High-integrity extraction method of DNA in FFPE sample

Abstract

The invention discloses a high-integrity extraction method of DNA in an FFPE sample, and on the basis of an existing FFPE magnetic bead method, an optimal combination relationship between environmental conditions and magnetic bead particle sizes is defined, and the DNA extraction method with long nucleic acid fragment and high integrity is obtained through pH setting of a lysis solution and a washing solution and selection of the magnetic bead particle sizes.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA extraction, and in particular to a method for extracting DNA from FFPE samples with high integrity. Background Art

[0002] FFPE (Formalin Fixed Paraffin Embedded) is a common method for processing tissue samples. By fixing the tissue with formalin and embedding it with paraffin, the tissue can maintain its integrity after long-term storage and sectioning, making FFPE samples one of the most commonly used tissue sample types in the fields of cancer diagnosis, treatment, and prognosis assessment.

[0003] However, formalin fixation and high-temperature paraffin infiltration can easily cause varying degrees of degradation and molecular cross-linking of nucleic acids in tissues, making it difficult to extract high-quality DNA from FFPE samples. With the continuous advancement of next-generation sequencing (NGS) technology, by optimizing nucleic acid extraction methods, researchers can make better use of FFPE samples and deeply explore the precious information resources therein, bringing more possibilities and opportunities for disease research and personalized treatment.

[0004] Invention patent CN103725672B discloses a kit and method for separating DNA and RNA from formalin-fixed paraffin-embedded tissues, which involves first dewaxing FFPE sections, then lysing the tissues, and finally using a column extraction method to achieve DNA separation.

[0005] Invention patent application CN108841920A proposed the FFPE magnetic bead method, which can confine cell tissues in a certain spatial range through a magnetic separation device, effectively preventing the tissues from being sucked away along with the dewaxing agent or other solutions. By comparison, its extraction effect is better than the column extraction method.

[0006] However, after testing, the DNA extraction effect of the FFPE magnetic bead method given in the invention patent application CN108841920A is still not very ideal. The nucleic acid extracted from the FFPE sample is highly degraded, the fragments are small, and the integrity is low. Excessive DNA breaks will introduce additional sequence mutations, which are specifically manifested as some base changes in the DNA sequence in the NGS results that did not exist before fixation. These artificial mutations cause false negatives and false positives in NGS detection. Summary of the invention

[0007] In view of the technical defects in DNA extraction from FFPE samples, the present invention aims to provide a high-integrity extraction method for DNA from FFPE samples. By changing the magnetic bead particle size and pH environment, the extracted nucleic acid fragments are longer and more complete, and the FFPE nucleic acid extraction method is easy to automate.

[0008] A method for extracting DNA from FFPE samples with high integrity, comprising the following steps:

[0009] Step 1, dewaxing the FFPE samples and collecting the dewaxed sample tissues;

[0010] Step 2, add lysis buffer and protease to lyse the sample tissue, incubate at 50-70°C for 1 hour until the sample is completely lysed, then raise the temperature to 75-95°C and continue incubation for 1 hour to de-crosslink;

[0011] The lysis solution is 10-30 mM Tris-HCl, 10-50 mM EDTA, 1-5 M guanidine thiocyanate, 2-10 M NaCl, 2-10% Triton X-100, pH = 6.0-7.0;

[0012] Step 3, separating DNA by magnetic bead method: first, collecting the lysed DNA solution, adding magnetic beads to absorb DNA; then, collecting the magnetic beads that absorb DNA, adding detergent I to wash protein impurities; then, collecting the magnetic beads that absorb DNA, adding detergent II to wash salt ion impurities, repeating for multiple times; then, aspirating the residual liquid and drying; finally, adding elution solution to dissolve the DNA and collect it;

[0013] The particle size of the magnetic beads is 100-1000 nm; the washing solution I is 10-50 mM Tris-base, 2-25 mM EDTA, 1-20 M NaCl, 10-30% ethanol, pH = 6.0-7.0; the washing solution II is 10-50 M Tris-base, 60-80% ethanol, pH = 6.0-7.0; the elution solution is 1-10 mM Tris-base, 1-5 mM EDTA, pH = 8.0.

[0014] Furthermore, the step 1 is specifically as follows: taking an appropriate amount of FFPE slices, placing them in a clean 1.5 mL centrifuge tube, adding 400 μL of dewaxing solution, vortexing vigorously for 5 seconds, briefly centrifuging to allow the sample to be immersed in the dewaxing solution, incubating at 50-70° C. for 2-8 minutes, and then centrifuging at 10200 rpm (10000×g) for 2 minutes, discarding the supernatant, and obtaining the dewaxed sample tissue.

[0015] Furthermore, the dewaxing liquid used in the dewaxing treatment in step 1 is one or more liquid reagents that can dissolve paraffin, such as mineral oil, silicone oil, liquid paraffin, undecane, and 1,5-cyclooctadiene.

[0016] Furthermore, the step 2 specifically includes adding 200 μL of lysis solution and 20 μL of protease, vortexing for 10 seconds, incubating at 50-70° C. for 1 hour until the sample is completely lysed; heating to 75-95° C., and continuing to incubate for 1 hour to decrosslink at high temperature.

[0017] Furthermore, the step 3 comprises the following steps:

[0018] Step 3.1, draw 200 μL of solution from the bottom of the tube, place it into another clean 1.5 mL centrifuge tube, add 20 μL of magnetic bead suspension, vortex mix for 20 seconds, place it on a rotary mixer and incubate for 10 minutes. After a short centrifugation, place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are completely adsorbed, and discard the supernatant;

[0019] Step 3.2, add 700 μL of washing solution I, oscillate and mix for 1 min to ensure that the magnetic beads are dispersed, centrifuge briefly, place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are completely adsorbed, then aspirate and discard the liquid;

[0020] Step 3.3, add 700 μL washing solution II, shake and mix for 1 min to ensure that the magnetic beads are dispersed, and after a short centrifugation, place the centrifuge tube on the magnetic rack and let it stand until the magnetic beads are completely adsorbed, aspirate the liquid and discard it, and repeat several times;

[0021] Step 3.4, aspirate the residual liquid, place the centrifuge tube on the magnetic rack to dry for 5-10 minutes, remove the centrifuge tube from the magnetic rack, add 50-100 μL of elution buffer, oscillate and mix, if there are magnetic beads clumping, blow with a pipette; after a short centrifugation, incubate at 50-70℃ for 5 minutes, oscillate and mix during this period to improve the elution effect;

[0022] Step 3.5, after a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are completely adsorbed, then transfer the DNA solution to another clean centrifuge tube for testing.

[0023] Based on the existing FFPE magnetic bead method, the present invention clarifies the preferred combination relationship between environmental conditions and magnetic bead particle size, and obtains a DNA extraction method with long nucleic acid fragments and high integrity by setting the pH of the lysis solution and the washing solution and selecting the magnetic bead particle size. DETAILED DESCRIPTION

[0024] For ease of understanding of the present invention, the present invention will be described more fully below in conjunction with specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The technical scheme of the present invention is described more clearly and completely below in conjunction with specific embodiments and comparative examples.

[0026] Example 1

[0027] A method for extracting DNA from FFPE samples with high integrity, comprising the following steps:

[0028] 1. Dewax the FFPE samples and collect the dewaxed sample tissues.

[0029] Take an appropriate amount of FFPE sections, place them in a clean 1.5mL centrifuge tube, add 400μL of dewaxing solution, vortex vigorously for 5s, centrifuge briefly to allow the sample to soak in the dewaxing solution, incubate at 56℃ for 5min, then centrifuge at 10200rpm (10000×g) for 2min, discard the supernatant, and obtain the dewaxed sample tissue.

[0030] The dewaxing liquid used in the dewaxing treatment is one or more liquid reagents that can dissolve paraffin, such as mineral oil, silicone oil, liquid paraffin, undecane, and 1,5-cyclooctadiene.

[0031] 2. Add lysis buffer and protease to lyse the sample tissue, incubate at 50-70°C for 1 hour until the sample is completely lysed, then raise the temperature to 75-95°C and continue incubating for 1 hour to reverse the cross-linking; wherein the lysis buffer is 10-30mM Tris-HCl, 10-50mM EDTA, 1-5M guanidine thiocyanate, 2-10M NaCl, 2-10% TritonX-100, pH = 7.0.

[0032] In this embodiment, 200 μL of lysis solution and 20 μL of protease were added, vortexed for 10 seconds, and incubated at 56° C. for 1 hour until the sample was completely lysed; the temperature was raised to 90° C. and incubated for another 1 hour to decrosslink at high temperature.

[0033] 3. Magnetic bead method to separate DNA: First, collect the lysed DNA solution and add magnetic beads to absorb DNA; then, collect the magnetic beads that absorb DNA, add detergent I to wash protein impurities; then, collect the magnetic beads that absorb DNA, add detergent II to wash salt ion impurities, and repeat several times; then, aspirate the residual liquid and dry it; finally, add eluent to dissolve the DNA and collect it;

[0034] The particle size of the magnetic beads is 100-1000 nm; the washing solution I is 10-50 mM Tris-base, 2-25 mM EDTA, 1-20 M NaCl, 10-30% ethanol, pH = 7.0; the washing solution II is 10-50 M Tris-base, 60-80% ethanol, pH = 7.0; the elution solution is 1-10 mM Tris-base, 1-5 mM EDTA, pH = 8.0.

[0035] In this embodiment, the following steps are specifically included:

[0036] Step 3.1, draw 200 μL of solution from the bottom of the tube, place it into another clean 1.5 mL centrifuge tube, add 20 μL of magnetic bead suspension (1000 nm), vortex mix for 20 seconds, place it on a rotary mixer and incubate for 10 minutes. After a brief centrifugation, place the centrifuge tube on a magnetic stand and let it stand until the magnetic beads are completely adsorbed, and discard the supernatant;

[0037] Step 3.2, add 700 μL of washing solution I, oscillate and mix for 1 min to ensure that the magnetic beads are dispersed, centrifuge briefly, place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are completely adsorbed, then aspirate and discard the liquid;

[0038] Step 3.3, add 700 μL washing solution II, shake and mix for 1 min to ensure that the magnetic beads are dispersed, and after a short centrifugation, place the centrifuge tube on the magnetic rack and let it stand until the magnetic beads are completely adsorbed, aspirate the liquid and discard it, and repeat several times;

[0039] Step 3.4, aspirate the residual liquid, place the centrifuge tube on the magnetic rack to dry for 5-10 minutes, remove the centrifuge tube from the magnetic rack, add 50-100 μL of elution buffer, oscillate and mix, if there are magnetic beads clumping, blow with a pipette; after a short centrifugation, incubate at 50-70℃ for 5 minutes, oscillate and mix during this period to improve the elution effect;

[0040] Step 3.5, after a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are completely adsorbed, then transfer the DNA solution to another clean centrifuge tube for testing.

[0041] Example 2

[0042] In this embodiment, the particle size of the magnetic beads in the magnetic bead suspension is 500 nm, and other operations are the same as in Embodiment 1.

[0043] Example 3

[0044] In this embodiment, the particle size of the magnetic beads in the magnetic bead suspension is 200 nm, and other operations are the same as in Embodiment 1.

[0045] Example 4

[0046] In this embodiment, the pH of the lysate is 6.0, the pH of the washing solution I is 6.0, the pH of the washing solution II is 6.0, and other operations are the same as those in embodiment 3.

[0047] Example 5

[0048] In this embodiment, the pH of the lysate is 5.0, the pH of the washing solution I is 5.0, the pH of the washing solution II is 5.0, and other operations are the same as those in embodiment 3.

[0049] Example 6

[0050] In this embodiment, the pH of the lysate is 6.0, the pH of the washing solution I is 6.0, the pH of the washing solution II is 6.0, and other operations are the same as in embodiment 2.

[0051] Example 7

[0052] In this embodiment, the pH of the lysate is 6.0, the pH of the washing solution I is 6.0, the pH of the washing solution II is 6.0, and other operations are the same as in embodiment 1.

[0053] Comparative Example 1

[0054] Use the commercially available magnetic bead-based FFPE sample DNA extraction kit (Xinbaiji, catalog number: M2015) to extract DNA according to the instructions.

[0055] Experimental verification: 8 groups of the same FFPE samples were used to extract DNA using the methods of Examples 1-7 and Comparative Example 1, respectively. The results are recorded in Table 1.

[0056] Table 1

[0057] Experimental Group Concentration (ng / μL) Main peak of fragment (bp) Example 1 55 4530 Example 2 80 4451 Example 3 101 4423 Example 4 98 5916 Example 5 85 5025 Example 6 70 4857 Example 7 64 4362 Comparative Example 1 81 4805

[0058] It can be seen from the data in Table 1 that in Example 4, when the environment of the magnetic beads is pH = 6.0 and the particle size of the magnetic beads is 200nm, the DNA integrity is the highest, and the obtained DNA concentration and fragment main peak are significantly better than those of Qiagen, which further shows that the binding of nucleic acids to magnetic beads is not only related to the particle size of the magnetic beads themselves, but also closely related to the environment (pH value).

[0059] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0060] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for extracting DNA with high integrity from FFPE samples, characterized in that: The following steps are involved: Step 1, dewaxing the FFPE samples and collecting the dewaxed sample tissues; Step 2, add lysis buffer and protease to lyse the sample tissue, incubate at 50-70°C for 1 hour until the sample is completely lysed, then raise the temperature to 75-95°C and continue incubation for 1 hour to de-crosslink; The lysis solution is 10-30 mM Tris-HCl, 10-50 mM EDTA, 1-5 M guanidine thiocyanate, 2-10 M NaCl, 2-10% TritonX-100, pH = 6.0-7.0; Step 3, separating DNA by magnetic bead method: first, collecting the lysed DNA solution, adding magnetic beads to absorb DNA; then, collecting the magnetic beads that absorb DNA, adding detergent I to wash protein impurities; then, collecting the magnetic beads that absorb DNA, adding detergent II to wash salt ion impurities, repeating for multiple times; then, aspirating the residual liquid and drying; finally, adding elution solution to dissolve the DNA and collect it; The particle size of the magnetic beads is 100-1000 nm; the washing solution I is 10-50 mM Tris-base, 2-25 mM EDTA, 1-20 M NaCl, 10-30% ethanol, pH = 6.0-7.0; the washing solution II is 10-50 M Tris-base, 60-80% ethanol, pH = 6.0-7.0; the elution solution is 1-10 mM Tris-base, 1-5 mM EDTA, pH = 8.

0.

2. The method for extracting DNA from FFPE samples with high integrity according to claim 1, characterized in that: Specifically, step 1 comprises taking an appropriate amount of FFPE sections, placing them in a clean 1.5 mL centrifuge tube, adding 400 μL of dewaxing solution, vortexing vigorously for 5 seconds, briefly centrifuging to allow the sample to be immersed in the dewaxing solution, incubating at 50-70° C. for 2-8 minutes, and then centrifuging at 10200 rpm (10000×g) for 2 minutes, discarding the supernatant, and obtaining the dewaxed sample tissue.

3. The method for extracting DNA from FFPE samples with high integrity according to claim 1, characterized in that: The dewaxing liquid used in the dewaxing treatment in step 1 is one or more liquid reagents that can dissolve paraffin, such as mineral oil, silicone oil, liquid paraffin, undecane, and 1,5-cyclooctadiene.

4. The method for extracting DNA from FFPE samples with high integrity according to claim 1, characterized in that: Specifically, step 2 includes adding 200 μL of lysis solution and 20 μL of protease, vortexing for 10 seconds, incubating at 50-70° C. for 1 hour until the sample is completely lysed; heating to 75-95° C., and continuing to incubate for 1 hour to decrosslink at high temperature.

5. The method for extracting DNA from FFPE samples with high integrity according to claim 1, characterized in that: The step 3 comprises the following steps: Step 3.1, draw 200 μL of solution from the bottom of the tube, place it into another clean 1.5 mL centrifuge tube, add 20 μL of magnetic bead suspension, vortex mix for 20 seconds, place it on a rotary mixer and incubate for 10 minutes. After a short centrifugation, place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are completely adsorbed, and discard the supernatant; Step 3.2, add 700 μL of washing solution I, oscillate and mix for 1 min to ensure that the magnetic beads are dispersed, centrifuge briefly, place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are completely adsorbed, then aspirate and discard the liquid; Step 3.3, add 700 μL washing solution II, shake and mix for 1 min to ensure that the magnetic beads are dispersed, and after a short centrifugation, place the centrifuge tube on the magnetic rack and let it stand until the magnetic beads are completely adsorbed, aspirate the liquid and discard it, and repeat several times; Step 3.4, aspirate the residual liquid, place the centrifuge tube on the magnetic rack to dry for 5-10 minutes, remove the centrifuge tube from the magnetic rack, add 50-100 μL of elution buffer, oscillate and mix, if there are magnetic beads clumping, blow with a pipette; after a short centrifugation, incubate at 50-70℃ for 5 minutes, oscillate and mix during this period to improve the elution effect; Step 3.5, after a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are completely adsorbed, then transfer the DNA solution to another clean centrifuge tube for testing.