CN113088515A

CN113088515A - Kit and method for extracting FFPE tissue sample DNA and application thereof

Info

Publication number: CN113088515A
Application number: CN202110496661.8A
Authority: CN
Inventors: 雷小军; 高玉晓; 张瑞; 王琛
Original assignee: Hangzhou Kangdai Sirui Biotechnology Co ltd
Current assignee: Hangzhou Kangdai Sirui Biotechnology Co ltd
Priority date: 2021-05-07
Filing date: 2021-05-07
Publication date: 2021-07-09

Abstract

The invention discloses a kit and a method for extracting FFPE tissue sample DNA and application thereof. The kit comprises a dewaxing lysis buffer and proteinase K, wherein the dewaxing lysis buffer comprises: 1-6% of sodium dodecyl sulfate, 50-300mM of Tris-HCl, 1-30mM of EDTA-2Na, 0.1-4% of Triton X-100, 0.1-2M of NaCl, 0.1-8% of Tween 20 and 0.1-10% of PEG 6000; the percentages are volume percentages. The method adopts a unique dewaxing lysis buffer solution, can realize the dewaxing and tissue lysis of the FFPE tissue sample without independently adding a dewaxing agent, simplifies the dewaxing step, reduces the manual operation flow and improves the nucleic acid extraction and purification efficiency.

Description

Kit and method for extracting FFPE tissue sample DNA and application thereof

Technical Field

The invention belongs to the field of biological tissue preservation, and particularly relates to a kit and a method for DNA extraction and application thereof, in particular to a kit and a method for FFPE tissue sample DNA extraction and application thereof.

Background

The tumor tissue retains the specific genetic information thereof, is very precious and irreplaceable, is an important research material for medical workers, but the tumor tissue must be stored under the condition of ultralow temperature refrigeration after being isolated, otherwise the original structure and function of the tumor tissue are easily lost, and the inconvenience is brought to the subsequent research due to the poor storage. FFPE (Formalin-Fixed and Parrffin-Embedded) is a tissue sample Fixed by Formalin and Embedded by paraffin, can be preserved for a long time at normal temperature, researchers carry out paraffin embedding and other treatments on tumor tissue blocks, and then treat the tumor tissue blocks into tissue samples with the thickness of 5-10 mu m by an ultrathin microtome, and the purpose of long-term preservation at normal temperature can be achieved, so that the FFPE becomes the most common sample preservation means in hospitals gradually.

The cell nucleus protein structure of the tissue can be maintained by formalin fixation and paraffin embedding, in order to protect the integrity of the cell structure, formalin and protein amino are subjected to irreversible crosslinking, but the crosslinking effect of the formalin is harmful to nucleic acid, so that the nucleic acid is degraded to different degrees and intermolecular crosslinking is caused, meanwhile, the paraffin embedding treatment affects the extraction of the nucleic acid, high-quality DNA is difficult to obtain from FFPE tissue by using a conventional DNA extraction method, and components such as residual inhibitors affect the activity of DNA polymerase, PCR amplification and the like.

The magnetic bead method is a novel nucleic acid extraction technology with nano biological magnetic beads as carriers, nucleic acid molecules can be specifically identified and combined with groups modified on the surfaces of the magnetic beads in a special buffer system, then are aggregated or dispersed under the action of an external magnetic field, and adsorbed DNA can be reversibly released when the buffer condition is changed, so that the aim of quickly separating and purifying the DNA is fulfilled. The magnetic bead method for extracting nucleic acid can get rid of the manual operation processes of centrifugation, supernatant removal and the like in the traditional nucleic acid extraction process, and can be applied to various automatic nucleic acid extraction platforms.

Due to the specificity of FFPE samples, in order to extract DNA therefrom, a dewaxing process is required in the processing of the samples, and different dewaxing modes have a great influence on the quality of the product. Most of the kits sold in the market at present separate dewaxing solution from lysis solution, and dewaxing solution needs to be added for dewaxing, and then the subsequent cracking extraction step is carried out. The traditional dewaxing method is to use xylene, wash tissue slices with xylene repeatedly for several times to dissolve paraffin, then wash residual xylene with ethanol before DNA extraction, although xylene dewaxing is widely used in commercial kits, it still has some problems, firstly, xylene is a toxic organic reagent, which has certain danger to the personal safety of operators, secondly, in order to remove xylene, the steps of repeated centrifugation, washing and the like are needed, which increases the workload of operators and causes loss to target DNA.

At present, a relatively simple one-step dewaxing method is disclosed in chinese patent application CN109022418A (2018, 12/18), which discloses a rapid dewaxing method for FFPE sample nucleic acid, and the specific steps are to use an oil phase reagent capable of dissolving paraffin and a water phase reagent capable of cleaving a tissue sample to achieve simple and rapid FFPE sample dewaxing by single temperature incubation, add the reagent into a sample tube with FFPE added, incubate at 56 ℃ for 30 minutes to 2 hours, take out the liquid in the lower water phase layer by a mechanical device or a liquid-moving device, and the water phase liquid is the liquid which can be used for subsequent nucleic acid extraction operation.

However, since only a single temperature (about 56 ℃) is used for incubation, nucleic acid and protein cannot be completely de-crosslinked, the incubation time is also affected by the type of the FFPE sample, if the incubation time at the single temperature is too long, excessive fragmentation of DNA is easily caused, the extraction yield of DNA is affected, and in addition, the residue of oil phase reagent in the operation process also affects the subsequent extraction and purification of DNA.

Disclosure of Invention

The invention aims to overcome the defects of low DNA extraction amount and low purity of FFPE tissue sample extraction caused by long time consumption of oil phase reagent for dewaxing and dewaxing in the prior art, and provides a kit and a method for DNA extraction and application thereof, in particular to the kit and the method for FFPE tissue sample DNA extraction and application thereof. By adopting the invention, the FFPE tissue sample can be efficiently and rapidly extracted without using an oil phase reagent, the extracted DNA quality is high, and the extraction process is convenient and safe.

In the study of extracting DNA from FFPE tissue samples, the inventors have conducted diligent attempts on dewaxing lysis buffer, and unexpectedly found that dewaxing lysis treatment of FFPE tissue samples can be achieved by using a dewaxing lysis buffer with a specific ratio in combination with conventional proteinase K. On the basis, the inventors tried to adjust the conditions of dewaxing and DNA extraction, and unexpectedly obtained a dewaxing method and a DNA extraction method having a good DNA extraction effect, and obtained DNA with a high extraction amount and good purity.

The invention solves the problems through the following technical scheme:

in a first aspect the invention provides a kit for FFPE tissue sample DNA extraction, the kit comprising a deparaffinization lysis buffer and proteinase K, the deparaffinization lysis buffer comprising:

1-6% of sodium dodecyl sulfate, 50-300mM of Tris-HCl, 1-30mM of EDTA-2Na, 0.1-4% of Triton X-100, 0.1-2M of NaCl, 0.1-8% of Tween 20 and 0.1-10% of PEG 6000;

the percentages are volume percentages.

Preferably, the deparaffinization lysis buffer comprises: 1-4% Sodium Dodecyl Sulfate (SDS), 80-250mM Tris-HCl, 4-20mM EDTA-2Na, 0.1-2.5% Triton X-100, 0.1-1M NaCl, 0.1-5% Tween 20 and 0.1-5% PEG 6000; the percentages are volume percentages.

More preferably, the deparaffinized lysis buffer comprises: 2-3% of sodium dodecyl sulfate, 100-200mM of Tris-HCl, 5-10mM of EDTA-2Na, 0.5-1% of Triton X-100, 0.1-0.2M of NaCl, 0.1-1% of Tween 20 and 0.1-2% of PEG 6000.

In a specific embodiment of the invention, the dewaxed lysis buffer comprises: 2% sodium dodecyl sulfate, 200mM Tris-HCl, 10mM EDTA-2Na, 1% Triton X-100, 0.1M NaCl, 1%

Tween

20 and 2% PEG 6000; the percentages are volume percentages.

In a specific embodiment of the invention, the dewaxed lysis buffer comprises: 3% sodium dodecyl sulfate, 100mM Tris-HCl, 5mM EDTA-2Na, 0.5% Triton X-100, 0.2M NaCl, 0.1% Tween 20 and 0.1% PEG 6000; the percentages are volume percentages.

The proteinase K (proteinase K) may be conventional in the art, with a working concentration of 1.25 mg/mL.

In a preferred embodiment of the present invention, the kit further comprises magnetic beads.

Preferably, the working concentration of the magnetic beads is 3-15%, more preferably 5-12%; the percentages are volume percentages.

Preferably, the kit further comprises a binding solution, a washing solution I, a washing solution II and an eluent; wherein: the binding liquid includes: 50-95% isopropanol and 0.1-3M NaCl; the washing solution I comprises: 50-85% ethanol and 10-50mM Tris-HCl; the washing solution II comprises: 50-80% ethanol; the eluent comprises: 10-50mM Tris-HCl and 1-5mM EDTA-2Na, wherein the pH of the eluent is 7.0-9.0; the percentages are volume percentages.

In a specific embodiment of the present invention, the concentration of the magnetic beads is 9%; the percentages are volume percentages.

In some embodiments of the invention, the binding liquid comprises: 50. 60, 80 or 95% isopropanol and 0.1, 0.2, 0.4 or 3M NaCl; the percentages are volume percentages.

In some embodiments of the invention, wash I comprises: 50. 70, 75 or 85% ethanol and 20mM Tris-HCl; the percentages are volume percentages.

In some embodiments of the invention, wash II comprises: 50. 70, 75 or 80% ethanol; the percentages are volume percentages.

In some embodiments of the invention, the eluent comprises: 10. 20 or 50mM Tris-HCl and 1, 2 or 5mM EDTA-2Na, the eluent having a pH of 7.5 or 8.0; the percentages are volume percentages.

A second aspect of the invention provides a method for DNA extraction of an FFPE tissue sample, the method using the kit of the first aspect for dewaxing, comprising:

mixing the FFPE tissue sample, the dewaxing lysis buffer solution and proteinase K, and sequentially performing incubation in a first heating stage and incubation in a second heating stage to obtain a DNA solution of the FFPE tissue sample; the incubation in the first heating stage is carried out at 40-70 ℃ for 15-30 min; the incubation of the second heating stage is at 60-100 deg.C for 15-30 min.

The DNA solution is the aqueous fraction containing DNA after dewaxing.

Preferably, the mixing is oscillatory mixing.

Preferably, the temperature of the incubation of the first heating stage is 55 or 56 ℃ and the time of the incubation is 15min or 30 min; the temperature of the incubation in the second heating stage is 80 ℃ and the incubation time is 15min or 30 min.

In a preferred embodiment of the present invention, the FFPE tissue sample is a paraffin section or a paraffin block.

The thickness of the paraffin section is preferably 5-10 μm; or the mass of the paraffin mass is less than 15 mg.

In a specific embodiment of the invention, the dewaxed lysis buffer and the proteinase K are added sequentially to the FFPE tissue sample.

In a preferred embodiment of the present invention, the method further comprises extracting DNA by a magnetic bead method.

The magnetic bead method may be conventional in the art, and preferably comprises:

(1) adding a binding solution into the DNA solution, uniformly mixing, and carrying out magnetic bead adsorption;

(2) adding a washing solution I into the DNA solution adsorbed by the magnetic beads in the step (1) for first washing; then adding a washing solution II, carrying out second washing and drying to obtain a DNA extract;

performing magnetic bead adsorption after the first washing and before the second washing and drying;

(3) and (3) adding the eluent into the DNA extract obtained in the step (2), uniformly mixing, and carrying out magnetic bead adsorption to obtain a DNA extraction solution.

Preferably, the mixing in the step (1) is sucking mixing, and the frequency of sucking mixing is mixing once every 2min or 3 min; the adsorption time of the magnetic beads is 30 s.

Preferably, the first washing and the second washing in (2) are both shaking washing, and the time of the shaking washing is 1 min; the adsorption time of the magnetic beads is 30 s; the drying temperature is room temperature or 37 ℃, when the drying temperature is room temperature, the drying time is 2-3min, and when the drying temperature is 37 ℃, the drying time is 3-5 min.

The room temperature may be conventional in the art.

Preferably, the mixing in the step (3) is sucking, beating and mixing for 1 min; the adsorption time of the magnetic beads is 30-60 s.

A third aspect of the invention provides the use of a kit as described in the first aspect in DNA extraction of FFPE tissue samples.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

(1) the method adopts a unique dewaxing lysis buffer solution, does not need to separately add dewaxing agents such as organic reagents of xylene, phenol, chloroform and the like, can directly reverse formalin crosslinking and shorten dewaxing time, effectively removes paraffin, PCR inhibitors and the like, and can realize dewaxing and tissue lysis of FFPE tissue samples.

(2) The invention improves the dewaxing treatment process, shortens the heating time in the early stage, realizes the dewaxing and tissue cracking of the FFPE tissue sample by adopting the temperature difference, simplifies the dewaxing step, reduces the manual operation flow, can be conveniently combined with a related nucleic acid automatic extraction platform, has sufficient yield of extracted DNA and high purity, can meet the requirements of downstream experiments, and realizes the fast and efficient extraction of the FFPE sample nucleic acid.

(3) The invention adopts the magnetic bead method to purify and extract the DNA, does not need a centrifuge, is simple and convenient to operate, adopts the nano magnetic bead with the surface modified with special chemical groups, has strong affinity to the target DNA under a special buffer system, and can reversibly release the adsorbed DNA when the conditions are changed, thereby achieving the aim of rapidly separating and purifying the DNA.

(4) The invention adopts the buffer solution which integrates dewaxing and cracking into a whole during extraction, can complete the dewaxing and cracking process within half an hour, can obtain the DNA of the FFPE tissue sample with high purity within 1 hour, greatly reduces the extraction time of nucleic acid, reduces the workload, is safe and nontoxic, has reliable result and improves the extraction and purification efficiency of nucleic acid.

Drawings

FIG. 1 shows the qPCR results for the DNA extracted in example 1.

FIG. 2 shows the qPCR results for the DNA extracted in example 2.

FIG. 3 shows the qPCR results for the DNA extracted in example 3.

FIG. 4 shows the qPCR results for the DNA extracted in example 4.

FIG. 5 shows the qPCR results for the DNA extracted in example 5.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The reagents and materials used in the examples are shown in table 1.

Table 1 reagents and materials used in the examples

Name (R)	Suppliers of goods	Goods number
			Magnetic bead	SUZHOU NANOMICRO TECHNOLOGY Co.,Ltd.	MSRSH-050
Proteinase K	YEASEN BIOLOGICAL TECHNOLOGY (SHANGHAI) Co.,Ltd.	10401ES60
			FFPE tissue samples	Horizon Discovery	HD141
Mineral oil	SHANGHAI TITAN TECHNOLOGY Co.,Ltd.	84237A

The FFPE tissue samples used in the examples may remove the paraffin portion of the long term exposure to air as appropriate, and are less than 15 mg.

Example 1

1. Scraping FFPE tissue samples (5-10 μm thick, 1X 1 cm)²Size) 1-4 pieces into a 1.5mL centrifuge tube, 300. mu.L of deparaffinized lysis buffer (2% SDS, 200mM Tris-HCl, 10mM EDTA-2Na, 1% Triton X-100, 0.1M NaCl, 1

% Tween

20, 2% PEG6000) was added, 20. mu.L of proteinase K was added, mixed with shaking, and incubated in a 55 ℃ metal bath for 15 min.

2. The metal bath was warmed to 80 ℃ and incubated for a further 15min (note: the temperature was raised directly from 55 ℃ to 80 ℃ without removing the tube), after incubation, the lower aqueous phase was removed by pipetting gun and the lower aqueous solution (about 250. mu.L) was transferred to a new 1.5mL centrifuge tube by pipetting.

3. And sequentially adding 30 mu L of magnetic beads and 300 mu L of binding solution (65% isopropanol and 0.2M NaCl) into the centrifuge tube, sucking, uniformly mixing once every 2min, and uniformly mixing for 3 times to avoid aggregation of the magnetic beads.

4. The centrifuge tube was placed on a magnetic stand for 30 seconds to allow the magnetic beads to be completely adsorbed. The supernatant was carefully removed to avoid touching or breaking up the beads.

5. Adding 500. mu.L of washing solution I (70% ethanol, 20mM Tris-HCl), washing with shaking for 1min, and placing the centrifuge tube on a magnetic frame for 30s to make the magnetic beads be completely adsorbed. The supernatant was removed to avoid touching or breaking up the beads.

6. Adding 500. mu.L of washing solution II (70% ethanol), washing with shaking for 1min, and placing the centrifuge tube on a magnetic frame for 30s to make the magnetic beads be completely adsorbed. The supernatant was removed to avoid touching or breaking up the beads.

7. And opening the cover for 2-3min at room temperature, and airing the magnetic beads.

8. 40-50. mu.L of eluent (TE buffer) (10mM Tris-HCl, 1mM EDTA-2Na, pH 8.0) was added and pipetted for 1min, followed by 30-60s adsorption with a magnet, and the supernatant was collected in a new 1.5mL EP tube to ensure that the collected supernatant was free of magnetic beads.

The collected supernatant is the extracted genome DNA, and can be stored at-20 ℃ for later use.

Example 2

1. Scraping FFPE tissue samples (5-10 μm thick, 1X 1 cm)²Size) 1-4 pieces into a 1.5mL centrifuge tube, 300. mu.L of deparaffinized lysis buffer (3% SDS, 100mM Tris-HCl, 5mM EDTA-2Na, 0.5% Triton X-100, 0.2M NaCl, 0.1% Tween 20, and 0.1% PEG6000), 20. mu.L of proteinase K were added, mixed by shaking, and incubated at 56 ℃ for 15 min.

2. The metal bath was warmed to 80 ℃ and incubated for a further 15min (note: the temperature was raised from 55 ℃ to 80 ℃ directly without removing the tube), after incubation, the lower aqueous phase was removed with a pipette and the lower aqueous solution (about 250. mu.L) was transferred to a new 1.5mL centrifuge tube by aspiration.

3. Adding 20 μ L of magnetic beads and 300 μ L of binding solution (80% isopropanol and 0.4M NaCl) in sequence, shaking, mixing, and mixing once every 3min for 3 times to avoid aggregation of magnetic beads.

5. Add 500. mu.L of washing solution I (75% ethanol, 20mM Tris-HCl), shake and wash for 1min, place the centrifuge tube on the magnetic frame for 30s, so that the magnetic beads are completely adsorbed. The supernatant was carefully removed to avoid touching or breaking up the beads.

6. Adding 500. mu.L of washing solution II (75% ethanol), washing with shaking for 1min, and placing the centrifuge tube on a magnetic frame for 30s to make the magnetic beads be completely adsorbed. The supernatant was removed to avoid touching or breaking up the beads.

7. Uncovering the cover for 3-5min at room temperature or 37 ℃, and airing the magnetic beads (note that the magnetic beads cannot be dried excessively so as not to influence the elution effect).

8. Adding 40-50 μ L of eluent (20mM Tris-HCl, 2mM EDTA-2Na, pH 7.5), sucking and mixing for 1min, adsorbing with magnet for 30-60s, collecting supernatant in new 1.5mL EP tube, and ensuring that there is no magnetic bead in the collected supernatant.

Example 3

1. Scraping FFPE tissue samples (5-10 μm thick, 1X 1 cm)²Size) 1-4 pieces to 1.5mL centrifuge tubes, 300. mu.L of deparaffinized lysis buffer (2% SDS, 200mM Tris-HCl, 10mM EDTA-2Na, 1% Triton X-100, 0.1M NaCl, 1

% Tween

20, 2% PEG6000) was added, 20. mu.L of proteinase K was added, mixed with shaking, and incubated at 55 ℃ in a metal bath for 30 min.

2. The metal bath was warmed to 80 ℃ and incubated for a further 30min (note: the temperature was raised from 55 ℃ to 80 ℃ directly without removing the tube), after incubation, the lower aqueous phase was removed with a pipette and the lower aqueous solution was transferred to a new 1.5mL centrifuge tube by aspiration.

The subsequent steps were the same as in example 1.

Example 4

1. Scraping FFPE tissue samples (5-10 μm thick, 1X 1 cm)²Size) 1-4 pieces into a 1.5mL centrifuge tube, 300. mu.L of deparaffinized lysis buffer (6% SDS, 50mM Tris-HCl, 1mM EDTA-2Na, 4% Triton X-100, 2M NaCl, 0.1

% Tween

20, 10% PEG6000) was added, 20. mu.L of proteinase K was added, mixed with shaking, and incubated in a 55 ℃ metal bath for 15 min.

3. And sequentially adding 30 mu L of magnetic beads and 300 mu L of binding solution (50% isopropanol and 0.1M NaCl) into the centrifuge tube, sucking, uniformly mixing once every 2min, and uniformly mixing for 3 times to avoid aggregation of the magnetic beads.

5. Add 500. mu.L of washing solution I (50% ethanol, 20mM Tris-HCl), wash with shaking for 1min, place the centrifuge tube on the magnetic frame for 30s, so that the magnetic beads are completely adsorbed. The supernatant was removed to avoid touching or breaking up the beads.

6. Adding 500. mu.L of washing solution II (50% ethanol), washing with shaking for 1min, and placing the centrifuge tube on a magnetic frame for 30s to make the magnetic beads be completely adsorbed. The supernatant was removed to avoid touching or breaking up the beads.

8. 40-50. mu.L of eluent (TE buffer) (50mM Tris-HCl, 5mM EDTA-2Na, pH 8.0) was added and pipetted for 1min, followed by 30-60s adsorption with a magnet, and the supernatant was collected in a new 1.5mL EP tube to ensure that the collected supernatant was free of magnetic beads.

Example 5

1. Scraping FFPE tissue samples (5-10 μm thick, 1X 1 cm)²Size) 1-4 pieces into a 1.5mL centrifuge tube, 300. mu.L of deparaffinized lysis buffer (1% SDS, 300mM Tris-HCl, 30mM EDTA-2Na, 0.1% Triton X-100, 0.1M NaCl, 8% Tween 20, 0.1% PEG6000) was added, 20. mu.L of proteinase K was added and mixed with shaking, and the mixture was incubated in a metal bath at 55 ℃ for 15 min.

3. And sequentially adding 30 mu L of magnetic beads and 300 mu L of binding solution (95% isopropanol and 3M NaCl) into the centrifuge tube, sucking, uniformly mixing once every 2min, and uniformly mixing for 3 times to avoid aggregation of the magnetic beads.

5. Add 500. mu.L of washing solution I (85% ethanol, 20mM Tris-HCl), wash with shaking for 1min, place the centrifuge tube on the magnetic frame for 30s, so that the magnetic beads are completely adsorbed. The supernatant was removed to avoid touching or breaking up the beads.

6. Adding 500. mu.L of washing solution II (80% ethanol), washing with shaking for 1min, and placing the centrifuge tube on a magnetic frame for 30s to make the magnetic beads be completely adsorbed. The supernatant was removed to avoid touching or breaking up the beads.

Comparative example 1 dewaxing extraction Using conventional oil phase reagents

1. Scraping FFPE tissue samples (5-10 μm thick, 1X 1 cm)²Size) 1-4 pieces into a 1.5mL centrifuge tube, 300. mu.L of deparaffinized lysis buffer (2% SDS, 200mM Tris-HCl, 10mM EDTA-2Na, 1% Triton X-100, 0.1M NaCl, 1% Tween, 2% PEG6000), 20. mu.L of proteinase K (working concentration 1.25mg/mL) and 50. mu.L of oil phase reagent (mineral oil) were added, mixed with shaking, and incubated at 55 ℃ for 15 min.

The subsequent steps were the same as in example 1.

Comparative example 2

Extraction was performed using the Magnetic Universal Genomic DNA Kit (DP705) from Tiangen Biochemical technology (Beijing) Ltd, the method of Tiangen Kit instructions is as follows.

1. Slicing paraffin (5-10 μm thick, 1 × 1 cm)²Size) 2-8 pieces, placing into a 1.5mL sterile centrifuge tube, adding 300 μ L environmental protection dewaxed oil DPR (TIANGEN, RK208, available), 300 μ L tissue digestive juice GHA and 20 μ L protease K, respectively, vigorously vortexing for 10sec, and digesting at 75 deg.C for 30-60min until the tissue mass disappears.

2. Digesting at 90 deg.C for 1h (adding sample after temperature of temperature control equipment is raised to 90 deg.C).

3. The lower layer, 300. mu.L, was transferred to a new 1.5mL centrifuge tube for further experiments.

4. Adding 900 μ L buffer GDZ (before use, checking whether absolute ethanol has been added), and shaking and mixing for 2 min.

5. The centrifuge tube was placed on a magnetic stand and allowed to stand for 30sec, and after the magnetic beads were completely adsorbed, the liquid was carefully aspirated.

6. Add 500. mu.L of buffer GDZ and mix well for 2min with shaking.

7. The centrifuge tube was placed on a magnetic stand and allowed to stand for 30sec, and after the magnetic beads were completely adsorbed, the liquid was carefully aspirated.

8. Taking off the centrifuge tube from the magnetic frame, adding 900 μ L rinsing liquid PWD (before use, checking whether absolute ethanol has been added), and shaking and mixing for 2 min.

9. The centrifuge tube was placed on a magnetic stand and allowed to stand for 30sec, and after the magnetic beads were completely adsorbed, the liquid was carefully aspirated.

10. Taking off the centrifugal tube from the magnetic frame, adding 300 μ L of rinsing liquid PWD, and shaking and mixing for 2 min.

11. The centrifuge tube was placed on a magnetic stand and allowed to stand for 30sec, and after the magnetic beads were completely adsorbed, the liquid was carefully aspirated.

12. Placing the centrifuge tube on a magnetic frame, and air drying at room temperature for 10-15 min.

Note that: the ethanol residue can inhibit subsequent enzyme reaction, so that the ethanol is completely volatilized during air drying. But also not dried for too long a time to avoid difficulty in eluting the DNA.

13. Taking off the centrifuge tube from the magnetic frame, adding 50-100 μ L of elution buffer TB, shaking, mixing, incubating at 56 deg.C for 10min, and mixing 3 times by reversing each time for 3-5 times.

14. The centrifuge tube was placed on a magnetic rack and allowed to stand for 2min, after the magnetic beads were completely adsorbed, the DNA solution was carefully transferred to a new centrifuge tube and stored under appropriate conditions.

Results examples

The results of examples 1 to 5 and comparative examples 1 to 2 are shown in Table 2.

TABLE 2 DNA content of paraffin tissue specimens extracted in each example

As shown in tables 3 and 4, the DNA extracted in the examples and comparative examples, which is HBB gene (human β -globin gene), was verified by qPCR.

The results of qPCR are shown in figures 1-5. As can be seen from the graphs, examples 1 to 5 are superior to comparative examples 1 and 2 in effect, and examples 1 to 5 using no oil phase agent are superior to comparative examples 1 and 2 using an oil phase agent in effect.

TABLE 3 amplification of primer sequences of HBB Gene Using Universal primers

TABLE 4 PCR amplification procedure

SEQUENCE LISTING

<110> Hangzhou Kongdairui Biotechnology Co., Ltd

<120> kit and method for FFPE tissue sample DNA extraction and application thereof

<130> P21012751C

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> HBB-F

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tgggtttctg ataggcactg act 23

<210> 2

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> HBB-R

<400> 2

aacagcatca ggagtggaca gat 23

<210> 3

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> HBB-P

<220>

<221> misc_feature

<222> (1)..(1)

<223> FAM Modification

<220>

<221> misc_feature

<222> (26)..(26)

<223> BHQ1 Modification

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tctacccttg gacccagagg ttcttt 26

Claims

1. A kit for FFPE tissue sample DNA extraction, comprising a deparaffinization lysis buffer and proteinase K, the deparaffinization lysis buffer comprising:

the percentages are volume percentages.

2. The kit of claim 1, wherein the deparaffinized lysis buffer comprises: 1-4% of sodium dodecyl sulfate, 80-250mM of Tris-HCl, 4-20mM of EDTA-2Na, 0.1-2.5% of Triton X-100, 0.1-1M of NaCl, 0.1-5% of Tween 20 and 0.1-5% of PEG 6000;

preferably, the deparaffinization lysis buffer comprises: 2-3% of sodium dodecyl sulfate, 100-200mM of Tris-HCl, 5-10mM of EDTA-2Na, 0.5-1% of Triton X-100, 0.1-0.2M of NaCl, 0.1-1% of Tween 20 and 0.1-2% of PEG 6000;

more preferably, the deparaffinized lysis buffer comprises: 2% sodium dodecyl sulfate, 200mM Tris-HCl, 10mM EDTA-2Na, 1% Triton X-100, 0.1M NaCl, 1% Tween 20 and 2% PEG 6000; alternatively, the first and second electrodes may be,

the deparaffinized lysis buffer comprises: 3% sodium dodecyl sulfate, 100mM Tris-HCl, 5mM EDTA-2Na, 0.5% Triton X-100, 0.2M NaCl, 0.1% Tween 20 and 0.1% PEG 6000;

the percentages are volume percentages.

3. The kit of claim 1 or 2, wherein the working concentration of proteinase K is 1.25 mg/mL.

4. The kit of claim 1, wherein the kit further comprises magnetic beads;

preferably, the working concentration of the magnetic beads is 3-15%, preferably 5-12%; and/or, the kit further comprises a binding solution, a washing solution I, a washing solution II and an eluent; wherein: the binding liquid includes: 50-95% isopropanol and 0.1-3M NaCl; the washing solution I comprises: 50-85% ethanol and 10-50mM Tris-HCl; the washing solution II comprises: 50-80% ethanol; the eluent comprises: 10-50mM Tris-HCl and 1-5mM EDTA-2Na, wherein the pH of the eluent is 7.0-9.0; the percentages are volume percentages.

5. The kit of claim 4, wherein the working concentration of the magnetic beads is 9%;

and/or, the binding liquid comprises: 50. 60, 80 or 95% isopropanol and 0.1, 0.2, 0.4 or 3M NaCl;

and/or, the wash liquor I comprises: 50. 70, 75 or 85% ethanol and 20mM Tris-HCl;

and/or, the wash solution II comprises: 50. 70, 75 or 80% ethanol;

and/or, the eluent comprises: 10. 20 or 50mM Tris-HCl and 1, 2 or 5mM EDTA-2Na, the eluent having a pH of 7.5 or 8.0;

the percentages are volume percentages.

6. A method for FFPE tissue sample DNA extraction using the kit of any one of claims 1-5 for dewaxing, comprising:

mixing the FFPE tissue sample, the dewaxing lysis buffer solution and proteinase K, and sequentially performing incubation in a first heating stage and incubation in a second heating stage to obtain a DNA solution of the FFPE tissue sample; the incubation in the first heating stage is carried out at 40-70 ℃ for 15-30 min; the incubation in the second heating stage is carried out at 60-100 ℃ for 15-30 min;

preferably, the mixing is oscillatory mixing; and/or the temperature of the incubation of the first heating stage is 55 or 56 ℃ and the time of the incubation is 15min or 30 min; the temperature of the incubation in the second heating stage is 80 ℃ and the incubation time is 15min or 30 min.

7. The method of claim 6, wherein the FFPE tissue sample is a paraffin section or a paraffin block;

preferably, the thickness of the paraffin section is 5-10 μm; or the mass of the paraffin mass is less than 15 mg.

8. The method of claim 6 or 7, further comprising extracting DNA by magnetic bead method; preferably, it comprises:

9. The method of claim 8,

the uniform mixing in the step (1) is sucking and beating uniform mixing, and the frequency of sucking, beating and uniform mixing is once per 2min or 3 min; the adsorption time of the magnetic beads is 30 s; and/or the presence of a gas in the gas,

the first washing and the second washing in the step (2) are both oscillating washing, and the oscillating washing time is 1 min; the adsorption time of the magnetic beads is 30 s; the drying temperature is room temperature or 37 ℃, when the drying temperature is room temperature, the drying time is 2-3min, and when the drying temperature is 37 ℃, the drying time is 3-5 min; and/or the presence of a gas in the gas,

the uniformly mixing in the step (3) is sucking, beating and uniformly mixing, and the sucking, beating and uniformly mixing time is 1 min; the adsorption time of the magnetic beads is 30-60 s.

10. Use of the kit of any one of claims 1 to 5 for DNA extraction from FFPE tissue samples.