Preparation and application of efficient paramagnetic particle method FFPE sample DNA nucleic acid extraction kit

Abstract

The invention discloses preparation and application of an efficient paramagnetic particle method FFPE sample DNA nucleic acid extraction kit, and belongs to the technical field of molecular biology. According to the method, the non-toxic and environment-friendly dewaxing liquid is used for dewaxing, and the FFPE digestive juice containing chaotropic salt, a nucleic acid settling accelerator and other components is used for full digestion, so that the yield of nucleic acid is increased; moreover, the anionic surfactant and chaotropic salt in the FFPE digestive juice are matched with the protease K liquid to fully destroy and digest protein in the sample, so that the purity of nucleic acid is improved, and the integrity of the extracted nucleic acid is improved. The kit is simple to prepare, automation can be realized, and the cost is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to the preparation and application of an efficient magnetic bead method FFPE sample DNA nucleic acid extraction kit. Background Art

[0002] FFPE (formalin fixation and paraffin embedding) samples are formalin-fixed paraffin-embedded tissue samples. Due to their long-term preservation characteristics, they are important resources in pathological research. At present, traditional DNA extraction methods have disadvantages such as low extraction efficiency, short DNA fragments, and low purity. In addition, toxic reagents such as xylene, phenol, and chloroform are often used in the extraction process, which need to be removed additionally, which can easily cause the loss of nucleic acids, thereby affecting the sensitivity and accuracy of subsequent experiments; or methods such as the centrifugal column method have defects such as small sample processing volume and large sample demand during the extraction process, which limit their application in subsequent experiments. For example, Chinese patent CN116694618A discloses a reagent and method for extracting nucleic acids from FFPE samples. This method only uses a single temperature (37°C) for incubation, and is unable to decrosslink nucleic acids and proteins, which can easily lead to unsatisfactory purity of nucleic acids obtained in subsequent extractions. Chinese patent CN115322988A also discloses a method for rapidly extracting DNA from FFPE samples. Although it solves the problem of low DNA extraction efficiency, it does not determine the purity data of the extracted nucleic acids, nor can it determine whether the purity of the extracted DNA meets the requirements of downstream sequencing, library construction and other applications.

[0003] With the rise of precision medicine and personalized treatment, the demand for high-quality DNA is increasing. Therefore, an effective method for extracting nucleic acid from FFPE samples is urgently needed. Summary of the invention

[0004] In view of the defects of the prior art, the present invention designs a magnetic bead method FFPE sample DNA nucleic acid extraction kit with environmentally friendly dewaxing, higher nucleic acid integrity and higher extraction efficiency, which can extract DNA from FFPE samples in an environmentally friendly, efficient and convenient manner.

[0005] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions: On the one hand, the present invention provides an efficient magnetic bead method FFPE sample DNA nucleic acid extraction kit, the kit comprising a dewaxing solution, an FFPE digestion solution, a lysis and binding solution, a washing solution 1, a washing solution 2, an elution solution, a proteinase K solution, and a magnetic bead solution; Wherein, the dewaxing solution comprises 97-99% organic solvent and 1-2% anionic surfactant; The FFPE digestion solution includes 2-5% anionic surfactant, 5-30 mM ion chelator, 10-80 mM pH buffer, 2.5-4M chaotropic salt and 0.5%-10% nucleic acid precipitation promoter, and the balance is water; The lysis and binding solution comprises 3-5M chaotropic salt, 1-3% nonionic surfactant, 1-3% anionic surfactant, 40-60 mM pH buffer, 45-50% alcohol and 6-14 mM ion chelating agent; The washing solution 1 comprises 2.5-3.5M chaotropic salt, 1.5-2% nonionic surfactant, 70-80mM pH buffer and 35-50% alcohol; The washing solution 2 includes 50-90 mM pH buffer and 40-60% alcohol; The eluent comprises 1-2% nonionic surfactant and 25-70 mM pH buffer; The proteinase K solution includes proteinase K, glycerol, calcium chloride, tris(hydroxymethyl)aminomethane) and water, and the concentration is 20 mg / mL; The magnetic bead liquid is silanol magnetic beads with a particle size of 200-400 nm and a concentration of 100 mg / mL; The percentages are percentages by volume.

[0006] The chaotropic salt and anionic surfactant contained in the digestion solution can fully digest the dewaxed sample, and the nucleic acid precipitation promoter can better separate the digested nucleic acids, thereby improving the yield of nucleic acids.

[0007] Preferably, the chaotropic salt is one or more of guanidine hydrochloride, guanidine isothiocyanate, sodium chloride, sodium periodate, and sodium iodide.

[0008] Preferably, the nonionic surfactant is one or more of Triton X-100, PEG 200, NP-40, Tween-20, and Tween-80.

[0009] Preferably, the anionic surfactant is one or more of SDS (sodium dodecyl sulfate), SLS (sodium dodecyl sulfate), TLS (sodium tallow dodecyl sulfate), and SDBS (sodium dodecylbenzene sulfonate).

[0010] Preferably, the pH buffer is one or more of Tris-HCl buffer, acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, and PBS buffer.

[0011] Preferably, the alcohol is one or more of ethanol, isopropanol, and isopentanol.

[0012] Preferably, the ion chelating agent is one or more of EDTA, EDTA•Na2, and EGTA.

[0013] Preferably, the organic solvent is one or more of mineral oil, C7-C20 straight-chain / branched alkane or a mixture thereof, C10-C20 straight-chain / branched alkane or a mixture thereof, and polyorganosiloxane.

[0014] Specifically, the C7~C20 straight-chain / branched alkane or a mixture thereof includes any one of n-heptane, n-octane, n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, n-octadecane, n-nonadecane, n-eicosane, isoheptane, isooctane, isononane, isodecane, etc., or a combination of at least two thereof.

[0015] Preferably, the nucleic acid precipitation promoting agent comprises one or more of Acryl polymer solution, glycogen, and Poly A potassium salt.

[0016] On the other hand, the present invention provides a method for extracting nucleic acid from FFPE sample DNA, using the magnetic bead method FFPE sample DNA nucleic acid extraction kit.

[0017] Preferably, the method comprises the following steps: S1. Add 1 mL of dewaxing solution to the FFPE sample and mix well. Centrifuge briefly and place in a 56°C metal bath or water bath for 5 minutes. S2. Centrifuge at 14,000 x g for 2 min, discard the supernatant, add 1 mL of organic solvent and mix, centrifuge at 14,000 x g for 2 min, discard the supernatant and remove the organic solvent; S3. Add 300 µL FFPE digestion solution and 20 µL proteinase K solution, mix well, centrifuge briefly, heat in a 56°C metal bath or water bath until the sample is completely lysed, heat at 90°C for 1 h, centrifuge at 1,000 x g for 1 min, and collect the supernatant; S4. Add 600 μL of lysis binding solution and sample to the first well of the extraction plate, add 900 μL of washing solution 1 to the second well, add 500 μL of washing solution 1 to the third well, add 900 μL of washing solution 2 to the fourth well, add 300 μL of washing solution 2 and 20 μL of magnetic bead solution to the fifth well, and add 70 μL of elution solution to the sixth well; S5. First, take the magnetic rod cover in the 5th well and immerse it in the 1st well at room temperature for 10 minutes, and then immerse it in the 2nd well, 3rd well, 4th well, and 5th well in turn at room temperature, with the immersion time in each well being 2 minutes; then air-dry the magnetic beads for 5 minutes, and then immerse them in the 6th well and mix for 10 minutes at 60°C. Finally, discard the magnetic beads, and the resulting eluate is the final nucleic acid product.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The invention uses an environmentally friendly and non-toxic dewaxing solution for dewaxing treatment, which cooperates with a FFPE digestion solution containing components such as a nucleic acid precipitation promoter and a chaotropic salt, thereby improving the yield of nucleic acids.

[0019] First, the use of anionic surfactants in FFPE digestion solution can open the hydrogen bonds and hydrophobic bonds of proteins. Combined with proteinase K solution, it can fully digest the proteins in FFPE. At the same time, the combination of high-concentration chaotropic salts in FFPE digestion solution will be more effective than using them alone, enhancing its effect on protein denaturation and having a strong impact on RNase activity, thereby protecting RNA nucleic acids from degradation; then, the digested DNA and RNA can be better separated under the action of nucleic acid precipitation promoters, and the electrostatic effect of consumables can be removed, reducing the loss of nucleic acids.

[0020] In addition, the use of high concentrations of non-ionic surfactants can fully dissolve lipid substances in the sample, allowing other active ingredients in the reagent to play a greater role.

[0021] The kit designed by the present invention not only reduces the risk of operators being exposed to toxic reagents during the experiment, but also improves the integrity of the extracted nucleic acid. It also uses readily available chemicals, is low-priced, has stable chemical properties, and is simple to prepare. It can be automated in conjunction with the use of an extractor during the extraction process, saving time and manpower, and is conducive to providing strong support for the fields of tumor research, genetic disease diagnosis, forensic medicine, and the construction of biological sample libraries. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is the GADPH amplification curve of different magnetic bead-based FFPE sample genomic DNA extraction kits.

[0024] Figure 2 This is the PABPN1 amplification curve of different magnetic bead-based FFPE sample genomic DNA extraction kits.

[0025] Figure 3 These are the RNase P amplification curves of different magnetic bead-based FFPE sample genomic DNA extraction kits.

[0026] Figure 4This is the mtDNA:1555A amplification curve of different magnetic bead-based FFPE sample genomic DNA extraction kits. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0029] Example 1 1. An efficient magnetic bead method FFPE sample DNA nucleic acid extraction kit, the kit includes dewaxing solution, FFPE digestion solution, lysis and binding solution, washing solution 1, washing solution 2, elution solution, proteinase K solution, and magnetic bead solution. The specific formula is shown in Table 1 below: Table 1 Formula of FFPE sample DNA nucleic acid extraction kit in Example 1

[0030] 2. A method for extracting DNA nucleic acid from FFPE samples, using the above kit, specifically comprising the following steps: (1) Take no more than five paraffin sections with a thickness of about 10 µm and an area of ​​about 0.5 cm × 0.5 cm and place them in a sterile 1.5 mL centrifuge tube; (2) Add 1 mL of dewaxing solution to the 1.5 mL centrifuge tube containing the FFPE sample, vortex mix for 10 seconds, centrifuge briefly, and then place in a 56°C water bath for 5 min; (3) Centrifuge at 14,000 x g for 2 min, discard the supernatant, add 1 mL of anhydrous ethanol, vortex mix for 10 s, centrifuge at 14,000 x g for 2 min, discard the supernatant, open the tube cap and place at room temperature or in a 37°C oven for 10 min until the ethanol is completely evaporated; (4) Add 300 µL FFPE digestion buffer and 20 µL proteinase K solution. Vortex to mix, centrifuge briefly, and heat in a 56°C metal bath or water bath for more than 2 h until the sample is completely lysed (it can be lysed overnight). Gently mix the sample several times during this period. (5) Heat at 90°C for 1 h (if RNA needs to be removed, after heating, wait for the liquid to cool to room temperature, add 5 µL RNase A (self-prepared or purchased separately) to the sample, vortex to mix, let stand for 5 min, and then proceed to the next step); (6) Centrifuge the heated product at 1,000 x g for 1 min and transfer all the supernatant to a new 2 mL centrifuge tube as a sample to be tested; (7) Add 600 μL of lysis binding solution and all samples to be tested to the first well of the extraction plate, add 900 μL of washing solution 1 to the second well, add 500 μL of washing solution 1 to the third well, add 900 μL of washing solution 2 to the fourth well, add 300 μL of washing solution 2 and 20 μL of magnetic bead solution to the fifth well, and add 70 μL of elution solution to the sixth well; (8) First, take the magnetic rod cover from the 5th well, then immerse it in the 1st well and mix for 10 min at room temperature, then immerse it in the 2nd well and mix for 2 min at room temperature, then immerse it in the 3rd well and mix for 2 min at room temperature, then immerse it in the 4th well and mix for 2 min at room temperature, then immerse it in the 5th well and mix for 2 min at room temperature, then air-dry the magnetic beads for 5 min, then immerse it in the 6th well and mix for 10 min at 60°C, finally discard the magnetic beads and perform UV spectrophotometer detection, Qubit detection and qPCR detection on the obtained eluate.

[0031] In this example, the nucleic acid extraction operation shown in Table 2 below was performed in the Eas Equ 32 purifier: Table 2 Nucleic acid extraction process chart

[0032] Example 2 1. An efficient magnetic bead method FFPE sample DNA nucleic acid extraction kit, the kit includes dewaxing solution, FFPE digestion solution, lysis and binding solution, washing solution 1, washing solution 2, elution solution, proteinase K solution, and magnetic bead solution. The specific formula is shown in Table 3 below: Table 3 Formula of the FFPE sample DNA nucleic acid extraction kit of Example 2

[0033] 2. A method for extracting DNA from FFPE samples is the same as in Example 1.

[0034] Example 3 1. An efficient magnetic bead method FFPE sample DNA nucleic acid extraction kit, the kit includes dewaxing solution, FFPE digestion solution, lysis and binding solution, washing solution 1, washing solution 2, elution solution, proteinase K solution, and magnetic bead solution. The specific formula is shown in Table 4 below: Table 4 Formula of FFPE sample DNA nucleic acid extraction kit of Example 3

[0035] 2. A method for extracting DNA from FFPE samples is the same as in Example 1.

[0036] Comparative Example 1 Comparative Example 1 is a column extraction kit for FFPE sample genomic DNA sold commercially by Q Company.

[0037] Comparative Example 2 Comparative Example 2 is a commercially available magnetic bead-based FFPE sample genomic DNA extraction kit from Company T, which uses xylene for dewaxing.

[0038] Comparative Example 3 Comparative Example 3 is a magnetic bead method FFPE sample genomic DNA extraction kit commercially available from Company B, which uses xylene for dewaxing.

[0039] Comparative Example 4 Comparative Example 4 is a magnetic bead-based FFPE sample genomic DNA extraction kit commercially available from Company M, which uses an environmentally friendly dewaxing solution for dewaxing.

[0040] Test Example 1 Two pieces of commercial paraffin sections were taken respectively, and extracted with each extraction kit (Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Example 1). Example 1 used the method of the present invention to extract and purify nucleic acid, and the extracted nucleic acid was detected by UV spectrophotometer, Qubit detection and qPCR detection.

[0041] Table 5 FFPE sample DNA extraction-UV spectrophotometer and Qubit test results

[0042] Table 6 FFPE sample DNA extraction-qPCR test results

[0043] From Table 5-6 and Figure 1-4As shown, in the DNA extraction of FFPE samples, the purity of the nucleic acid extracted in Example 1 meets the downstream application requirements, the extraction effect is stable, the obtained nucleic acid concentration is high, and the detection rate in the nucleic acid detection of GADPH, PABPN1, RNase P, and mtDNA: 1555A is 100%, while Comparative Examples 2 and 4 did not detect the target gene in the detection of mtDNA: 1555A, and Comparative Example 3 missed detection in PABPN1 and did not detect mtDNA: 1555A. There was no obvious difference in the detection results of Comparative Example 1 and the embodiment in qPCR, and the nucleic acid concentration extracted in Example 1 was higher, that is, in the DNA extraction of FFPE samples in Example 1, the extracted DNA has better integrity, stable extraction performance, and its nucleic acid concentration and nucleic acid purity can better meet downstream applications.

[0044] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An efficient magnetic bead method FFPE sample DNA nucleic acid extraction kit, characterized in that: The kit includes dewaxing solution, FFPE digestion solution, lysis and binding solution, washing solution 1, washing solution 2, elution solution, proteinase K solution, and magnetic bead solution; Wherein, the dewaxing solution comprises 97-99% organic solvent and 1-2% anionic surfactant; The FFPE digestion solution includes 2-5% anionic surfactant, 5-30 mM ion chelator, 10-80 mM pH buffer with a pH of 7.5-8.5, 2.5-4 M chaotropic salt and 0.5%-10% nucleic acid precipitation promoting agent, and the balance is water; The lysis and binding solution includes 3-5M chaotropic salt, 1-3% nonionic surfactant, 1-3% anionic surfactant, 40-60 mM pH buffer with a pH of 7.5-8.5, 45-50% alcohol and 6-14 mM ion chelating agent; The washing solution 1 comprises 2.5-3.5M chaotropic salt, 1.5-2% nonionic surfactant, 70-80mM pH buffer and 35-50% alcohol; The washing solution 2 comprises 50-90 mM pH buffer and 40-60% alcohol; The eluent comprises 1-2% nonionic surfactant and 25-70 mM pH buffer; The proteinase K solution is used at a concentration of 20 mg / mL; The magnetic bead liquid is silanol magnetic beads with a particle size of 200-400 nm and a concentration of 100 mg / mL; The percentages are percentages by volume.

2. The magnetic bead method FFPE sample DNA nucleic acid extraction kit according to claim 1, characterized in that: The chaotropic salt is one or more of guanidine hydrochloride, guanidine isothiocyanate, sodium chloride, sodium periodate, and sodium iodide.

3. The magnetic bead method FFPE sample DNA nucleic acid extraction kit according to claim 1, characterized in that: The nonionic surfactant is one or more of Triton X-100, PEG 200, NP-40, Tween-20, and Tween-80; the anionic surfactant is one or more of SDS, SLS, TLS, and SDBS.

4. The magnetic bead method FFPE sample DNA nucleic acid extraction kit according to claim 1, characterized in that: The pH buffer is one or more of Tris-HCl buffer, acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, and PBS buffer.

5. The magnetic bead method FFPE sample DNA nucleic acid extraction kit according to claim 1, characterized in that: The alcohol is one or more of ethanol, isopropanol and isopentanol.

6. The magnetic bead method FFPE sample DNA nucleic acid extraction kit according to claim 1, characterized in that: The ion chelating agent is one or more of EDTA, EDTA•Na2, and EGTA.

7. The magnetic bead method FFPE sample DNA nucleic acid extraction kit according to claim 1, characterized in that: The organic solvent is one or more of mineral oil, C7-C20 straight-chain / branched alkane or a mixture thereof, C10-C20 straight-chain / branched alkane or a mixture thereof, and polyorganosiloxane.

8. The magnetic bead method FFPE sample DNA nucleic acid extraction kit according to claim 1, characterized in that: The nucleic acid precipitation promoting agent includes one or more of Acryl polymer solution, glycogen, and PolyA potassium salt.

9. A method for extracting DNA nucleic acid from FFPE samples, characterized in that: A magnetic bead method FFPE sample DNA nucleic acid extraction kit is used as described in any one of claims 1 to 9.

10. The method for extracting nucleic acid from FFPE samples according to claim 9, characterized in that: The method comprises the following steps: S1. Add 1 mL of dewaxing solution to the FFPE sample and mix well. Centrifuge briefly and place in a 56°C metal bath or water bath for 5 minutes. S2. Centrifuge at 14,000 x g for 2 min, discard the supernatant, add 1 mL of organic solvent and mix, centrifuge at 14,000 x g for 2 min, discard the supernatant and remove the organic solvent; S3. Add 300 µL FFPE digestion solution and 20 µL proteinase K solution, mix well, centrifuge briefly, heat in a 56°C metal bath or water bath until the sample is completely lysed, heat at 90°C for 1 h, centrifuge at 1,000 x g for 1 min, and collect the supernatant; S4. Add 600 μL of lysis binding solution and sample to the first well of the extraction plate, add 900 μL of washing solution 1 to the second well, add 500 μL of washing solution 1 to the third well, add 900 μL of washing solution 2 to the fourth well, add 300 μL of washing solution 2 and 20 μL of magnetic bead solution to the fifth well, and add 70 μL of elution solution to the sixth well; S5. First, take the magnetic rod cover in the 5th well and immerse it in the 1st well at room temperature for 10 minutes, and then immerse it in the 2nd well, 3rd well, 4th well, and 5th well in turn at room temperature, with the immersion time in each well being 2 minutes; then air-dry the magnetic beads for 5 minutes, and then immerse them in the 6th well and mix for 10 minutes at 60°C. Finally, discard the magnetic beads, and the resulting eluate is the final nucleic acid product.

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