DNA extraction kit and extraction method for paraffin-embedded tissues

文档序号：445922 发布日期：2021-12-28 浏览：7次中文

阅读说明：本技术 一种石蜡包埋组织的dna提取试剂盒以及提取方法 (DNA extraction kit and extraction method for paraffin-embedded tissues ) 是由赵瑾盛巧芸蒋亚辉张丽缪其杰于 2021-09-26 设计创作，主要内容包括：本发明涉及一种石蜡包埋组织的DNA提取试剂盒以及提取方法,属于基因检测技术领域。提取试剂盒中包含：脱蜡剂,用于对样本进行脱蜡处理；裂解缓冲液和蛋白酶,用于对样本进行消化处理；结合缓冲液和磁珠、用于对消化产物中的DNA进行结合；洗涤液和乙醇,用于对结合了DNA的磁珠进行洗涤；洗脱液,用于将磁珠上结合的DNA进行洗脱。本专利的方法可以有效地对石蜡包埋组织中的DNA进行提取,具有提取效率高、提取质量好的优点。(The invention relates to a DNA extraction kit and an extraction method for paraffin-embedded tissues, belonging to the technical field of gene detection. The extraction kit comprises: the dewaxing agent is used for carrying out dewaxing treatment on the sample; lysis buffer and protease for digesting the sample; the binding buffer solution and the magnetic beads are used for binding the DNA in the digestion product; a washing solution and ethanol for washing the magnetic beads to which the DNA is bound; and the eluent is used for eluting the DNA bound on the magnetic beads. The method can effectively extract the DNA in the paraffin-embedded tissue, and has the advantages of high extraction efficiency and good extraction quality.)

1. A DNA extraction kit for paraffin-embedded tissues, comprising:

the dewaxing agent is used for carrying out dewaxing treatment on the sample;

lysis buffer and protease for digesting the sample;

the binding buffer solution and the magnetic beads are used for binding the DNA in the digestion product;

a washing solution and ethanol for washing the magnetic beads to which the DNA is bound;

and the eluent is used for eluting the DNA bound on the magnetic beads.

2. The DNA extraction kit for wax-embedded tissue according to claim 1, wherein the dewaxing agent is xylene.

3. The kit for extracting DNA from wax-embedded tissue according to claim 1, wherein the lysis buffer comprises: 5-50mmol/L Tris-HCl; 1-5mmol/L NaCl; 5-20mmol/L EDTA; 8-50mmol/L DTT; 3-5mol/L of guanidinium isothiocyanate; 0.5% -2% SDS; 1% -5% of TritonX-100.

4. The kit for extracting DNA from wax-embedded tissue according to claim 1, wherein the protease is proteinase K.

5. The DNA extraction kit for wax-embedded tissue according to claim 1, wherein the combination buffer comprises: 100-200mmol/L Tris-HCl; 1-5mmol/L NaCl; 1% -5% of TritonX-100; 2-4mol/L of guanidinium isothiocyanate; 20-40% of isopropanol.

6. The kit for extracting DNA from wax-embedded tissue according to claim 1, wherein the washing solution comprises a first washing solution and a second washing solution, the first washing solution comprising: 100-200mmol/L Tris-HCl; 1-5mmol/L NaCl; 40-60% ethanol; 2-4mol/L of guanidinium isothiocyanate; the second washing solution comprises: 25-75mmol/L Tris-HCl; 75-85% ethanol.

7. The DNA extraction kit for wax-embedded tissue according to claim 1, wherein the ethanol is absolute ethanol; the eluent is free of nuclease water.

8. A method for extracting DNA of paraffin-embedded tissues is characterized by comprising the following steps:

step 1, adding a dewaxing agent into a tissue sample, heating after vortexing, centrifuging, removing supernatant, adding ethanol, vortexing, removing supernatant after centrifugation, and drying;

step 2, adding a lysis buffer solution and protease into the sample obtained in the step 1, and incubating;

step 3, adding a binding buffer solution and magnetic beads into the sample obtained in the step 2, and incubating;

step 4, removing the supernatant of the sample obtained in the step 4 on a magnetic frame, washing the sample by a washing solution and ethanol in sequence, and removing the supernatant on the magnetic frame;

and 5, adding an eluent into the sample obtained in the step 4, incubating and taking a supernatant.

9. The method for extracting DNA from paraffin-embedded tissue as claimed in claim 8, wherein in step 1, the vortex time is 5-20s, the centrifugal force is 10000-; the heating treatment is carried out at 35-60 deg.C for 5-20 min;

in the step 2, the incubation treatment process is carried out at 45-65 ℃ and at a rotation speed of 500-.

10. The method for extracting DNA from paraffin-embedded tissue according to claim 8, wherein in step 3, the incubation treatment is performed at room temperature for 1-30 min;

in the step 4, a first washing solution, a second washing solution and ethanol are adopted for washing in sequence in the washing process, the vortex is carried out for 1-30s in each washing process, and the incubation is carried out for 1-5 min;

and in the step 5, incubating for 1-10 min.

Technical Field

The invention relates to a DNA extraction kit and an extraction method for paraffin-embedded tissues, belonging to the technical field of gene detection.

Background

Malignant gliomas include anaplastic oligodendroglioma, anaplastic astrocytoma, anaplastic oligoastrocytoma, and glioblastoma. The prognosis of anaplastic oligodendroglioma with heterozygous deletion of chromosome 1 short arm (1p) and combined heterozygous deletion of chromosome 19 long arm (19q) is obviously better than that of same-grade astrocytoma when malignant glioma is treated by a combination of procarbazine/lomustine/vincristine (PCV); in subsequent studies, the combined deletion of 1p/19q was found to correlate with sensitivity to chemotherapy, and the anaplastic oligodendroglioma with the combined deletion of 1p/19q had a longer survival time. Therefore, the 1p/19q hybrid deletion can be used for the management of oligodendrocyte tumors and can be clinically used as an index for evaluating diagnosis, treatment and prognosis judgment. Temozolomide is a second generation alkylating agent and has been used for chemotherapy of malignant gliomas. The temozolomide can easily pass through a blood brain barrier, and has small toxic and side effects and adverse reactions. 06-methylguanine-DNA-methyltransferase (O6-methyl guanine-DNA-methyl transferase, MGMT) has a DNA repair effect and can cause temozolomide resistance, i.e. removal of alkylated adducts of guanine O6 site on DNA to restore damaged guanine. The MGMT gene silencing in tumor cell strains is usually accompanied with the methylation of a gene promoter CpG island, which indicates that the methylation of the gene promoter mediates the expression of the MGMT protein in tumor cells. The MGMT gene promoter methylation is clinically used for predicting the sensitivity and prognosis of temozolomide chemotherapy of glioblastoma multiforme patients.

In the prior art, the detection method for MGMT gene promoter methylation can be mainly divided into: (1) pyrosequencing, (2) methylation-sensitive restriction enzyme method (MS-RE), (3) fluorescent PCR method, (4) high-throughput sequencing method; among the above methods, the pyrosequencing method is a "gold standard" method; in high-throughput sequencing, the problems of complicated operation, poor amplification efficiency of primers, high cost and the like exist at present. In order to solve the above problems, the present invention provides a method for detecting methylation of 99 CpG sites of the MGMT promoter.

Disclosure of Invention

The purpose of the invention is: a method for detecting the methylation level of 99 CpG sites on the MGMT promoter and related reagents are provided.

The technical scheme is as follows:

a primer group for amplifying CpG sites on an MGMT promoter comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1-4, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 5-8.

A construction method of a methylation detection library of an MGMT promoter region comprises the following steps:

step 1, obtaining a sample, extracting DNA, and treating the DNA by a methylation modification conversion reagent;

step 2, performing multiple PCR amplification on the nucleotide obtained in the step 1 through a primer group to obtain an amplification product;

and 3, performing terminal repair on the amplification product, adding a base A at the terminal, adding a connector, performing amplification, and purifying to obtain the library.

In the step 1, the sample is an FFPE tissue sample, and the extracted DNA is obtained by a magnetic bead method.

In step 2, the multiplex PCR amplification process performs the following amplification procedures:

pre-denaturation at 92-97 deg.C for 5-15 min;

denaturation at 92-97 deg.C for 20-40s, annealing at 40-65 deg.C for 20-40s, and extension at 70-75 deg.C for 0.5-5min for 30-48 cycles;

final extension at 70-75 deg.C for 3-8 min.

In the step 3, the reaction solution in the end repairing process includes: 30-70ul of DNA, 5-15ul of phosphorylation reaction buffer solution, 10nM dNTP, 1-10ul of T4DNA polymerase, 0.5-2ul of Klenow Escherichia coli polymerase fragment, 1-10ul of T4 polynucleotide kinase and nuclease-free water, so that the total volume is supplemented to 100 ul.

In the step 3, the reaction solution with the base A added at the end comprises: 20-40ul DNA, 1mM dATP, 1-10ul 10XKlenow E.coli polymerase buffer, 1-5ul Klenow E.coli polymerase fragment, and nuclease-free water to make up the total volume to 50 ul.

In the step 3, the amplification procedure comprises pre-denaturation at 95-99 ℃ for 20-40 seconds, annealing at 60-70 ℃ for 20-40 seconds, extension at 70-75 ℃ for 20-40 seconds, co-circulation for 3-10 times, and extension at 70-75 ℃ for 3-6 minutes.

In the step 4, the reaction solution composition in the amplification process is as follows: adding 20-40ul of the DNA library after the joint, 2-6ul of 10X high-fidelity DNA polymerase buffer solution, 0.5-2ul of DNA polymerase buffer solution, 1ul of the positive primer of the joint, 1ul of the reverse primer of the joint and nuclease-free water to supplement the total volume to 50 ul.

A methylation detection method of an MGMT promoter comprises the following steps:

the sequencing library was obtained by the above method, and the number of reads of methylated CpG sites and unmethylated CpG sites was obtained by the second generation sequencing method, and the methylation rate was calculated.

A kit comprises the primer group.

A DNA extraction kit for paraffin-embedded tissue comprising:

the dewaxing agent is used for carrying out dewaxing treatment on the sample;

lysis buffer and protease for digesting the sample;

the binding buffer solution and the magnetic beads are used for binding the DNA in the digestion product;

a washing solution and ethanol for washing the magnetic beads to which the DNA is bound;

and the eluent is used for eluting the DNA bound on the magnetic beads.

The dewaxing agent is xylene.

The lysis buffer comprises: 5-50mmol/L Tris-HCl; 1-5mmol/L NaCl; 5-20mmol/L EDTA; 8-50mmol/L DTT; 3-5mol/L of guanidinium isothiocyanate; 0.5% -2% SDS; 1% -5% of TritonX-100.

The protease is proteinase K.

The combination buffer comprises: 100-200mmol/L Tris-HCl; 1-5mmol/L NaCl; 1% -5% of TritonX-100; 2-4mol/L of guanidinium isothiocyanate; 20-40% of isopropanol.

The washing liquid comprises a first washing liquid and a second washing liquid, wherein the first washing liquid comprises: 100-200mmol/L Tris-HCl; 1-5mmol/L NaCl; 40-60% ethanol; 2-4mol/L of guanidinium isothiocyanate; the second washing solution comprises: 25-75mol/L Tris-HCl; 75-85% ethanol.

The ethanol is absolute ethanol.

The eluent is free of nuclease water.

A method for extracting DNA of paraffin-embedded tissues comprises the following steps:

step 1, adding a dewaxing agent into a tissue sample, heating after vortexing, centrifuging, removing supernatant, adding ethanol, vortexing, removing supernatant after centrifugation, and drying;

step 2, adding a lysis buffer solution and protease into the sample obtained in the step 1, and incubating;

step 3, adding a binding buffer solution and magnetic beads into the sample obtained in the step 2, and incubating;

and 5, adding an eluent into the sample obtained in the step 4, incubating and taking a supernatant.

In the step 1, the vortex time is 5-20s, the centrifugal force is 10000-; the heating treatment is carried out at 35-60 deg.C for 5-20 min.

In the step 2, the incubation treatment process is carried out at 45-65 ℃ and at a rotation speed of 500-.

In the step 3, the incubation treatment is carried out for 1-30min at room temperature.

In the step 4, the washing process sequentially adopts a first washing solution, a second washing solution and ethanol for washing, the vortex is carried out for 1-30s in each washing, and the incubation is carried out for 1-5 min.

And in the step 5, incubating for 1-10 min.

Advantageous effects

The method can effectively detect the methylation levels of 99 CpG sites on the MGMT promoter, and has the advantages of high detection accuracy and wide coverage range.

Drawings

FIG. 1 is a flow chart of the present patent;

FIG. 2 is a DNA sample extraction procedure;

FIG. 3 is a depth map of sequencing at various sites of example 1;

FIG. 4 shows the results of the methylation level assay of example 1;

FIG. 5 is a graph showing the sequencing depth at each site in comparative example 1;

FIG. 6 shows the results of detecting the methylation level in comparative example 1;

Detailed Description

The sample is derived from FFPE tissue of a patient with glioma, the methylation negative control sample is healthy FFPE tissue of a human, and the methylation positive control sample is a cell line SW 620.

First, DNA is extracted from a sample, and genomic DNA is extracted from the sample. The tissue sample extraction kit and the extraction steps are as follows:

1) dewaxing the FFPE sample with a dewaxing agent to obtain a dewaxed sample; the method further comprises the step of washing by using ethanol to remove the dewaxing agent, so that the dewaxing agent can be effectively removed, and the subsequent treatment is easy. The dewaxing agent can adopt xylene. The ethanol is preferably absolute ethanol,

2) deparaffinized FFPE samples or fresh tissues and cells were subjected to digestion with lysis buffer and proteinase K to obtain a digest containing released DNA. The composition of the lysis buffer included: 5-50mmol/L Tris-HCl; 1-5mmol/L NaCl; 5-20mmol/L EDTA; 8-50mmol/L DTT; 3-5mol/L of guanidinium isothiocyanate; 0.5% -2% SDS; 1% -5% of TritonX-100. Wherein the dosage of proteinase K is 0.5-2.5 mg. Preferably, the lysate composition: 25mmol/L Tris-HCl; 2.5mmol/L NaCl; 10mmol/L EDTA; 25mmol/L DTT; 4mol/L of guanidine isothiocyanate; 1% SDS; 5% TritonX-100, and protease K1.5mg.

3) And adding a binding buffer solution and magnetic beads into the digestion product to obtain magnetic bead DNA binding products so as to facilitate the binding of the magnetic beads to DNA. Wherein the composition of the binding buffer comprises: 100-200mmol/L Tris-HCl; 1-5mmol/L NaCl; 1% -5% of TritonX-100; 2-4mol/L of guanidinium isothiocyanate; 20-40% of isopropanol. According to a specific example of the present invention, the binding liquid preferably: 150mmol/L Tris-HCl; 2.5mmol/L NaCl; 2.5 percent of TritonX-100; 3mol/L of guanidine isothiocyanate; 30% isopropyl alcohol.

4) The magnetic bead DNA binding products are washed with washing solution 1, washing solution 2 and absolute ethanol, respectively, to recover and purify DNA. Wherein the composition of washing solution 1: 100-200mmol/L Tris-HCl; 1-5mmol/L NaCl; 40-60% ethanol; 2-4mol/L of guanidine isothiocyanate. According to a specific example of the present invention, the binding liquid preferably: 150mmol/L Tris-HCl; 2.5mmol/L NaCl; 60% ethanol; 3mol/L of guanidinium isothiocyanate. Wherein the composition of washing solution 2: 25-75mol/L Tris-HCl; 75-85% ethanol. According to a specific example of the present invention, the binding liquid preferably: 50mmol/L Tris-HCl; 80% ethanol.

5) The DNA was eluted from the magnetic beads using nuclease-free water to obtain purified DNA.

The operation steps are as follows:

1) FFPE tissue of a patient with glioma and FFPE tissue samples of healthy people are deparaffinized. Sections were cut from paraffin-embedded tissue blocks using a microtome and collected in 1.5mL EP tubes. ② adding 1mL dimethylbenzene into the slices, whirling for 15s, placing the sample on a 50 ℃ metal bath for heating for 10min, centrifuging for 5min at 18000g speed, and removing the supernatant. ③ adding 1mL 100% ethanol, whirling for 15s, and centrifuging for 5min at 18000g speed, and removing the supernatant. And step three is repeated. And fourthly, vacuum drying for 5 to 10 minutes at the temperature of 45 ℃ by using a vacuum drying instrument, and taking out the sample.

2) Cell line SW620 was centrifuged. Cells were resuspended using PBS and collected in 1.5mL EP tubes. ② centrifuging for 5min at the speed of 300g, and removing the supernatant.

3) To the treated FFPE and cell samples 380. mu.L lysis buffer and 20. mu.L proteinase K were added, vortexed for 5s, and incubated at 56 ℃ at 800rpm for 1-2h, depending on the time of complete digestion of the tissue.

4) The transfer was incubated for 10min with the temperature having risen to 90 ℃ at 350 rpm.

5) And (4) carrying out short-time centrifugation, adding 380 mu L of binding buffer and 20 mu L of magnetic beads after the sample returns to the room temperature, carrying out vortex mixing for 30 seconds, and carrying out rotary incubation for 10min at the room temperature.

6) Standing on a magnetic frame for 3min, and turning the sample tube upside down once when standing for 1.5min, and removing the supernatant.

7) Remove from the magnetic frame, add 500. mu.L of Wash buffer 1, vortex and mix for 10s, incubate for 2min at room temperature.

8) Standing on a magnetic frame for 3min, and turning the sample tube upside down once when standing for 1.5min, and removing the supernatant.

9) Removed from the magnetic frame, 500. mu.L of washing buffer 2 was added, vortexed for 10s and incubated at room temperature for 2 min.

10) Standing on a magnetic frame for 3min, and turning the sample tube upside down once when standing for 1.5min, and removing the supernatant.

11) Taking down from the magnetic frame, adding 500 μ L100% ethanol, vortexing and mixing for 10s, and incubating at room temperature for 2 min.

12) Standing on a magnetic frame for 3min, and turning the sample tube upside down once when standing for 1.5min, and removing the supernatant.

13) And after the magnetic beads are dried, taking the sample tube down from the magnetic frame, adding 100 mu L of nuclease-free water above the magnetic beads along the tube wall, uniformly mixing by vortex to separate the magnetic beads from the tube wall, completely mixing in the nuclease-free water, and incubating at room temperature for 5 min.

14) The sample tube was placed on a magnetic stand and allowed to stand for 2min, after which the supernatant was carefully transferred to a new 1.5mL centrifuge tube.

After obtaining the sample DNA, carrying out bisulfite conversion and recovery, adding an internal control into the extracted genomic DNA sample, and then converting the DNA sample containing the internal control by using an EZ DNA Methylation-Gold Kit (Zymo research company) Kit, wherein the specific extraction steps refer to Kit operation instructions;

taking the transformed genome DNA as a template, and performing MGMT promoter region methylation specificity multiplex PCR amplification by using a primer combination, wherein the amplified MGMT promoter region CpG island sequence combination comprises 4 upstream primers and 4 downstream primers, and the specific sequences are as follows:

the design concept of the primers in the patent is as follows: the amplification regions of the 4 pairs of primers cover 99 CpG sites of an MGMT promoter and are positioned at-552 bp- +289bp positions of a 5' regulation region of the promoter (the sites are defined as that the upstream is negative and the downstream is positive relative to the distance of a transcription initiation Site (Tss) — 552 bp- +289bp, the length of the primers is 20-25 bp, the primers need to avoid covering methylated CpG sites, the annealing temperature difference between the primer pairs is less than or equal to 5 ℃, and primer dimer is avoided to be formed between the primer pairs.

TABLE 1 primer design

TABLE 2 multiplex PCR amplification procedure

3) Performing magnetic bead purification on the obtained multiple PCR amplification product;

library construction is the process of adding adapters to the sequenced fragments. And (3) adding adapters at two ends of the amplified DNA fragment to perform on-machine sequencing. The library construction mainly comprises the following steps:

1) DNA end repair. The end repair of the DNA fragment can be performed using Klenow fragment, T4DNA polymerase and T4 polynucleotide kinase. Filling in cDNA/DNA5 'protruding sticky ends and flattening 3' protruding sticky ends using T4 polymerase and Klenow E.coli polymerase fragments to generate blunt ends for subsequent blunt end ligation. According to an embodiment of the present invention, a step of purifying the DNA fragment having the sticky end A may be further included, thereby enabling convenient subsequent processing. The reaction is carried out in a PCR amplification instrument for 20-30 minutes. The reaction system is as follows:

TABLE 3 DNA end repair reaction solution composition

2) Base A was added to the 3' end of the cDNA/DNA sample. A base A is added to the 3' -end of the DNA fragment subjected to end repair so as to obtain a DNA fragment having a cohesive end A. According to an embodiment of the present invention, a step of purifying the DNA fragment having the sticky end A may be further included, thereby enabling convenient subsequent processing.

The reaction was carried out in a PCR amplification apparatus at 37 ℃ for 30 minutes. The reaction system is as follows:

TABLE 4DNA end-to-A reaction solution composition

3) Linkers are added to both ends of the DNA. Adaptors were ligated to both ends of the DNA fragments using T4 ligase for subsequent library amplification.

TABLE 5 adaptor reaction solution composition

4) And (3) amplifying the DNA library.

Polymerase Chain Reaction (PCR), performed in a PCR amplificator, reaction program: pre-denaturation at 98 ℃ for 30 seconds, annealing at 65 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds, wherein the cycle is 4-6 times. Finally extension was carried out at 72 ℃ for 5 minutes. The reaction system is as follows:

TABLE 6 library amplification reaction solution composition

5) And (5) purifying the library. And purifying the amplified library DNA fragments by using magnetic beads, and using the purified library DNA fragments for a sequencer. Magnetic bead purification was performed using the Beckman Coulter Ampure Beads kit (cat # A63880).

5. Methylation detection method

The methylation library was sequenced using the Illumina sequencing platform. After the sequencing is finished, generating a fastq original sequence by using bcl2fastq, and then performing quality control on original data by using trimmatic to remove joints and low-quality bases. The resulting cleardata was aligned using bismark for genome (hg 19). And obtaining methylated CpG sites after comparison, and determining the methylated reads number of each CpG site and the unmethylated reads number of the site region according to the obtained sites to obtain the methylation rate of the MGMT promoter region.

Percent methylation ═ methylation sequence/(methylation sequence + non-methylation sequence) × 100%

The depth of sequencing of the MGMT promoter region is shown in fig. 3. The methylation sequencing result of a clinical sample is shown in figure 4, the methylation level detection can be performed on 99 CpG sites in an MGMT promoter region, the average methylation level of the sample is calculated, and the methylation level of 8 CpG sites in the MGMT promoter region is verified by pyrosequencing.

TABLE 6 methylation level test results

The results of primer combination optimization for both clinical samples are shown above.

Comparative example 1

The primer sequence combination for detecting the methylation sites of the amplified MGMT promoter region CpG island sequence comprises 3 pairs of primer combinations which are respectively as follows: the specific sequences of the upstream primer F1, the downstream primer R1, the upstream primer F2, the downstream primer R2, the upstream primer F3 and the downstream primer R4 are as follows:

TABLE 7 primer design

The remaining conditions were as in example 1.

The depth of sequencing of the MGMT promoter region is shown in FIG. 5. The methylation level of each CpG island sequence in the MGMT promoter region was calculated as the mean methylation level of the sample, as shown in fig. 6.

TABLE 8 methylation test results

The optimized primer combination can obviously improve the total sequencing depth of NGS sequencing, and has smaller deviation with the data obtained in the pyrophosphate method.

Sequence listing

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