CN116656792A - Methylation level detection method based on Massarray - Google Patents
Methylation level detection method based on Massarray Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The application discloses a methylation level detection method based on Massarray, which comprises the following steps of: (1) obtaining DNA of a sample to be detected through extraction; (2) bisulfite conversion of DNA; (3) Carrying out specific multiplex PCR amplification reaction on the methylated fragment and the unmethylated fragment simultaneously; (4) performing an alkaline phosphatase reaction on the shrimp; (4) The extension reaction simultaneously obtains extension products of methylated sites and unmethylated sites; (5) purification desalting with resin; (6) massaray time-of-flight mass spectrometry; (7) Calculating the methylation rate of the CpG sites according to HEIGHT data of the methylation sites and the unmethylated sites, and judging whether the methylation is abnormal or not; the application is suitable for detecting the methylation level of genes, is a novel detection method, can detect a plurality of CpG sites and a plurality of samples of a plurality of genes with high flux, and reduces the cost.
Description
Technical Field
The application relates to a methylation level detection method based on Massary, in particular to a novel method for detecting the methylation level of CpG sites by amplifying multiple PCR reactions of methylated and unmethylated fragments simultaneously and obtaining a methylation and unmethylated Massary flight time mass spectrum peak by using the same extension primer.
Background
DNA methylation (DNA methylation) refers to a chemical modification process in which active methyl groups are transferred to specific bases in a DNA strand under the catalysis of DNA methyltransferase (DNMT) with S-adenosylmethionine (SAM) as a methyl donor. In mammalian genomes, DNA methylation occurs mostly at the 5-position carbon atom of cytosine in CpG island dinucleotides. DNA methylation is an epigenetic modification that plays an important regulatory role in the growth, development, gene expression pattern, and stability of the genome of an individual without altering the DNA sequence, and is stably transmitted during development and cell proliferation. In recent years, a great deal of research shows that abnormal methylation of DNA has a close relation with the occurrence, development and canceration of tumors.
Current DNA methylation detection techniques mainly include three-generation sequencing, whole genome methylation sequencing (WGBS) based on two-generation sequencing, methylated DNA immunoprecipitation sequencing (media-Seq), simplified methylation sequencing (RRBS), gene chip detection techniques (MicroArray), mass spectrometry (MassArray), methylation-specific PCR (MSP), and bisufite post-processing sequencing (BSP) based on one-generation sequencing techniques. However, in the methods, the detection method based on the sequencing technology and the chip has the defects of long time and high cost; PCR-based methods have the disadvantage of low throughput.
At present, methylation detection based on Massary detects the methylation rate of one gene fragment, only 1 gene can be detected in each hole, the flux is low, analysis cannot be carried out one by one CpG site, and the analysis is complex. Therefore, development of a new detection method is needed, which can detect methylation level in high throughput, and has the advantages of low cost and rapid detection.
Disclosure of Invention
In order to solve the problems, the application discloses a methylation level detection method based on Massarray, which combines creatively multiple PCR (polymerase chain reaction) and Massarray time-of-flight mass spectrometry for simultaneously amplifying methylated/unmethylated fragments, can detect a plurality of CpG sites and a plurality of samples of a plurality of genes at high flux, and reduces cost. The result of the methylation level of the gene provides the change of human body more comprehensively, perfects the gene detection information from epigenetic science, and is helpful for assisting doctors in diagnosis.
In order to achieve the above purpose, the technical scheme of the application is as follows:
a methylation level detection method based on Massary comprises the following steps:
(1) Extracting DNA of a sample to be detected by using a DNA extraction kit;
(2) Performing Bisulfite (Bisulfite) conversion on the extracted DNA, and detecting the concentration of single-stranded DNA (ssDNA) after the conversion;
(3) Taking the DNA after the Bisulfite conversion as a template, and obtaining methylated and unmethylated amplification products through multiplex PCR amplification reaction;
(4) Removing redundant deoxyribonucleoside triphosphates from the amplified products by shrimp alkaline phosphatase reaction;
(5) Carrying out an extension reaction on the product of the step (4) to obtain an extension product of the detected CpG site, wherein the methylated product is a C peak, and the unmethylated product is a T peak;
(6) Purifying the product of the extension reaction by resin, and desalting;
(7) The desalted product is spotted on a mass spectrum chip matrix for co-crystallization, then the crystal is transferred into a vacuum tube of a mass spectrometer, and laser excitation is used for Massarray flight time mass spectrum detection;
(8) Methylation rate was calculated from the methylation C-peak heigh data and the heigh data of the unmethylated T-peak detected by massaray time-of-flight mass spectrometry.
As an improvement of the present application, each pair of PCR primers of the multiplex PCR in the step (3) simultaneously amplifies methylated and unmethylated fragments.
As an improvement of the application, the multiplex PCR in the step (3) comprises a target detection gene fragment and a quality control fragment.
As an improvement of the application, the length of the amplified fragment is 100-400 bp, and the quality is controlled to be a imprinting gene H19.
As an improvement of the application, the system of the multiplex PCR amplification reaction in the step (3) comprises a PCR Buffer and MgCl 2 The PCR primer Pool is a mixture of target detection gene amplification primers and quality control amplification primers.
As an improvement of the application, the PCR amplification primer sequence is as follows:
as an improvement of the present application, the same extension primer in the step (5) may extend out of the C peak of the methylated product and the T peak of the unmethylated product.
As an improvement of the application, the system of the extension reaction comprises Buffer Plus, termination Mix, an extension primer mixture and an iPLEX Enzyme, wherein the extension primer mixture is matched with the sequence of the amplification product of the target detection gene and the quality control amplification product in the step (3).
As an improvement of the present application, the extended primer sequence is as follows:
as an improvement of the application, the methylation ratio of CpG sites can be calculated according to the following formula by methylation C peak HEIGHT data and unmethylated T peak HEIGHT data in the step (8):
CpG site methylation rate = C peak heigh value/(C peak heigh value + T peak heigh value)
The beneficial effects of the application are as follows: the methylation level detection method based on the Massary provided by the application, which is creatively combined with multiple PCR and Massary time-of-flight mass spectrometry for simultaneously amplifying methylated and unmethylated fragments, can detect a plurality of CpG sites and a plurality of samples of a plurality of genes with high flux, has the characteristics of large flux, high accuracy, low cost and the like, provides the change of human bodies more comprehensively according to the result of the methylation level of the genes, perfects the gene detection information from epigenetic science, and is beneficial to assisting doctors in diagnosis.
Drawings
FIG. 1 is a schematic representation of a comparison of methylated and unmethylated DNA after bisulfite conversion according to the application.
FIG. 2 is a graph showing the results of Massary of the present application.
FIG. 3 is a graph showing the methylation ratio calculated by the Massary method and the second generation sequencing of the target gene according to the present application.
Detailed Description
The present application is further illustrated in the following drawings and detailed description, which are to be understood as being merely illustrative of the application and not limiting the scope of the application.
Example 1
Detection of CpG site methylation rates of pax1 and SOX1 genes:
(1) Preparation of DNA samples
Cervical scraping specimens were taken and human genomic DNA was extracted using a commercial DNA extraction kit. The quality of the extracted DNA is detected by Nano300, which is required to meet OD 260 /OD 280 The concentration is more than 5 ng/Mul between 1.8 and 2.0. And placing the DNA with qualified quality at 4 ℃ for standby.
(2) Bisulphite conversion
Bisulphite conversion of human genomic DNA was performed using the Zymo kit (cat No. D5006). The quality of the DNA obtained by detection with the Qubit ssDNA reagent should meet the concentration of > 5 ng/mu l. And (5) placing the qualified conversion product at the temperature of-20 ℃ for standby.
(3) PCR amplification reaction
The multiplex PCR reaction system used in the application contains PCR Buffer and MgCl 2 dNTPs and PCR enzymes (purchased from Agena Bioscience, cat. No. 21327L), and PCR primer Pool. Preferred CpG sites for PAX1 in the present application are PAX1-1 (chr 20: 2170567)0) And PAX1-2 (chr 20: 21706091), the CpG sites of SOX1 are SOX1-1 (chr 13: 112067245) and SOX1-2 (chr 13: 112067399), and the reference genome of the positional information is hg38. Preferred primers designed according to CpG sites in the present application are shown in Table 1, and comprise specific amplification primers for 2 CpG sites of PAX1 gene (SEQ ID NO: 1-4), specific amplification primers for 2 CpG sites of SOX1 gene (SEQ ID NO: 5-8), and specific amplification primers for CpG sites of quality control H19 (SEQ ID NO: 9-10). The PCR primers Pool were prepared by mixing at a final concentration of 0.5. Mu.M. The PCR amplification reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.
TABLE 1 PCR amplification primer sequences
TABLE 2 PCR amplification reaction System
TABLE 3 PCR amplification reaction procedure
(4) SAP reaction
The SAP reaction system described above contained an SAP enzyme and an SAP Buffer (available from Agena Bioscience, inc., cat. 10141). After PCR amplification, 2ul of SAP reaction solution was added to the amplified product. The SAP reaction system is shown in Table 4 and the reaction procedure is shown in Table 5.
TABLE 4 SAP reaction System
TABLE 5 SAP reaction procedure
(5) Extension reaction
The extension reaction system used in the present application contains Buffer Plus, termination Mix, and iPLEX Enzyme (available from Agena BioScience, cat# 10141), and an extension primer mixture. The preferred sequence of the extension primer combination in the application is shown in Table 6, and the extension primers are specific extension primers (SEQ ID NO: 11-12) of 2 CpG sites of the PAX1 gene, specific extension primers (SEQ ID NO: 13-14) of 2 CpG sites of the SOX1 gene and specific extension primers (SEQ ID NO: 15) of CpG sites of the quality control H19. The extension primers (UEPs) were mixed at a final concentration of 5. Mu.M to prepare a UEP mixture. After the SAP reaction was completed, 2ul of extension reaction solution was added to the SAP product. The elongation reaction system is shown in Table 7, and the reaction procedure is shown in Table 8.
TABLE 6 extended primer sequences
TABLE 7 extension reaction System
TABLE 8 extension reaction procedure
(6) Desalination
Adding 19ul of ultrapure water into the product of the extension reaction, putting the product into a Massary ARRAY-CPM machine, setting the machine to add 10 mul of resin, desalting and purifying, and standing for sedimentation.
(7) Mass spectrometry detection
The MassARRAY-CPM automatically spotted the product on a chip and transferred into a vacuum tube, which was then excited with a laser for time-of-flight mass spectrometry.
2. Data analysis
The experimental results were opened with Typer software and the Data table was exported by clicking on View-Plate Data Pane to give HEIGHT values for the methylated C peak and the unmethylated T peak (Table 9).
TABLE 9 HEIGHT values
Methylation rates were calculated according to the following formula based on the HEIGHT parameters of the methylated C peak and unmethylated T peak in the data table, with the results shown in Table 10.
CpG site methylation rate = C peak heigh value/(C peak heigh value + T peak heigh value)
TABLE 10 methylation Rate calculated by Massarray method
From the methylation results, it can be seen that the methylation rate of the 3 cervical cancer samples (CC 1, CC2 and CC 3) tested was significantly higher than that of the inflammatory sample (INF).
3. Quality control standard
And controlling the quality, wherein the methylation rate of CpG sites of QC is 40-60%, and if the methylation rate exceeds the methylation rate, the experiment is considered to be unqualified and the experiment needs to be reworked.
Example 2
The same bisulfite converted samples were compared to the Massary results using the method of second generation sequencing of the target gene. The results are shown in Table 11.
TABLE 11 Massarray method and target gene second generation sequencing methylation Rate
According to the results, the detection result of the Massary method is consistent with the result of the second generation sequencing of the target genes, and the methylation rate of 3 cervical cancer samples is higher than that of an inflammation sample. Methylation comparison results of Massary and target gene second generation sequencing are shown in FIG. 3. The overall methylation rate trend was consistent across different samples compared to both methods. However, as can be seen from the methylation rate of inflammatory samples (INF), the background value of the massaray method is lower, making it easier to distinguish between methylation rate changes. This is seen from the PAX1 and PAX2 results of cervical cancer sample CC3, which have slightly lower methylation rates, the massaray method can better distinguish cervical cancer samples from inflammatory samples. Is more favorable for clinically assisting doctors in diagnosing cervical cancer and judging the course of the disease.
The application develops a novel method for detecting the methylation level of genes, based on Massary time-of-flight mass spectrometry, creatively adopts multiple PCR to detect a plurality of CpG sites in one hole, can detect the methylation level of genes in a large flux, has low cost and high aging, and is beneficial to more comprehensively providing gene detection information to assist doctors in diagnosis.
It should be noted that the foregoing description is only a preferred embodiment of the present application, and is not intended to limit the scope of the present application, and it will be apparent to those skilled in the art that modifications and variations can be made in the above-described embodiment without departing from the principles of the present application, and the modifications and variations fall within the scope of the appended claims.
Claims (8)
1. A methylation level detection method based on massaray, comprising the steps of:
(1) Extracting DNA of a sample to be detected by using a DNA extraction kit;
(2) Performing Bisulfite (Bisulfite) conversion on the extracted DNA, and detecting the concentration of single-stranded DNA (ssDNA) after the conversion;
(3) Taking the DNA after the Bisulfite conversion as a template, and obtaining methylated and unmethylated amplification products through multiplex PCR amplification reaction;
(4) Removing redundant deoxyribonucleoside triphosphates from the amplified products by shrimp alkaline phosphatase reaction;
(5) Carrying out an extension reaction on the product of the step (4) to obtain an extension product of the detected CpG site, wherein the methylated product is a C peak, and the unmethylated product is a T peak;
(6) Purifying the product of the extension reaction by resin, and desalting;
(7) The desalted product is spotted on a mass spectrum chip matrix for co-crystallization, then the crystal is transferred into a vacuum tube of a mass spectrometer, and laser excitation is used for Massarray flight time mass spectrum detection;
(8) Methylation rate was calculated from the methylation C-peak heigh data and the heigh data of the unmethylated T-peak detected by massaray time-of-flight mass spectrometry.
2. The method for detecting the methylation level based on the Massary according to claim 1, wherein the method comprises the following steps: each pair of PCR primers for multiplex PCR in step (3) simultaneously amplifies methylated and unmethylated fragments.
3. The method for detecting the methylation level based on the Massary according to claim 1, wherein the method comprises the following steps: the multiplex PCR in the step (3) comprises target detection gene fragments and quality control fragments.
4. A method for detecting methylation level based on massaray according to claim 3, characterized in that: the length of the amplified fragment is 100-400 bp, and the quality is controlled to be a imprinting gene H19.
5. The method for detecting the methylation level based on the Massary according to claim 1, wherein the method comprises the following steps: the system of the multiplex PCR amplification reaction in the step (3) comprises a PCR Buffer, mgCl2, dNTPs, a PCR primer Pool and a PCR enzyme, wherein the PCR primer Pool is a mixture of a target detection gene amplification primer and a quality control amplification primer.
6. The method for detecting the methylation level based on the Massary according to claim 1, wherein the method comprises the following steps: the same extension primer in step (5) may extend the C peak of the methylated product and the T peak of the unmethylated product.
7. The method for detecting the methylation level based on the Massary according to claim 1, wherein the method comprises the following steps: the system of the extension reaction comprises Buffer Plus, termination Mix, an extension primer mixture and an iPLEX Enzyme, wherein the extension primer mixture is matched with the sequence of the amplification product and the quality control amplification product of the target detection gene in the step (3).
8. The method for detecting the methylation level based on the Massary according to claim 1, wherein the method comprises the following steps: the methylation ratio of CpG sites can be calculated according to the following formula by using the methylation C peak HEIGHT data and the non-methylation T peak HEIGHT data in the step (8):
CpG site methylation rate = C peak heightvalue/(C peak heightvalue + T peak heightvalue).
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