at 40 C. to obtain the nano-microsphere powder. Adding octane solution at 1:1 w/v to obtain oleic acid-embedded Fe.sub.3O.sub.4 nano-microsphere mixture. Then, adding ddH.sub.2O and surfactant, sonicating for 30 min to form a fine emulsion bl. Preparing a 0.1 wt % SDS solution, adding styrene at 1:1 v/v, and the solution is emulsified to form solution b2 after being stirred evenly. Mixing b1 and b2 at 1:3v/v, adding ddH2O-soluble initiator and stirring for 0.5 hour, transferring to a ddH2O bath at 75 C. and reacting for 18 hours, then magnetic composite microspheres can be obtained. Next, adding 0.5 wt % Tween-20 solution to the above magnetic composite microspheres, sonicating for 30 minutes, discarding the supernatant after magnetic separation. The sonication/magnetic separation is performed alternately for 3 times to obtain a stable and uniformly dispersed nano-microsphere polymer suspension pre-LibNorm of 1 wt %. Under the catalysis of ammonia, adding glycerol and pre-LibNorm to ethyl ortho silicate at 1:1:1 v/v, and reacting at room temperature for 24 hours. Washing the product with 100% ethanol/ddH2O alternately for three times, and vacuum-drying at 40 C. to obtain the final product. A hydrophilic, monodisperse, high magnetic nano magnetic composite microsphere LibNorm (nano-microspheres for short) is obtained. The test result indicates that the average particle diameter of the nano-microspheres is 0.5 to 2 m.

[0036] 1.2 Due to the excellent hydrophilic properties, the LibNorm magnetic microspheres can be stably dispersed as a uniform nano-microsphere suspension in a pre-prepared diluted matrix LibNorm buffer.

Embodiment 2

Automated Nano-Microsphere Dilution Gradient Verification

[0037] Diluting the nano-microspheres in embodiment 1 with the dilution gradients of: 10, 30, 90, 120, 240, 360, 480, 540, 600, 720, 900 (one duplicate well per gradient). Equal proportional dilution is performed on 20 L, nucleic acid of 20 ng/L with a programmed iNaSP automatic instrument (adding 454, of nano-microspheres to the nucleic acid, shaking and capturing for 5 min at room temperature, discarding the supernatant, adding 90 L of 10 mM Tris-HCL pH8.5 elution buffer, and adding the same amount of solvent), and using Qubit3.0 to detect the concentration.

[0038] The results are as follows:

TABLE-US-00001 TABLE 1 Equal proportional dilution results of LibNorm beads nano- microspheres by iNaSP automatic operation (unit: ng/L) Dilution ratio Parallel 1 Parallel 2 Ave. 10x 6.68 6.72 6.70 30x 5.16 5.08 5.12 90x 3.88 4.12 4.00 120x 4.48 3.98 4.23 240x 2.81 3.05 2.93 360x 2.92 2.46 2.69 480x 2.46 2.77 2.615 540x 2.28 2.44 2.36 600x 2.12 2.44 2.28 720x 1.66 2.02 1.84 900x 2.02 1.85 1.935

[0039] According to the results in Table 1, in the test of equal proportional diluting nucleic acid with nano-microspheres by iNaSP automatic instrument, the saturated adsorption dilution ratio of LibNorm beads is 120.

Embodiment 4

Experiment of Equal Proportional Dilution of Original Samples with Different Concentrations with Nano-Microspheres Operated by iNaSP Automatic Instrument

[0040] Using 120-diluted nano-microspheres to verify the equal proportional dilution of 8 nucleic acids with different concentrations (Ct values ranging from small to large). Adding 45 L, of nano-microspheres to each nucleic acid, after shaking and capturing at room temperature for 5 min, discarding the supernatant, adding 90 L of 10 mM Tris-HCL pH8.5 elution buffer, and then adding the same amount of solvent). Each sample employs three duplicate wells.

[0041] Qubit3.0 test results are as follows:

TABLE-US-00002 TABLE 2 Results of automated equal proportional dilution of cervical secretion nucleic acid (unit: ng/L) Initial Ct iNaSP automatic operation Manual operation value Parallel 1 Parallel 2 Parallel 3 Parallel 1 Parallel 2 19.0 1.45 1.80 1.69 1.31 1.36 21.4 1.52 1.59 1.43 1.38 1.46 24.9 1.18 1.39 1.67 1.12 1.37 28.2 1.12 1.44 1.50 1.44 1.28 28.5 1.22 1.41 1.28 1.32 1.29 31.4 1.25 1.52 1.30 1.28 1.33 31.6 1.21 1.25 1.43 1.41 1.28 32.6 1.21 1.26 1.53 1.30 1.18

[0042] The results in Table 2 indicate that for the starting template with different concentrations, the requirement of equal proportional dilution can be achieved after the equal proportional dilution by 120 diluted nanometer microspheres (CV<0.05). That is, the iNaSP automated operating system can successfully achieve automation of LibNorm beads nano-microspheres with good stability. At the same time, the results also show that the iNaSP automated equal proportional dilution of nucleic acid (automation group, the same below) can be equivalent to manual operation.

Embodiment 5

Equal Proportional Dilution of Nucleic Acid of Mouse Small Intestine Tissue by Libnorm Beads

[0043] 1) Weighing 8 part of small intestine tissue of mice, each part has a weight of 20 mg, and extracting total nucleic acid with NucleoMag 96 Tissue;

[0044] 2) Adding 90 L of distilled ddH.sub.2O to 10 L of the nucleic acid extracted in the step 1), completing a 10-fold dilution, and obtaining a nucleic acid sample of 200 ng/L Taking 20 L of the 10-fold diluted nucleic acid, and adding 45 L of 120-fold diluted LibNorm beads nano-microspheres. After shaking and capturing for 5min at room temperature, discarding the supernatant, adding 90L, of 10 mM Tris-HCL solution with pH8.5 to elute, and adding solvent of the same amount. Finally, detecting the nucleic acid concentration after being diluted in equal proportion by using Qubit3.0;

[0045] 3) non-nano-microsphere group and nano-microsphere automation group are used as control groups.

[0046] The quantitative results of Qubit3.0 are shown in Table 3.

TABLE-US-00003 TABLE 3 Concentration detection results of nucleic acid of mouse small intestine tissue after equal proportional dilution (unit: ng/L) Non-nano- Serial microsphere Nano-microsphere group Nano-microsphere number group (manual operation) automation group 1 1.39 1.46 1.58 2 1.40 1.48 1.55 3 1.45 1.45 1.54 4 1.43 1.52 1.57 5 1.39 1.49 1.55 6 1.50 1.51 1.57 7 1.47 1.47 1.54 8 1.46 1.45 1.56

[0047] The results in Table 3 show that the nano-microsphere groups (manual operation groups and automation groups) can achieve the effect of diluting nucleic acids in equal proportions as the non-nano-microsphere group. The nano-microsphere group (manual operation) and nano-microsphere automation group are basically equivalent, and the latter has smaller differences in concentration between different equal proportional dilution treatments, that is, it is more stable.

Embodiment 6

Equal Proportional Dilution Results of Human Saliva Sample Microbial 16S rDNA Community Microecological High-throughput Sequencing Library

[0048] 1) Taking 2004, of freshly collected saliva and using QIAamp DNA Mini Kit to extract total nucleic acid;

[0049] 2) 16S rDNA PCR: Preparing a PCR system-10*buffer 5 Mg.sup.2+ (25 mM) 4 L dNTP (10 mM) 1 L, Taq enzyme 0.5 L, ddH.sub.2O 12.5 L, Amplicon PCR Forward/Reverse Primer (1 M) 0.5 L each, DNA template 1 L (all for each person); PCR conditions are95 C. for 3 minutes; 95 C. for 30 seconds, 55 C. for 30 seconds, 72 C. for 30 seconds, 25 cycles; another 72 C. for 5 minutes.

[0050] 3) The PCR products are purified by AMPure XP beads and then linked with Illumina index linkers. The linker PCR system is 10*buffer 5 Mg.sup.2+ (25 mM) 4 L, dNTP (10 mM) 1 L, Taq enzyme 0.5 L, ddH.sub.2O 24.5 L, 5 L each for Index 1 and Index 2, 5 L for DNA template. The PCR condition is 95 C. for 3 minutes; 95 C. for 30 seconds, 55 C. for 30 seconds, 72 C. for 30 seconds, 8 cycles; another 72 C. for 5 minutes;

[0051] Taking 20 L of sequencing library with different concentrations, respectively, and adding 45 L of 120-fold diluted LibNorm beads nano-microspheres. After shaking and capturing for 5min at room temperature, discarding the supernatant, adding 90L, of 10 mM Tris-HCL solution with pH8.5 to elute, and adding solvent of the same amount of into each sample, finally, detecting the concentration for each sequencing library that has been diluted in equal proportion by using Qubit3.0;

[0052] 5) non-nano-microsphere group and nano-microsphere automation group are used as control groups.

[0053] The quantitative results of Qubit3.0 are shown in Table 4:

TABLE-US-00004 TABLE 4 Concentration detection results of high-throughput sequencing library of saliva samples (unit: ng/L) Serial Non- Nano microsphere group Nano-microsphere number nanospheres (manual operation) automation group 1 1.48 1.60 1.61 2 1.50 1.59 1.58 3 1.51 1.55 1.57 4 1.33 1.54 1.54 5 1.29 1.44 1.58 6 1.47 1.52 1.60 7 1.51 1.55 1.55 8 1.38 1.46 1.59

[0054] The results in Table 4 show that the nano-microsphere groups (manual operation groups and automation groups) can achieve the effect of diluting the high throughput sequencing libraries in equal proportions as the non-nano-microsphere group. The nano-microsphere group (manual operation) and nano-microsphere automation group are basically equivalent, and the latter has smaller differences in concentration between different equal proportional dilution treatments, that is, it is more stable.

Embodiment 7

Equal Proportional Dilution of Nucleic Acid of Mouse Small Intestine Tissue by Glass Beads

[0055] 1) Taking 8 parts of mouse small intestine total nucleic acid with different concentrations;

[0056] 2) adding 90L, of distilled ddH.sub.2O to 10 L, of the nucleic acid extracted in the step 1), completing a 10-fold dilution. Taking 20 L of 10-fold diluted nucleic acid, and adding 45 L of 150-fold diluted glass beads (diameter of 0.5 to 2 m), such that the nucleic acid samples absorbed by glass beads in each sample are saturated. After shaking and capturing for 5 min at room temperature, discarding the supernatant after centrifugation at 12000rpm for 5 min, adding 90 L, of 10 mM Tris-HCL pH8.5 solution to elute, finally, detecting the concentration for each nucleic acid that has been diluted in equal proportion by using Qubit3.0;

[0057] 3) non-glass beads group and glass beads automation group are used as control groups.

[0058] Qubit3.0 quantitative results are shown in Table 5:

TABLE-US-00005 TABLE 5 Concentration detection results of nucleic acid of mouse small intestine tissue by equal proportional dilution by glass beads (unit: ng/L) Initial Ct Non-glass Glass beads group Glass bead value beads group (manual operation) automation group 18.0 1.43 1.47 1.54 20.2 1.42 1.51 1.57 22.5 1.46 1.49 1.56 26.2 1.44 1.51 1.54 28.1 1.52 1.54 1.58 30.4 1.49 1.53 1.56 31.8 1.44 1.49 1.55 33.2 1.46 1.55 1.54

[0059] The results in Table 5 show that the glass beads groups (manual operation and automation groups) can achieve the effect of diluting nucleic acids in equal proportions as the non-glass beads group. The glass beads group (manual operation) and the glass beads automation group are basically equivalent, and the latter has smaller differences in concentration between different equal proportional dilution treatments, that is, it is more stable.

[0060] The above embodiments are intended to illustrate the disclosed embodiments of the disclosure and are not understood as restrictions on the disclosure. In addition, various modifications of the present disclosure, as well as variations of the methods and compositions of the disclosure, will be apparent to those skilled in the art without departing from the scope of the disclosure. While the disclosure has been described in detail in connection with various specific preferred embodiments thereof, however, it should be understood that the present disclosure should not be limited to these specific embodiments. In fact, various modifications to the disclosure as apparent to those skilled in the art are intended to be included within the scope of the disclosure.