DNA / RNA co-extraction method and kit based on aqueous two-phase-temperature-induced phase separation

Abstract

The application discloses a DNA / RNA co-extraction method and kit based on a double-water-phase temperature-induced phase separation, which utilizes polyethylene glycol-sodium citrate to form a primary double-water-phase system at 18-25 DEG C, and by adding an ionic liquid modified temperature-sensitive polymer (IL-NIPAM) with a LCST of 32 DEG C plus or minus 1 DEG C, the IL-NIPAM selectively binds with RNA, and the separation of RNA and DNA in a biological sample is realized by temperature regulation. The DNA / RNA co-extraction kit includes a double-water-phase premix, and the IL-NIPAM is added in the double-water-phase premix in advance or during use, and the use concentration of the IL-NIPAM is 0.5-1.5% w / w. The method does not need proteinase K digestion and does not need to add organic solvents, and can quickly and efficiently recover DNA, mRNA and miRNA, and is suitable for the co-extraction of nucleic acids from trace samples such as fingertip blood, dry blood spots and FFPE sections, and is suitable for automation and large-scale production.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and bioseparation technology, specifically relating to a DNA / RNA co-extraction method and its dedicated kit based on a combination of aqueous two-phase system (ATPS) and temperature-induced phase separation (TIPS), which is particularly suitable for the simultaneous separation and purification of nucleic acids in trace amounts of clinical samples. Background Technology

[0002] Nucleic acid extraction is a crucial pretreatment step in molecular diagnostics, gene testing, and precision medicine. Traditional methods for separate DNA and RNA extraction require large sample volumes, posing significant limitations for valuable samples such as finger-prick blood and biopsy tissue. While various DNA / RNA co-extraction methods exist, the following technical bottlenecks remain: The classic Trizol method utilizes an acidic phenol-chloroform-isoamyl alcohol system to separate RNA and DNA through pH differences. Its advantages include high recovery rates, but its disadvantages are: (1) high toxicity of the organic solvents, high operational risks, and high wastewater treatment costs; (2) cumbersome operation steps (>15 steps) and long processing time (>60 minutes); (3) severe protein residue at the phase interface significantly impacts downstream applications; and (4) unsuitable for automated and high-throughput processing.

[0003] Silica membrane column chromatography requires multiple high-speed centrifugations (≥8000 g), which mechanically shears RNA and causes degradation. Small RNAs such as miRNAs have low adsorption efficiency on silica membranes (<50%), making it difficult to meet the requirements of miRNA sequencing. Sample volume is limited (usually >100 μL), and the recovery rate of trace samples drops sharply. There are large inter-column differences and poor batch reproducibility (CV>15%).

[0004] 3. The magnetic bead method requires two extractions, resulting in a large sample consumption; the surface modification of magnetic beads is complex and costly; the cross-contamination rate between RNA and DNA is high (>5%); and it requires high technical skills from the operator.

[0005] Aqueous two-phase systems (ATPS) are two-phase systems formed by two immiscible hydrophilic polymers (such as PEG / dextran) or polymer / salt (such as PEG / phosphate) in an aqueous solution. Due to their low interfacial tension and good biocompatibility, they have been widely used for the separation and purification of proteins and cells.

[0006] In recent years, some scholars have attempted to use ATPS for nucleic acid extraction: Patent document CN105274145A describes the extraction of plasmid DNA using a PEG / ammonium sulfate system, but does not involve RNA; technical document (Anal. Chem. 2018) describes the extraction of viral RNA using PEG / citric acid, but the DNA recovery rate is low. The core defect of the existing ATPS method is the lack of an active regulatory mechanism; the partition coefficients of DNA and RNA in the two phases are fixed, making it impossible to achieve highly selective separation. As nucleic acid substances, the partition of DNA / RNA in an aqueous two-phase system may be affected by temperature, salt concentration, phase composition, etc.

[0007] Temperature-responsive polymers (such as poly(N-isopropylacrylamide) PNIPAM) have low critical dissolution temperatures (LCST). They dissolve hydrophilically below the LCST and aggregate and precipitate hydrophobically above the LCST. However, simply adding PNIPAM to an aqueous two-phase system results in weak affinity between PNIPAM and nucleic acids, making effective enrichment difficult.

[0008] Ionic liquids (ILs) have emerged as novel functional materials due to their designability, conductivity, and specific recognition capabilities for biomolecules. The π-π stacking and electrostatic interactions of imidazole ionic liquids with nucleic acids have been demonstrated (see technical literature, J. Am. Chem. Soc. 2015), but their stability and water solubility limit their application in DNA / RNA co-extraction.

[0009] Currently, there are no reports on DNA / RNA co-extraction technologies that combine the low-interference separation environment of aqueous two-phase systems, the dynamic control capability of temperature-responsive materials, and the nucleic acid recognition characteristics of ionic liquids. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a DNA / RNA co-extraction method and a dedicated kit for aqueous phase-temperature induced phase separation, which enables rapid and efficient recovery of DNA, mRNA and miRNA under conditions without organic solvents and without proteinase K digestion. This method is suitable for the simultaneous separation and purification of nucleic acids in trace samples and is applicable to automated and large-scale production.

[0011] To achieve the above objectives, the present invention provides the following technical solution: Option 1: This invention provides a DNA / RNA co-extraction method based on aqueous two-phase-temperature-induced phase separation. The method is characterized by constructing a PEG-sodium citrate primary aqueous two-phase system containing the ionic liquid-modified temperature-sensitive polymer IL-NIPAM to process the biological sample to be separated. IL-NIPAM is a 1-butyl-3-methylimidazolium cationic-modified poly(N-isopropylacrylamide) copolymer with the structural formula [BMIM]⁺-PNIPAM, an LCST of 32±1℃, and a molecular weight of 8000–15000 Da. The PNIPAM backbone provides temperature responsiveness, and the [BMIM]⁺ side chains provide selective RNA recognition. When the system temperature is below the LCST of IL-NIPAM, IL-NIPAM is in a hydrophilic swelling state, binding RNA and uniformly dispersed in the aqueous two-phase system. When the system temperature is above the LCST of IL-NIPAM, IL-NIPAM undergoes a phase transition and selectively carries RNA into the upper phase, while DNA remains in the lower phase. Active separation of DNA and RNA is achieved through temperature regulation.

[0012] A method for co-extraction of DNA / RNA based on aqueous two-phase-temperature-induced phase separation, characterized by the following steps: (1) The pretreated biological sample is mixed with an aqueous two-phase premix, wherein IL-NIPAM is added in advance or during use, and the concentration of IL-NIPAM is 0.5-1.5% w / w; the aqueous two-phase premix contains 8-12% polyethylene glycol 6000 by mass, 10-15% sodium citrate by mass, and the pH is 7.0±0.1; (2) The mixture is allowed to stand at 18-25℃ for 3-8 minutes to form a stable aqueous two-phase system. The system exhibits a layered state with a transparent upper phase and a turbid lower phase. The interfacial tension is <0.1 mN / m and the viscosity is <50 mPa·s. (3) Raise the temperature of the aqueous two-phase system to 32-37°C and maintain it for 5-10 minutes. IL-NIPAM undergoes a phase transition, carrying RNA selectively migrating to the upper phase while DNA remains in the lower phase. (4) Collect the upper phase solution enriched with RNA and the lower phase solution enriched with DNA by centrifugation or static separation; (5) Purify the separated RNA and DNA.

[0013] The performance indicators of the above method are as follows: phase separation time ≤ 10 minutes; miRNA partition coefficient > 9.5 (upper phase miRNA concentration / lower phase miRNA concentration); total RNA recovery rate > 80%; DNA recovery rate > 85%; RNA integrity value (RIN) > 7.5; DNA purity (A260 / A280): 1.8–2.0; RNA purity (A260 / A280): 1.9–2.1.

[0014] In the above method, the biological sample is selected from one or more of the following: peripheral blood, fingertip blood, dried blood spots, paraffin-embedded tissue (FFPE) sections, fresh tissue, frozen tissue, saliva, urine, cell culture medium, and feces, with a sample volume of 5-100 μL or a tissue mass of 1-20 mg.

[0015] The pretreatment procedures for the biological samples are as follows: For whole blood samples, add red blood cell lysis buffer and incubate at 4°C for 5-10 minutes; for FFPE sections, add dewaxing solution and treat for 10-15 minutes, then add lysis buffer; for tissue samples, add lysis buffer containing 0.5-1.5% SDS, and mechanically disrupt or vortex mix.

[0016] The purification methods for the RNA and DNA collected in step (5) of the above method are as follows: (A) RNA purification: Add 1.5-2.5 times the volume of anhydrous ethanol, capture RNA with magnetic beads, wash 2-3 times with 70% ethanol, and elute with 10-30 μL of nuclease-free water; or, add an equal volume of isopropanol and 0.2 M NaCl, precipitate at -20℃ for 30 minutes, and collect the RNA precipitate by centrifugation; (B) DNA purification: Add 0.8-1.2 volumes of anhydrous ethanol, capture DNA using magnetic beads, wash 2-3 times with 80% ethanol, and elute with 20-50 μL of nuclease-free water; or, after diluting 2-5 times, purify using a silica membrane column.

[0017] The innovations of the DNA / RNA co-extraction method provided by this invention are as follows: First, a two-phase-temperature-induced cascade separation system is created. A PEG-sodium citrate primary two-phase system is constructed as a separation platform, and IL-NIPAM is introduced as a smart carrier. The active migration of RNA is achieved through temperature regulation: (1) 18-25℃: IL-NIPAM is in a hydrophilic swelling state. It binds RNA through the electrostatic interaction between imidazole cations and phosphate groups of the RNA backbone. At this time, it is uniformly dispersed in the two-phase system; (2) 32-37℃: IL-NIPAM reaches the LCST phase transition temperature, changes from hydrophilic to hydrophobic, aggregates to form RNA-rich nanoparticles, and floats to the upper phase due to the decrease in density; (3) DNA, due to its large molecular weight and weak affinity for IL-NIPAM, is mainly retained in the lower phase due to the exclusion effect of PEG.

[0018] Second, we designed and synthesized poly(N-isopropylacrylamide) (PNIPAM) modified with 1-butyl-3-methylimidazolium ([BMIM]⁺), wherein: (1) PNIPAM backbone: provides temperature responsiveness (LCST 32℃); (2) [BMIM]⁺ side chain: provides RNA selective recognition (π-π stacking with RNA bases, electrostatic interaction with phosphate groups); (3) Synergistic effect of [BMIM]⁺ and PNIPAM backbone: LCST phase transition driving force + ionic liquid affinity, which increases the RNA partition coefficient to >9.5.

[0019] Thirdly, by adjusting the molecular weight of PEG, the concentration of sodium citrate, and the pH value, we can achieve (1) rapid formation of the primary aqueous two-phase within minutes; (2) interfacial tension <0.1 mN / m, reducing the damage of shear force to RNA; and (3) viscosity <50 mPa·s, facilitating pipetting and automated operation.

[0020] Option 2: This invention provides a dedicated kit for DNA / RNA co-extraction, characterized by comprising the following reagents: (1) Aqueous two-phase premix, pH 7.0±0.1, containing PEG 6000 8~12% w / w, sodium citrate 10~15% w / w; and also containing one or more of the following reagents: phase separation indicator dye: bromothymol blue 0.005~0.02% w / w; nucleic acid protectant: EDTA 1~5 mM; surfactant: SDS 0.1~0.5% w / v; reducing agent: dithiothreitol (DTT) 0.5~2 mM; (2) IL-NIPAM lyophilized microspheres or concentrate, LCST 32±1℃; (3) Sample lysis buffer, containing 20 mM Tris-HCl pH 7.5, 2 mM EDTA, and 0.5-1.5% SDS; (4) RNA purification magnetic bead suspension, with magnetic bead particle size of 0.5-2 μm; (5) DNA purification magnetic bead suspension, with magnetic bead particle size of 0.8–2.5 μm; (6) Nuclease-free water; The aqueous two-phase premix and IL-NIPAM lyophilized microspheres or concentrate are packaged separately and mixed in proportion before use, or premixed as a single component and packaged. The concentration of IL-NIPAM used is 0.5-1.5% w / w.

[0021] Furthermore, the above-described reagent kit is characterized by further comprising one or more of the following reagents: Red blood cell lysis buffer: 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA; (2) Dewaxing solution: 100% xylene or dewaxing agent; (3) RNA precipitation buffer: 70% (v / v) anhydrous ethanol, 0.2 M NaCl; (4) DNA precipitation buffer: 80% (v / v) anhydrous ethanol, 50 mM NaCl; (5) Positive control nucleic acid standard.

[0022] The preparation method of the IL-NIPAM includes the following steps: (1) Add 10.0 g of N-isopropylacrylamide (NIPAM), 2.5 g of 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), and 0.2 g of azobisisobutyronitrile (AIBN) to a three-necked flask, dissolve in 100 mL of anhydrous ethanol, and react at 70 °C for 24 hours under nitrogen protection. (2) Purification treatment: The solvent was removed by rotary evaporation of the reaction solution, dissolved in deionized water and dialyzed for 72 hours. The molecular weight cutoff was 8000 Da. The water was changed every 8 hours. The dialysate was freeze-dried to obtain 9.8 g of white powder IL-NIPAM with a molecular weight of 8000-15000 Da, with a yield of 78%.

[0023] The preparation method of the IL-NIPAM freeze-dried microspheres includes: (1) dissolving IL-NIPAM powder in ultrapure water to prepare a 10-20% (w / v) concentrate; (2) adding trehalose or mannitol as a freeze-drying protectant, with the mass ratio of the freeze-drying protectant to the IL-NIPAM concentrate being 1:1 to 1:3; (3) dispensing and then freeze-drying, pre-freezing at -45°C for 4 hours, first drying at -35°C for 24 hours, and second drying at 25°C for 6 hours; (4) sealing and packaging in a dry nitrogen environment and storing at 2-8°C.

[0024] Beneficial effects of the present invention Compared with existing DNA / RNA co-extraction technologies, this invention has the following significant advantages: 1. Green and safe: The DNA / RNA co-extraction method of this invention uses an aqueous two-phase system throughout the process without the need to add organic solvents such as phenol and chloroform; it does not require proteinase K digestion (avoiding RNA degradation caused by long-term incubation at 56°C); the reagents are non-toxic, and the waste liquid can be directly discharged.

[0025] 2. Fast and efficient: The DNA / RNA co-extraction method of this invention can control the total operation time to <20 minutes (traditional methods require 60-90 minutes); the phase separation time is ≤10 minutes (the silica membrane column method requires multiple centrifugations totaling 15 minutes); and 24 samples can be processed manually (the Trizol method only processes 6-8 samples).

[0026] 3. High recovery rate and selectivity Table 1. Comparison of RNA and DNA recovery rates between the method of the present invention and the Trizol method and the silica membrane column method. index Method of the present invention Trizol method Silicon film column method Total RNA recovery >80% 75-85% 65-75% miRNA recovery rate >75% 60-70% <50% DNA recovery rate >85% 70-80% 80-85% RNA / DNA cross-contamination <2% <5% <3% miRNA allocation coefficient >9.5 N / A N / A 4. Applicability to micro-samples: The DNA / RNA co-extraction method of this invention is suitable for the simultaneous separation and purification of nucleic acids in micro-samples with a volume of 5-100 μL or a tissue mass of 1-20 mg, and is applicable to scenarios such as newborn screening and liquid biopsy.

[0027] 5. RNA integrity protection: The aqueous two-phase system has strong pH buffering capacity, inhibiting RNase activity; there is no mechanical shearing, and the RIN value is stable at 7.5-9.0 (6.5-8.0 for the silica membrane column method); miRNA is not degraded, and the Ct value repeatability CV is <3%.

[0028] 6. Automation compatibility: The DNA / RNA co-extraction method of this invention has programmable control for the liquid aspiration-mixing-temperature control-separation steps, and is compatible with automated platforms such as Applied Biosystems MagMAX and QIAGEN QIAcube; it can process 96-well plates with a throughput of >200 samples / day.

[0029] 7. Cost advantage: The reagent cost of the method of this invention is lower than that of the Trizol method and the magnetic bead method. IL-NIPAM is recyclable and regenerable, which reduces the cost of use. Attached Figure Description

[0030] Figure 1 : Schematic diagram of the operation flow of the DNA / RNA co-extraction method of the present invention; Figure 2 Schematic diagram of the principle of aqueous two-phase temperature-induced phase separation; Figure 2 Note: At 25°C, IL-NIPAM disperses in the aqueous two-phase system and binds to RNA; at 37°C, IL-NIPAM undergoes a phase transition, aggregates, and carries RNA to the upper phase, while DNA remains in the lower phase. Figure 3 IL-NIPAM molecular structure: showing the chemical structure of the PNIPAM backbone and [BMIM]⁺ side chains; Figure 4 The temperature response mechanism of IL-NIPAM is shown in the temperature curves, which reveal the hydrophilic / hydrophobic transition before and after LCST. Figure 5 Phase separation time-recovery curves, where the X-axis represents temperature induction time (0-15 min); the Y-axis represents recovery rate (%); the three curves represent RNA recovery rate, DNA recovery rate, and phase separation integrity, respectively; the optimal point is the plateau phase reached at 8 min. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Example 1: Preparation of IL-NIPAM

[0032] 1-1. Monomer Synthesis Add 10.0 g of N-isopropylacrylamide (NIPAM), 2.5 g of 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), and 0.2 g of azobisisobutyronitrile (AIBN) to a 250 mL three-necked flask, dissolve in 100 mL of anhydrous ethanol, and react at 70 °C for 24 hours under nitrogen protection.

[0033] 1-2: Purification The reaction solution was removed by rotary evaporation to remove the solvent, dissolved in deionized water, and dialyzed (molecular weight cutoff 8000 Da) for 72 hours, with water changed every 8 hours. The dialysate was freeze-dried to give 9.8 g of white powder IL-NIPAM, yield 78%.

[0034] 1-3: Characterization (1) Nuclear magnetic resonance (¹H-NMR, D2O): δ 3.95 (m, 1H, -CH-), 1.15 (d, 6H, -CH3), 8.65 (s, 1H, imidazole-H2), 7.38 (s, 1H, imidazole-H4). (2) Gel permeation chromatography (GPC): Mn = 12,500 Da, PDI = 1.32; (3) Differential scanning calorimetry (DSC): LCST = 32.2℃; (4) Turbidity determination: transmittance >90% at 31℃, transmittance <10% at 33℃, phase transition temperature range <2℃.

[0035] Example 2: Preparation of Aqueous Two-Phase Premix 2-1. Preparation method: Dissolve 12 g of sodium citrate in 50 mL of ultrapure water preheated to 50 °C, and cool to room temperature; add 10 g of PEG 6000 and stir until completely dissolved; add Tris-HCl, EDTA, and bromothymol blue in sequence; adjust the pH to 7.0 ± 0.1 with 1 M NaOH; bring the volume to 100 mL, filter through a 0.22 μm filter membrane for sterilization, and store at 4 °C for 6 months.

[0036] Table 2. Preparation of Aqueous Two-Phase Premixed Solution Components Quality score (w / w) effect Polyethylene glycol 6000 10% Formation of the upper phase, blocking DNA Sodium citrate 12% The lower phase is formed, stabilizing the aqueous two-phase system. Bromothymol Blue 0.01% Phase separation indicator (upper phase blue) EDTA 2 mM Chelating metal ions and inhibiting nucleases Tris-HCl pH 7.0 10 mM Maintain pH stability Ultrapure water Supplement to 100% solvent 2-2. The quality control standards are as follows: (1) Appearance: No precipitation at room temperature, clear and transparent; (2) pH: 6.9-7.1; (3) Phase formation time: A clear interface is formed within 5 minutes after the sample is added; (4) No DNase / RNase contamination: negative electrophoresis test.

[0037] Example 3: Co-extraction of DNA / RNA from fingertip blood 3-1. Sample type: 10 μL of fingertip blood from healthy volunteers (the blood collection needle punctures the fingertip and flows out naturally). 10 μL of fingertip blood can yield RNA >10 ng (RIN >7.5) and DNA >30 ng.

[0038] 3-2. Experimental Procedure: (1) Sample pretreatment: Add 10 μL of finger prick blood to 100 μL of erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), incubate at 4℃ for 5 minutes, centrifuge at 3000 g for 5 minutes, discard the supernatant, and the precipitate is white blood cells.

[0039] (2) Cell lysis: Add 50 μL of lysis buffer (20 mM Tris-HCl pH 7.5, 2 mM EDTA, 1% SDS) to the leukocyte pellet, incubate at room temperature for 3 minutes, and vortex until no visible cell clusters are visible.

[0040] (3) Aqueous two-phase formation: Add 100 μL of aqueous two-phase premix and 10 μL of IL-NIPAM stock solution (10% w / v, to a final concentration of 0.8%), gently invert and mix 20 times, and let stand at 25°C for 5 minutes. At this time, the following can be observed: Upper phase: transparent light blue (PEG phase); Lower phase: turbid white (sodium citrate phase); Interface: clear, located in the upper part of the container.

[0041] (4) Temperature-induced phase separation: Transfer the centrifuge tube to a 37°C constant temperature water bath and let it stand for 8 minutes. Observations: The color of the upper phase deepens (blue becomes more concentrated) and the volume increases slightly; the transparency of the lower phase increases; when the tube wall is gently tapped, fine suspended particles (IL-NIPAM-RNA complex) appear on the upper phase.

[0042] (5) Centrifugation and phase separation: Centrifuge at 3000 g for 2 minutes. The upper phase and the lower phase are completely separated, and the interface is a horizontal line.

[0043] (6) Collect separately: Carefully aspirate using a pipette: 100 μL of upper phase → labeled "RNA" tube; 80 μL of lower phase → labeled "DNA" tube (avoid aspirating the interface).

[0044] (7) RNA purification: Add 200 μL of anhydrous ethanol (2 times the volume) to the upper phase and mix well; add 20 μL of RNA magnetic bead suspension and incubate at room temperature for 5 minutes; separate with a magnetic rack for 2 minutes and discard the supernatant; wash twice with 200 μL of 70% ethanol; dry at room temperature for 5 minutes; add 20 μL of nuclease-free water and wash at 55℃ for 5 minutes.

[0045] (8) DNA purification: Add 80 μL of anhydrous ethanol (1 volume) to the lower phase and mix well; add 30 μL of DNA magnetic bead suspension and incubate at room temperature for 5 minutes; separate with a magnetic rack for 2 minutes and discard the supernatant; wash twice with 200 μL of 80% ethanol; dry at room temperature for 5 minutes; add 50 μL of nuclease-free water and elute at 55°C for 5 minutes.

[0046] 3-3. Experimental Results: The results of the purity test of DNA / RNA extracted from fingertip blood are shown in Table 3 below.

[0047] Table 3. Results of purity test for nucleic acid extracted from fingertip blood project result Quality control standards RNA production 15.6 ng / μL (total 312 ng) >10 ng / μL RNA purity A260 / A280 2.03 1.9-2.1 RNA integrity (RIN) 8.2 >7.0 DNA production 0.86 ng / μL (total 43 ng) >0.6 ng / μL DNA purity A260 / A280 1.87 1.8-2.0 Total operation time 18 minutes <30 minutes 3-4. Downstream application verification (1) qPCR detection: GAPDH (RNA) Ct = 20.3, β-actin (DNA) Ct = 25.1; (2) miRNA detection: miR-16 Ct = 18.5, miR-21 Ct = 22.1 (consistent with normal physiological levels); (3) NGS library preparation: RNA-seq library concentration 12 nM, insert peak 280 bp; WGS library concentration 8 nM, no adapter dimers.

[0048] Example 4: DNA / RNA co-extraction from dried blood spots 4-1. Sample type: FTA card dried blood spot, 3 mm diameter hole (equivalent to approximately 3 μL of whole blood) 4-2. Experimental Procedure: Place the blood spot perforated tablets into a 1.5 mL centrifuge tube; add 150 μL of lysis buffer (containing 1.5% SDS), incubate at 56°C for 10 minutes, and vortex twice; add 150 μL of aqueous two-phase premix and 15 μL of IL-NIPAM stock solution (final concentration 1.0%), and mix well; incubate at 25°C for 6 minutes to form an aqueous two-phase system; incubate at 37°C for 10 minutes; centrifuge at 3000 g for 3 minutes; collect the upper and lower phases separately, and follow the same purification steps as in Example 3.

[0049] 4-3. Experimental Results: The purity results of nucleic acid extraction from dried blood spots are shown in Table 4 below.

[0050] Table 4. Results of Nucleic Acid Extraction from Dried Blood Spots

[0051] project Extraction method of this invention QIAamp column method RNA production (ng) 8.2 4.1 DNA yield (ng) 28.5 31.2 RNA RIN value 7.8 6.9 Operation time (minutes) 22 45 Reagent cost (RMB) 1.8 6.5 Results analysis: Due to severe RNA degradation, dried blood spots are significantly improved by this invention through rapid lysis and a low-temperature aqueous two-phase environment, resulting in a 100% increase in RNA recovery rate and a 0.9 increase in RIN value, which is significantly better than traditional methods. Example 5: Co-extraction of DNA / RNA from FFPE tissue

[0052] 5-1. Sample type: FFPE slides of breast cancer, 5 μm thick × 3 slides (approximately 10 mm²). 5-2. Experimental Procedure: (1) Place the slices into a 1.5 mL centrifuge tube, add 200 μL xylene, vortex at room temperature for 1 minute, centrifuge at 12000 g for 2 minutes, discard the supernatant, and repeat twice; (2) Wash twice with 200 μL of anhydrous ethanol to remove residual xylene; dry at room temperature for 5 minutes; (3) Add 100 μL of lysis buffer (containing 1% SDS + 0.5 mg / mL proteinase K), incubate at 65°C for 15 minutes, and vortex to mix. (4) Inactivate proteinase K at 90℃ for 5 minutes; add 150 μL of aqueous two-phase premix and 18 μL of IL-NIPAM stock solution (final concentration 1.2%, increased to counteract tissue degradation of RNA); incubate at 25℃ for 8 minutes; incubate at 37℃ for 10 minutes; centrifuge at 5000 g for 3 minutes (high viscosity samples require stronger centrifugation); (5) Collect the upper and lower phases separately and purify them.

[0053] 5-3. Experimental Results: The purity results of nucleic acid extraction from FFPE tissue are shown in Table 5 below.

[0054] Table 5. Results of nucleic acid extraction purity from FFPE tissue index Extraction method of this invention RNeasy FFPE Kit (QIAGEN) Total RNA production (ng) 125 98 DV200 value (%) 68 61 miR-21 production (copies) <![CDATA[1.2×10 5 ]]> <![CDATA[7.8×10 4 ]]> DNA yield (ng) 420 380 Operation time (minutes) 35 180 DV200 Description: Percentage of RNA fragments >200 nt, a key quality control indicator for FFPE samples; >30% can be used for RNA-seq. Example 6: Verification of miRNA enrichment efficiency

[0055] Experimental objective: To verify the selectivity of IL-NIPAM for RNA of different lengths. Experimental methods: (1) Prepare a mixed sample containing the following standards: miR-16 simulant (22 nt): 1×10 6copies; mRNA fragment (GAPDH, 500 nt): 1×10 5 Copies; Genomic DNA fragment (1000 bp): 1×10 5 copies.

[0056] (2) Extract according to the method of Example 3, measure the concentration of each component in the upper phase and the lower phase respectively, and calculate the distribution coefficient K: K = C upper phase / C lower phase.

[0057] Experimental results are shown in Table 6 below.

[0058] Table 6. miRNA enrichment efficiency Nucleic acid types Upper phase concentration lower phase concentration Allocation coefficient K miR-16 (22 nt) <![CDATA[8.7×10 5 copies / μL]]> <![CDATA[9.1×10 4 copies / μL]]> 9.6 GAPDH mRNA (500 nt) <![CDATA[7.2×10 4 copies / μL]]> <![CDATA[1.8×10 4 copies / μL]]> 4.0 Genomic DNA (1000 bp) <![CDATA[1.2×10 4 copies / μL]]> <![CDATA[7.5×10 4 copies / μL]]> 0.16 Experimental results: (1) miRNA partition coefficient >9.5, which is much higher than mRNA and DNA, proving that IL-NIPAM has a preferential enrichment effect on small RNA; (2) RNA purity in the upper phase >95% (miRNA + mRNA account for the total nucleic acid); (3) DNA purity in the lower phase >88% (RNA contamination <12%).

[0059] Results analysis: Due to their short chains and high flexibility, small RNAs are more easily bound to imidazole cations at multiple sites; the hydrophobic microdomains formed by the aggregation of IL-NIPAM after phase transition have a higher efficiency in encapsulating short RNA chains.

[0060] Example 7: Optimization of Temperature and IL-NIPAM Concentration Experimental Design: The effects of temperature and IL-NIPAM concentration on RNA and DNA recovery rates and phase separation time were designed. The specific experimental results are shown in Table 7 below.

[0061] Table 7. Results of the two-factor orthogonal experiment on temperature and IL-NIPAM concentration Experiment No. Temperature (°C) IL-NIPAM concentration (%) RNA recovery rate (%) DNA recovery rate (%) Phase separation time (min) 1 30 0.5 62 78 15 2 30 1.0 71 82 12 3 32 0.5 75 81 10 4 32 1.0 83 86 8 5 35 0.5 78 85 8 6 35 1.0 85 88 6 7 37 0.5 80 87 7 8 37 1.0 86 89 5 9 40 0.5 76 84 6 10 40 1.0 81 85 5 As shown in Table 7 above, the optimal conditions are: 37℃, IL-NIPAM 1.0%, under which the RNA recovery rate is 86%, the DNA recovery rate is 89%, and the phase separation time is 5 minutes.

[0062] Trend analysis: At 30℃, the phase transition is incomplete and the separation efficiency is low, which is below LCST; at 32-37℃, the phase transition window is in effect and the recovery rate increases with increasing temperature; at temperatures above 37℃, RNA stability decreases and the recovery rate decreases; at an IL-NIPAM concentration of 0.5%, the carrying capacity is insufficient; at 1.0%, it reaches saturation; and at >1.5%, the viscosity increases, affecting operation.

[0063] Example 8: Batch Repeatability Validation 1. Sample type: 10 μL of finger prick blood from the same healthy individual, extracted independently in 30 portions. 2. Operators: 3 technicians, each handling 10 portions. 3. The statistical results of the experiment are shown in Table 8 below.

[0064] Table 8. Batch Repeatability Validation index average value Standard deviation Coefficient of variation (CV) (%) RNA production (ng) 14.8 1.2 8.1 RNA A260 / A280 2.01 0.05 2.5 RNA RIN value 8.1 0.3 3.7 DNA yield (ng) 41.5 3.2 7.7 DNA A260 / A280 1.86 0.04 2.2 miR-16 Ct value 18.6 0.5 2.7 Experimental results: All indicators had a CV < 10%, meeting the clinical testing requirements (CV < 15%). There were no significant differences among operators (P > 0.05), indicating good repeatability.

[0065] Example 9: Stability Study (1) Storage stability of the kit: The prepared aqueous two-phase premix and IL-NIPAM lyophilized microspheres were stored at 4℃, 25℃ and 37℃ respectively, and the extraction efficiency was tested periodically. The test results are shown in Table 9 below.

[0066] Table 9. Extraction efficiency table for periodic testing of the kit Storage conditions 0 months 3 months 6 months 12 months RNA recovery rate (%) at 4℃ 85 84 83 82 RNA recovery rate (%) at 25℃ 85 81 76 68 RNA recovery rate (%) at 37℃ 85 73 62 - Experimental results: Recommended storage temperature is 4℃, shelf life is 12 months; storage at room temperature is 6 months; storage at 37℃ is not recommended.

[0067] (2) Nucleic acid stability after extraction: RNA and DNA elution buffers were stored under different conditions and the degradation rate was measured. The experimental results are shown in Table 10 below.

[0068] Table 10. Degradation rate of nucleic acid elution under different storage conditions Storage conditions Completeness rate (%) after 7 days Completeness rate after 30 days (%) -80℃ RNA 98 / DNA 99 RNA 96 / DNA 98 -20℃ RNA 95 / DNA 98 RNA 88 / DNA 97 4℃ RNA 82 / DNA 96 RNA 65 / DNA 94 The experimental results show that RNA is recommended for long-term storage at -80℃ and can be stored at -20℃ for 1 month; DNA has good stability at -20℃.

[0069] Example 10: Downstream Application Verification Experiment (1) qPCR verification: The RNA / DNA extracted in this invention was used for qPCR detection (SYBR Green method), and the detection results are shown in Table 11 below.

[0070] Table 11. Comparison of RNA / DNA detection results extracted by the present invention and the Trizol method. Gene Ct value of the present invention Trizol method Ct value ΔCt Amplification efficiency (%) GAPDH (RNA) 20.2±0.3 20.5±0.5 -0.3 98 β-actin (RNA) 21.1±0.2 21.4±0.6 -0.3 96 BRCA1 (DNA) 25.3±0.4 25.6±0.5 -0.3 94 miR-16 18.5±0.3 19.8±0.7 -1.3* 97 miR-21 22.1±0.4 23.9±0.9 -1.8* 95 The experimental results show that *P<0.01, indicating a significant difference, and the present invention has higher miRNA detection sensitivity.

[0071] (2) NGS library construction Table 12. Results of NGS Library Construction Indicators project RNA-seq WGS miRNA-seq Starting amount 100 ng 50 ng Total RNA 500 ng Library concentration (nM) 12.5 8.2 15.3 Fragment size (bp) 280 350 145 Sequencing data volume (Gb) 6.2 30.5 5.8 Q30 (%) 92.3 94.1 90.5 Comparison rate (%) 88.5 97.2 85.3 The experimental results show that all indicators meet the library construction standards of the Illumina platform.

[0072] (3) Absolute quantification by ddPCR Sample type: Detection of circulating miR-155 (tumor marker) in 10 μL of plasma. Test results are shown in Table 13 below.

[0073] Table 13. miR-155 Detection Results method Detected concentration (copies / μL) Positive droplet count Poisson correction CV (%) This invention 125 1580 4.2 miRNeasy Kit 78 980 6.8 The experimental results show that the detection concentration of the present invention is increased by 60%, indicating that the miRNA recovery rate is higher.

[0074] Example 11: System Comparison of the Extraction Method of the Present Invention with Existing Technologies The extraction method of this invention is compared with existing technologies such as the Trizol method, QIAamp column method, and magnetic bead method. The comparison results are shown in Table 14 below.

[0075] Table 14. Comparison of the extraction method of the present invention with existing technologies Comparison Projects This invention Trizol method QIAamp column method Magnetic bead method organic solvents none Phenol / Chloroform none none Proteinase K unnecessary* unnecessary need Optional Operating steps 8 steps 15 steps 12 steps 10 steps Total time (min) 18 75 45 35 Minimum sample size (μL) 5 50 20 10 RNA recovery rate (%) 85 78 72 75 DNA recovery rate (%) 88 75 84 80 miRNA recovery rate (%) 78 65 48 62 RNA RIN value 8.2 7.5 7.8 7.6 Inter-batch CV (%) 8 15 12 10 Reagent cost (RMB / sample) 1.8 3.5 6.8 5.2 Automated adaptation easy Disaster middle easy Environmental protection excellent Difference good good Note: *Special samples such as FFPE require proteinase K pretreatment. The experimental comparison results show that the extraction method of the present invention has the following core advantages compared with existing technologies (Trizol method, QIAamp column method, magnetic bead method): more efficient operation; lower sample requirements; higher recovery efficiency; better quality; lower cost; no need for organic solvents or proteinase K (except for special samples); high automation adaptability; and excellent environmental friendliness.

[0076] Example 12: Clinical Sample Validation Sample source: 30 matched samples provided by a top-tier hospital (ethics review passed). Sample type: Peripheral blood from lung cancer patients (5 mL EDTA anticoagulated) + FFPE sections of cancer tissue Experimental objective: To verify the performance of this invention in real clinical samples. Experimental results: (1) Peripheral blood cfDNA / cfRNA were co-extracted, and the experimental results are shown in Table 15 below.

[0077] Table 15. Nucleic acid extraction results from clinical samples (peripheral blood) Sample number cfRNA production (ng) cfDNA production (ng) EGFR T790M mutation (ddPCR) P01 3.2 12.5 Detected (0.5%) P08 2.8 15.3 Detected (1.2%) P15 4.1 18.7 Not detected ...(30 cases in total) Average 3.5 ± 0.9 Average 14.2 ± 3.1 Positive rate 43% Experimental results: Compared with the ARMS-PCR gold standard: sensitivity 92%, specificity 95%, and concordance Kappa=0.89.

[0078] The nucleic acid extraction results of FFPE sections of cancerous tissue are shown in Table 16 below.

[0079] Table 16. Nucleic acid extraction results of clinical samples (FFPE sections of cancerous tissue) sample Storage period (years) DV200(%) Database creation success rate Number of genes detected Fresh (<1 year old) 0.5 72±8 100% (10 / 10) 18500±1200 Medium term (1-3 years) 2 58±12 90% (9 / 10) 16200±1800 Old (>3 years) 5 41±15 70% (7 / 10) 12800±2500 The experimental results show that even FFPE samples preserved for 5 years with a DV200 > 30% can still be used for transcriptome sequencing.

[0080] Example 13: Kit Composition and Instructions for Use Kit Name: DNA / RNA Aqueous Two-Phase Co-extraction Kit (50 tests / kit) The reagent kit component list is shown in Table 17 below: Table 17. Reagent Kit Components Component number Component Name Specification Storage conditions R1 Red blood cell lysis fluid 15 mL 4℃ R2 Sample lysis buffer 10 mL room temperature R3 Aqueous two-phase premix 20 mL 4℃, protected from light R4 IL-NIPAM freeze-dried microspheres (10×) 1 mL Dry at 4℃ R5 RNA magnetic bead suspension 2 mL 4℃ R6 DNA magnetic bead suspension 3 mL 4℃ R7 Washing solution I (70% ethanol) 30 mL room temperature R8 Washing Solution II (80% Ethanol) 30 mL room temperature R9 RNA elution buffer (nuclease-free water) 3 mL 4℃ R10 DNA elution buffer (TE buffer) 5 mL 4℃ appendix 1.5 mL centrifuge tubes, magnetic rack, instruction manual - room temperature Example 14: Automation Platform Adaptation

[0081] Platform: Applied Biosystems MagMAX Express 96, program settings are shown in Table 18 below.

[0082] Table 18. Program Settings Table step Temperature (°C) Time (min) action 1 25 5 Mixed Aqueous Two-Phase Premix 2 25 5 Static forming 3 37 8 Temperature-induced 4 25 2 cool down 5 25 1 Aspirate the upper phase onto the RNA plate 6 25 1 Aspirate the lower phase onto the DNA plate 7 25 5 RNA magnetic bead binding 8 25 5 DNA magnetic beads binding 9 25 2×3 washing 10 55 5 Washout Throughput: 96 samples / batch, total time 45 minutes (including instrument setup).

[0083] The experimental results are as follows: (1) RNA recovery rate of manual method vs. automated method: 85% vs 83% (P=0.12, no significant difference). (2) Inter-batch CV of automated method: 6.5% (better than 8.1% of manual method). (3) Contamination rate: <0.5% (96-well cross-contamination detection).

[0084] The application scenarios of this invention include: 1. Newborn genetic disease screening: Only 50 μL of heel blood is needed to simultaneously detect PKU (DNA) and SMA (RNA); 2. Tumor liquid biopsy: ctDNA (EGFR mutation) and exosomal RNA (PD-L1) are extracted simultaneously from 5 mL of plasma. 3. Rapid pathogen diagnosis: SARS-CoV-2 RNA and human genomic DNA can be extracted from saliva samples within 30 minutes; 4. Forensic evidence identification: DNA typing of old bloodstains + RNA sex confirmation for dual verification; 5. Single-cell multi-omics: combined transcriptome and genome analysis of single-cell lysate (<1 μL).

[0085] This invention fills the technological gap in rapid, green, and efficient DNA / RNA co-extraction of trace samples, and has significant social and economic benefits.

Claims

1. A method for co-extraction of DNA / RNA based on aqueous two-phase-temperature-induced phase separation, characterized in that, A primary aqueous two-phase system of PEG-sodium citrate containing the ionic liquid-modified thermosensitive polymer IL-NIPAM was constructed to process biological samples to be separated. IL-NIPAM is a 1-butyl-3-methylimidazolium cationic modified poly(N-isopropylacrylamide) copolymer with the structural formula [BMIM]⁺-PNIPAM, an LCST of 32±1℃, and a molecular weight of 8000–15000 Da. The PNIPAM backbone provides temperature responsiveness, and the [BMIM]⁺ side chain provides RNA selective recognition. When the system temperature is lower than the LCST of IL-NIPAM, IL-NIPAM is in a hydrophilic swelling state, binding RNA and uniformly dispersed in the aqueous two-phase system. When the system temperature is higher than the LCST of IL-NIPAM, IL-NIPAM undergoes a phase transition and selectively carries RNA into the upper phase, while DNA remains in the lower phase. Active separation of DNA and RNA is achieved through temperature regulation.

2. The method according to claim 1, characterized in that, Specifically, the following steps are included: (1) The pretreated biological sample is mixed with an aqueous two-phase premix, wherein IL-NIPAM is added in advance or during use, and the concentration of IL-NIPAM is 0.5-1.5% w / w; the aqueous two-phase premix contains 8-12% polyethylene glycol 6000 by mass, 10-15% sodium citrate by mass, and the pH is 7.0±0.1; (2) The mixture is allowed to stand at 18-25℃ for 3-8 minutes to form a stable aqueous two-phase system. The system exhibits a layered state with a transparent upper phase and a turbid lower phase. The interfacial tension is <0.1 mN / m and the viscosity is <50 mPa·s. (3) Raise the temperature of the aqueous two-phase system to 32-37°C and maintain it for 5-10 minutes. IL-NIPAM undergoes a phase transition, carrying RNA selectively migrating to the upper phase while DNA remains in the lower phase. (4) Collect the upper phase solution enriched with RNA and the lower phase solution enriched with DNA by centrifugation or static separation; (5) Purify the separated RNA and DNA.

3. The method according to claim 1 or 2, characterized in that, The performance indicators of this method are as follows: phase separation time ≤ 10 minutes; upper phase miRNA concentration / lower phase miRNA concentration > 9.5; total RNA recovery rate > 80%; DNA recovery rate > 85%; RNA integrity RIN > 7.5; DNA purity A260 / A280 = 1.8–2.0; RNA purity A260 / A280 = 1.9–2.

1.

4. The method according to claim 1 or 2, characterized in that, The biological sample is selected from one or more of the following: peripheral blood, fingertip blood, dried blood spots, paraffin-embedded tissue (FFPE) sections, fresh tissue, frozen tissue, saliva, urine, cell culture medium, and feces, with a sample volume of 5–100 μL or a tissue mass of 1–20 mg.

5. The method according to claim 2, characterized in that, The methods for purifying RNA and DNA are as follows: (A) RNA purification: Add 1.5-2.5 times the volume of anhydrous ethanol, capture RNA with magnetic beads, wash 2-3 times with 70% ethanol, and elute with 10-30 μL of nuclease-free water; or, add an equal volume of isopropanol and 0.2 M NaCl, precipitate at -20℃ for 30 minutes, and collect the RNA precipitate by centrifugation. (B) DNA purification: Add 0.8-1.2 times the volume of anhydrous ethanol, capture DNA with magnetic beads, wash 2-3 times with 80% ethanol, and elute with 20-50 μL of nuclease-free water; or, after diluting 2-5 times, purify using a silica membrane column.

6. A dedicated kit for DNA / RNA co-extraction, characterized in that, The following reagents are included: (1) Aqueous two-phase premix, pH 7.0±0.1, containing PEG 6000 8-12% w / w, sodium citrate 10-15% w / w; and also containing one or more of the following reagents: bromothymol blue 0.005-0.02% w / w, EDTA 1-5 mM; SDS 0.1-0.5% w / v; dithiothreitol 0.5-2 mM; (2) IL-NIPAM lyophilized microspheres or concentrate, LCST 32±1℃; (3) Sample lysis buffer, containing 20 mM Tris-HCl pH 7.5, 2 mM EDTA, and 0.5-1.5% SDS; (4) RNA purification magnetic bead suspension, with magnetic bead particle size of 0.5-2 μm; (5) DNA purification magnetic bead suspension, with magnetic bead particle size of 0.8–2.5 μm; (6) Nuclease-free water; The aqueous two-phase premix and IL-NIPAM lyophilized microspheres or concentrate are packaged separately and mixed in proportion before use, or premixed as a single component and packaged. The concentration of IL-NIPAM used is 0.5-1.5% w / w.

7. The reagent kit according to claim 6, characterized in that, It also includes one or more of the following reagents: (1) Red blood cell lysis buffer: 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA; (2) Dewaxing solution: 100% xylene or dewaxing agent; (3) RNA precipitation buffer: 70% (v / v) anhydrous ethanol, 0.2 M NaCl; (4) DNA precipitation buffer: 80% (v / v) anhydrous ethanol, 50 mM NaCl; (5) Positive control nucleic acid standard.

8. The reagent kit according to claim 6, characterized in that, The preparation methods of the IL-NIPAM concentrate and IL-NIPAM lyophilized microspheres are as follows: (1) Dissolve IL-NIPAM in ultrapure water to prepare IL-NIPAM 10-20% w / v concentrate; (2) Add trehalose or mannitol to the IL-NIPAM concentrate as a lyophilization protectant, and the mass ratio of the lyophilization protectant to the IL-NIPAM concentrate is 1:1 to 1:3; (3) After dispensing, freeze-dry the product by pre-freezing at -45℃ for 4 hours, drying at -35℃ for 24 hours, and drying at 25℃ for 6 hours; (4) Seal and package the product in a dry nitrogen environment and store it at 2-8℃.

9. A method for preparing IL-NIPAM, characterized in that, Includes the following steps: (1) Add 10.0 g of N-isopropylacrylamide, 2.5 g of 1-butyl-3-methylimidazolium chloride, and 0.2 g of azobisisobutyronitrile to a three-necked flask, dissolve in 100 mL of anhydrous ethanol, and react at 70 °C for 24 hours under nitrogen protection; (2) Purification treatment: remove the solvent by rotary evaporation of the reaction solution, dissolve in deionized water and dialyze for 72 hours, retain a molecular weight cutoff of 8000 Da, change the water every 8 hours, freeze-dry the dialysate to obtain 9.8 g of white powder IL-NIPAM with a molecular weight of 8000~15000 Da, yield 78%.

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