CN110951872A - Method for detecting colorectal cancer gene DNA methylation level based on nucleic acid mass spectrometry technology and application thereof - Google Patents
Method for detecting colorectal cancer gene DNA methylation level based on nucleic acid mass spectrometry technology and application thereof Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The invention relates to the field of gene detection, in particular to a method for detecting DNA methylation level of colorectal cancer gene based on nucleic acid mass spectrometry technology and application thereof. The invention uses nucleic acid mass spectrum technology to detect the specific combination of 15 areas of 5 genes related to colorectal cancer, quantitatively analyzes the DNA methylation degree of nearly 120 CpG sites in the 15 areas, and establishes the characteristic DNA methylation spectrum of CRC patients. The invention detects the gene methylation by the nucleic acid mass spectrometry technology, and compared with TaqMan-PCR and second-generation platform sequencing, the detection method has the characteristics of simplicity, convenience, strong flexibility, high flux, low cost and the like. The invention can be used for early evaluation and large-scale screening of CRC, provides an important reference basis for evaluation and diagnosis of CRC disease risk, and realizes early warning of colorectal cancer diseases.
Description
Technical Field
The invention belongs to the field of gene detection, and particularly relates to a method for detecting DNA methylation level of colorectal cancer gene based on a nucleic acid mass spectrometry technology and application thereof.
Background
Colorectal cancer (CRC) is one of the most common malignant tumors in the digestive tract, the incidence and mortality rate of the CRC are on the rise year by year in the world, the situation in the large cities is particularly severe, the Colorectal cancer has become the first malignant tumor of the digestive system better than gastric cancer, and the incidence and mortality rate of the Colorectal cancer are only second to that of lung cancer in all malignant tumors. Although the treatment of colorectal cancer has been advanced in recent years, the overall curative effect is not greatly improved, and the 5-year survival rate after the operation is about 50%. The early-stage colorectal cancer patient has a good treatment effect, and the 5-year survival rate can be more than 90%. Factors that contribute to the onset of CRC include mainly environmental and genetic factors. By detecting the gene mutation of CRC, the risk of CRC diseases can be predicted in advance, and the method has great guiding significance for early diagnosis and effective prevention of CRC. In early CRC, tumor cells may be shed into the intestinal lumen and mixed with stool, and DNA extraction from stool samples is more stable and sensitive because the alkaline environment of the intestine is favorable for DNA preservation.
There are a number of early detection methods for CRC, including stool-based tests such as the stool occult blood test (FOBT), stool immunochemical test (FIT); endoscopy such as flexible sigmoidoscopy, colonoscopy and capsule endoscopy; imaging examinations such as colon pneumobarium double contrast, colon CT imaging, etc. Colonoscopy is the "gold standard" for CRC screening, but before the CRC screening, preparations such as fasting, taking laxatives and the like are required for patients, the examination process belongs to an invasive examination mode, the process is long and painful, and high-risk complications such as perforation and the like are possible, so that the colonoscopy is not easily accepted by the patients and is difficult to be used as an early screening means for CRC. The FOBT and FIT tests belong to noninvasive detection means, but the FOBT and FIT tests are low in sensitivity and specificity, repeated test verification is needed, the detection result is susceptible to the influence of factors such as medicines and the like to generate false positive, and the FOBT and FIT tests can only be used as primary screening means.
DNA methylation is one of the earliest discovered ways of modifying gene appearance, and methylation in eukaryotes occurs only in cytosine, i.e., cytosine at the 5 '-end of CpG dinucleotides is converted into 5' -methylcytosine by DNA methyltransferases (DNMTs). DNA methylation typically suppresses gene expression, and demethylation induces reactivation and expression of the gene. The DNA modification mode realizes the regulation and control of gene expression on the premise of not changing gene sequences. When tumors occur, the non-methylation degree of CpG sequences except the CpG island of the cancer suppressor gene is increased, and the CpG in the CpG island is in a high-methylation state, so that the expression of the cancer suppressor gene is reduced. The risk of CRC disease can therefore be judged by methylation detection.
There are a plurality of DNA methylation gene markers clearly related to the occurrence of CRC, and BMP3, NDRG4, SDC2, SFRP2, VIM and the like are selected, DNA methylation patterns of 3 regions in each gene are detected respectively, and colorectal cancer is identified.
The currently used method for detecting DNA methylation mainly adopts TaqMan-PCR and the next generation platform for sequencing, but has the problems of insufficient design flexibility, small detection capacity and high cost. The project develops a set of method for detecting the DNA methylation of the fecal gene on a MassRRAY flight mass spectrometer (MALDI-TOF) technical platform, and discovers CRC and CRC precancerous lesion adenoma at the early stage.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a method for identifying colorectal cancer by using gene DNA methylation patterns based on a nucleic acid mass spectrometry technology.
The purpose of the invention is realized by the following technical scheme:
a method for detecting DNA methylation level of colorectal cancer gene based on nucleic acid mass spectrometry technology, which comprises the following steps:
(1) screening genes and DNA methylation regions in the genes for assessing the risk of the individual suffering from colorectal cancer;
(2) designing a specific amplification primer of the colorectal cancer gene in the step (1), wherein the DNA fragment amplified by the primer is not more than 200 bp;
(3) carrying out bisulfite conversion on a DNA sample to be detected;
(4) in the step (2), all primers are represented by 7-bit numbers, the numbers are composed of letters and numbers, the 1 st to 6 th-bit numbers and the primers with the same letters are a pair, the 7 th bit is the letter F or R, the upstream and downstream primers of each pair of primers are mixed in an equimolar way to obtain corresponding amplification primer mixed liquor, and the final working concentration of each pair of amplification primer mixed liquor is 1 mu M;
(5) taking a DNA sample after bisulfite conversion as a template, and respectively carrying out PCR amplification reaction by using the amplification primers in the step (4) to obtain PCR product fragments of the target gene;
(6) SAP reaction digests the residual dNTP in the reaction system in the step (5);
(7) performing T-clean transcription and T-cutting on the DNA sample subjected to SAP digestion in the step (6);
(8) a clean resin desalination purification step (7) is carried out to finish products of T-clean transcription and T-cutting;
(9) and (3) quantitatively detecting DNA methylation sites of the gene related to the colorectal cancer risk by using the MassARRAY platform, and determining the methylation degree of each CpG site.
Wherein, in step (1), the genes which can be used for evaluating the correlation risk of the individual suffering from colorectal cancer are BMP3, NDRG4, SDC2, SFRP2 and VIM.
In the step (2), the PCR specific amplification primer pair for quantitatively detecting the DNA methylation sites of the genes related to the colorectal cancer risks of the individual is as follows: M00101F, M00101R; M00102F, M00102R; M00103F, M00103R; M00201F, M00201R; M00202F, M00202R; M00203F, M00203R; M00301F, M00301R; M00302F, M00302R; M00303F, M00303R; M00401F, M00401R; M00402F, M00402R; M00403F, M00403R; M00501F, M00501R; M00502F, M00502R; M00503F, M00503R.
In the step (3), the cycle conditions of the bisulfite conversion are as follows: (95 ℃ 30s → 50 ℃ 15 min). times.20 cycles → 4 ℃ Hold. The temperature of each step in the above amplification conditions is the middle of a range, where 2 ℃ above or below the middle is within the scope of the claims.
In the step (5), the PCR amplification conditions are as follows: 95 ℃ 4min → (95 ℃ 20s → 56 ℃ 30s → 72 ℃ 1min) × 45cycles → 72 ℃ 3min → 4 ℃ Hold. The temperature of each step in the above amplification conditions is the middle of a range, where 2 ℃ above or below the middle is within the scope of the claims.
In the step (6), the circulation conditions of SAP digestion are as follows: 20min at 37 ℃ → 5min at 85 ℃ → Hold at 4 ℃. The temperature of each step in the above amplification conditions is the middle of a range, where 2 ℃ above or below the middle is within the scope of the claims.
And (7) the circulation conditions of T-clean transcription and T-cutting are as follows: 37 ℃ for 3h → 4 ℃ for Hold. The temperature of each step in the above amplification conditions is the middle of a range, where 2 ℃ above or below the middle is within the scope of the claims.
Use of the above method for detecting the level of DNA methylation of a colorectal cancer gene based on nucleic acid mass spectrometry technology for the manufacture of a formulation for diagnosing whether a subject has colorectal cancer or is at risk of developing colorectal cancer.
And analyzing the detected methylation degree of each CpG site of the excrement DNA sample of the CRC patient and the normal volunteer sample, wherein the methylation degree of each CpG site of the two groups is different, and forming respective DNA methylation patterns of the CRC patient and the normal volunteer, so that the CRC patient can be identified.
The technical scheme of the invention uses a nucleic acid mass spectrometry detection technology to select 5 colorectal cancer related genes, each gene selects a medium number of CpG sites, and the methylation degree of each CpG site is detected. The degree of methylation at each CpG site in a sample constitutes the DNA methylation profile of the sample. The method is simple, the sample amount of the detection site is large, the accuracy is high, and the cost is low.
Detailed Description
The following examples are presented to illustrate certain embodiments of the invention in particular and should not be construed as limiting the scope of the invention. The present disclosure may be modified from materials, methods, and reaction conditions at the same time, and all such modifications are intended to be within the spirit and scope of the present invention. Specifically, the reagents used in the embodiments of the present invention are all commercially available products, and the databases used in the embodiments of the present invention are all public online databases.
Example (b): detection of DNA methylation characteristics of genes associated with colorectal cancer susceptibility by the technique of the invention
Detecting an object
From the stools of 50 patients whose pathology had been diagnosed as colorectal cancer, and 30 healthy volunteers, mutation sites of a group of genes related to early diagnosis of colorectal cancer were detected.
Secondly, extracting the DNA of the excrement sample by using a Qiagen QIAamp Fast DNA pool Mini kit to obtain the excrement person
DNA extract
1. A2.0 ml centrifuge tube was added to about 200mg of dry fecal sample and placed on an ice-box.
2. To each stool sample was added 1ml of the inhibiex buffer. Vortexing was continued for 1 minute until the fecal sample was completely homogeneous.
3. The mixed sample was centrifuged at 14500rpm (about 20000 Xg) for 1 min.
4. A clean 2.0ml centrifuge tube was then charged with 25. mu.l proteinase K.
5. Taking 600 mu l of supernatant of the centrifugation product in the step 3, and adding the supernatant into a centrifuge tube filled with the protease K.
6. Add 600 u l Buffer AL and vortex 15 s.
7. And placing the uniformly mixed sample in a metal bath at 70 ℃ for incubation for 10 min.
8. Then, 600. mu.l of absolute ethanol was added thereto, and the mixture was inverted upside down and mixed.
9. Taking the centrifugal column, adding 600 μ l of the mixed solution obtained in the step 8, centrifuging at 14500rpm for 1min, and discarding the collection tube containing the filtrate.
10. The clean 2.0ml collection tube is replaced, and the operation of the step 9 is repeated until all the mixed liquid is transferred (3 times in total).
11. The clean 2.0ml collection tube was replaced, 500. mu.l Buffer AW1 was added, the tube was centrifuged at 14500rpm for 1min, and the collection tube containing the filtrate was discarded.
12. The clean 2.0ml collection tube was replaced, 500. mu.l Buffer AW2 was added, the tube was centrifuged at 14500rpm for 3min, and the collection tube containing the filtrate was discarded.
13. The clean 2.0ml centrifuge tube (not provided in kit) was replaced, centrifuged at 14500rpm for 3min and the tube containing the filtrate was discarded.
14. Changing a clean 1.5ml centrifuge tube (not provided by a kit), adding 200 mul Buffer ATE, standing at room temperature for 2min, then centrifuging at 14500rpm for 1min, wherein the 1.5ml centrifuge tube is the DNA sample solution after extraction, can be directly used for the next operation, and can be sealed and stored at-20 ℃ if not used for the time.
Third, detecting the sample quality
1. The upper cover of the NANO DROP2000 instrument was opened and the outer edge of the well was measured with a dust free paper wipe clean instrument.
2. Pipette about 2. mu.l of Buffer ATE, drop into the small hole of the detection instrument, and cover the upper cover of the detection instrument.
3. Inputting the name of the sample to be tested in the computer operation software, clicking Blank, and then clicking the Measure key to read the experimental result.
4. And opening the upper cover of the detection instrument, and wiping the instrument with dust-free paper to detect the outer edge of the small hole.
5. The pipette is used to aspirate about 2. mu.l of DNA sample, and the DNA sample is dripped into the small hole of the detection instrument and the upper cover of the detection instrument is closed.
6. And inputting the name of the sample to be tested in the computer operation software, and clicking a Measure key to read the experimental result.
7. After the detection is finished, the DNA sample can be directly used for the next operation, and can be sealed and stored at the temperature of-20 ℃ if the DNA sample is not used for the moment.
Tetra, bisulfite conversion
1. CT Conversion Regent (CT Conversion reagent) was prepared: add 750. mu.l of water and 210. mu. l M-DiluenBuffer to a vial of CT Conversion reagent and vortex for 10 min.
2. A1.5 ml centrifuge tube was filled with 5. mu. l M-Dilution Buffer + DNA sample (2. mu.g) in a total amount of less than 50. mu.l, and water was added to make up.
3. The mixed sample was placed in a metal bath at 37 ℃ for 15 min.
4. 100 μ l of prepared CT Conversion reagent was added to each sample and pipetted up and down using a pipette.
5. Putting the mixture into a PCR amplification instrument. The following procedure was carried out:
(95℃30s→50℃15min)×20cycles→4℃∞。
6. the samples were placed in an ice box for 10 min.
7. Add 400. mu. l M-Binding Buffer to the sample and mix by pipetting up and down.
8. The samples were transferred in their entirety to a Zymo-Spin I Column and the Column was placed in a 2ml collection tube.
9. Centrifuge at 14500rpm (. gtoreq.10000 g) for 30s and discard the filtrate.
10. 200 μ l M-Wash Buffer was added to the column, centrifuged at 14500rpm for 30s and the filtrate discarded.
11. 200 μ l M-depletion Buffer was added to the column, left at room temperature (24 ℃) for 15min, and then centrifuged at 14500rpm for 30 s.
12. 200 μ l M-Wash Buffer was added to the column and centrifuged at 14500rpm for 30 s.
13. 200 μ l M-Wash Buffer was added to the column and centrifuged at 14500rpm for 1 min.
14. And replacing a new 1.5ml centrifuge tube as a collecting tube, eluting DNA by using 60 mu l of water, wherein the eluted DNA sample is the DNA sample after sulfite conversion, and can be directly used for the next operation, and if the sample is not used for the time, the sample can be sealed and placed at the temperature of-20 ℃ for storage.
Fifth, PCR amplification
Taking a DNA sample after bisulfite conversion as a template, mixing the upstream primer and the downstream primer of each pair of amplification primers in an equimolar way to obtain an amplification primer mixed solution, wherein the final concentration of each primer is 1 mu M, and carrying out PCR amplification reaction to obtain a PCR product fragment of a target site.
The PCR amplification reaction system (5. mu.l) is
PCR reaction procedure
95 ℃ 4min → (95 ℃ 20s → 56 ℃ 30s → 72 ℃ 1 min). times.45 cycles → 72 ℃ 3min → 4 ℃. infinity. Table 1: specific amplification primer sequence pair of gene locus to be detected
Sixthly, treating the PCR product SAP enzyme
1. And (3) processing the PCR product obtained in the previous step to eliminate the residual dNTP in the reaction system.
The SAP enzyme treatment reaction system was 7. mu.l (per reaction):
2. the digestion reaction conditions are as follows:
37℃20min→85℃5min→4℃∞。
seven, T Cleavage transcription and T Cleavage
After the SAP enzyme treatment is finished, T-Cleavage transcription and T-cutting are carried out on the site of the target product.
1. Adding the reagents in the following sequence to prepare a proper amount of a T transcription/RNase A mixed solution:
2. a new 384 well sample plate was prepared and 5. mu. L T transcription/RNase A mixture was added to each well.
3. The DNA sample plate (after PCR/SAP treatment) was centrifuged at 540 Xg for 1 min.
4. A2. mu. LDNA sample was transferred from each well of a DNA sample plate (after PCR/SAP treatment) and placed in each well of a new transcription sample plate, and the pipette tip was replaced for each transfer.
5. The transcription sample plate is sealed by a flat sealing film, and the good sealing performance of the edge of the sample plate is ensured.
6. The transcription sample plate was centrifuged at 540 Xg for 1 min.
7. The transcription sample plate was placed in a PCR amplification apparatus and incubated at 37 ℃ for 3 h.
8. After completion, the plate was removed from the PCR amplification apparatus and used directly for the next step, and if not used, it was sealed and stored at-20 ℃ overnight.
Eighthly, resin purification
And adding resin into the product in the previous step, desalting and purifying the extension product, avoiding the influence of ions on a sample to be detected in a mass spectrum, and eliminating salt peak interference. The method comprises the following specific steps:
to each well of the 384 well plate was added 20. mu.l of water and 6mg of dried clean resin. Sealing the plate with sealing plate film, and vertically rotating at low speed for 15min to make the resin fully contact with the reactant; 3200g was centrifuged for 5min to prepare for spotting.
Nine, chip sample application
The MassARRAY Nanodispenser RS1000 spotting instrument was started and the resin purified extension product was transferred to 384-well SpectroCHIP (sequenom) chips.
Ten, mass spectrometry
The spotted Spectro CHIP was analyzed by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight mass spectrometry), and the detection results were analyzed by EpiTyper software (sequenom) and outputted.
Eleven, conclusion
The results show that 50 cases of pathology examined in this example have been diagnosed with colorectal cancer patients, and 30 healthy volunteers, and that in total 80 cases of stool, the DNA methylation profile of colorectal cancer patients is different from that of healthy volunteers. Using methylation profiles characteristic of fecal DNA from colorectal cancer patients, colorectal cancer patient samples can be clearly distinguished from control samples of normal volunteers. Demonstrating that the present technology is effective in detecting DNA methylation profiles characteristic of colorectal cancer patients.
Claims (8)
1. A method for detecting the DNA methylation level of colorectal cancer genes based on a nucleic acid mass spectrometry technology is characterized by comprising the following steps:
(1) screening genes and DNA methylation regions in the genes for assessing the risk of the individual suffering from colorectal cancer;
(2) designing a specific amplification primer of the colorectal cancer gene in the step (1), wherein the DNA fragment amplified by the primer is not more than 200 bp;
(3) carrying out bisulfite conversion on a DNA sample to be detected;
(4) in the step (2), all primers are represented by 7-bit numbers, the numbers are composed of letters and numbers, the 1 st to 6 th-bit numbers and the primers with the same letters are a pair, the 7 th bit is the letter F or R, the upstream and downstream primers of each pair of primers are mixed in an equimolar way to obtain corresponding amplification primer mixed liquor, and the final working concentration of each pair of amplification primer mixed liquor is 1 mu M;
(5) taking a DNA sample after bisulfite conversion as a template, and respectively carrying out PCR amplification reaction by using the amplification primers in the step (4) to obtain PCR product fragments of the target gene;
(6) SAP reaction digests the residual dNTP in the reaction system in the step (5);
(7) performing T-clean transcription and T-cutting on the DNA sample subjected to SAP digestion in the step (6);
(8) a clean resin desalination purification step (7) is carried out to finish products of T-clean transcription and T-cutting;
(9) and (3) quantitatively detecting DNA methylation sites of the gene related to the colorectal cancer risk by using the MassARRAY platform, and determining the methylation degree of each CpG site.
2. The method for detecting DNA methylation level of colorectal cancer gene based on nucleic acid mass spectrometry technology according to claim 1, wherein the DNA methylation level of colorectal cancer gene is detected by: in step (1), the genes that can be used to assess the risk relevance of an individual for developing colorectal cancer are BMP3, NDRG4, SDC2, SFRP2, VIM.
3. The method for detecting DNA methylation level of colorectal cancer gene based on nucleic acid mass spectrometry technology according to claim 1, wherein the DNA methylation level of colorectal cancer gene is detected by: in the step (2), the PCR specific amplification primer pair for quantitatively detecting the DNA methylation sites of the genes related to the colorectal cancer risks of the individual is as follows: M00101F, M00101R; M00102F, M00102R; M00103F, M00103R; M00201F, M00201R; M00202F, M00202R; M00203F, M00203R; M00301F, M00301R; M00302F, M00302R; M00303F, M00303R; M00401F, M00401R; M00402F, M00402R; M00403F, M00403R; M00501F, M00501R; M00502F, M00502R; M00503F, M00503R.
4. The method for detecting DNA methylation level of colorectal cancer gene based on nucleic acid mass spectrometry technology according to claim 1, wherein the DNA methylation level of colorectal cancer gene is detected by: in the step (3), the cycle conditions of the bisulfite conversion are as follows: (95 ℃ 30s → 50 ℃ 15 min). times.20 cycles → 4 ℃ Hold. The temperature of each step in the above amplification conditions is the middle of a range, where 2 ℃ above or below the middle is within the scope of the claims.
5. The method for detecting DNA methylation level of colorectal cancer gene based on nucleic acid mass spectrometry technology according to claim 1, wherein the DNA methylation level of colorectal cancer gene is detected by: in the step (5), the PCR amplification conditions are as follows: 95 ℃ 4min → (95 ℃ 20s → 56 ℃ 30s → 72 ℃ 1min) × 45cycles → 72 ℃ 3min → 4 ℃ Hold. The temperature of each step in the above amplification conditions is the middle of a range, where 2 ℃ above or below the middle is within the scope of the claims.
6. The method for detecting DNA methylation level of colorectal cancer gene based on nucleic acid mass spectrometry technology according to claim 1, wherein the DNA methylation level of colorectal cancer gene is detected by: in the step (6), the circulation conditions of SAP digestion are as follows: 20min at 37 ℃ → 5min at 85 ℃ → Hold at 4 ℃. The temperature of each step in the above amplification conditions is the middle of a range, where 2 ℃ above or below the middle is within the scope of the claims.
7. The method for detecting DNA methylation level of colorectal cancer gene based on nucleic acid mass spectrometry technology according to claim 1, wherein the DNA methylation level of colorectal cancer gene is detected by: and (7) the circulation conditions of T-clean transcription and T-cutting are as follows: 37 ℃ for 3h → 4 ℃ for Hold. The temperature of each step in the above amplification conditions is the middle of a range, where 2 ℃ above or below the middle is within the scope of the claims.
8. Use of a method for detecting the DNA methylation level of a colorectal cancer gene according to any one of claims 1 to 7 based on nucleic acid mass spectrometry for the manufacture of a formulation for diagnosing whether a subject has colorectal cancer or is at risk of developing colorectal cancer.
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