阅读说明：本技术 飞行时间质谱多重靶向dna甲基化位点定量检测方法 (Flight time mass spectrum multiple target DNA methylation site quantitative detection method ) 是由 张方舟 廉欢欢 张起起 秦胜红 王新新 梁海泳 于 2021-02-02 设计创作，主要内容包括：本发明属生物技术领域,具体公开了一种飞行时间质谱多重靶向DNA甲基化位点定量检测方法,本发明提供了一种DNA甲基化位点的高通量检测方法,可以针对多重待检测的甲基化位点,设计特异性探针,在同一个反应体系中,得到定量甲基化程度检测结果。具有操作简单、可同时检测在不同区域内的甲基化位点,降低对应的实验成本等特点。(The invention belongs to the technical field of biology, and particularly discloses a flight time mass spectrum multiple-target DNA methylation site quantitative detection method. The method has the characteristics of simple operation, capability of simultaneously detecting methylation sites in different areas, reduction of corresponding experiment cost and the like.)

1. The flight time mass spectrum multiple target DNA methylation site quantitative detection method is characterized by comprising the following steps:

(1) designing specific primers for specific sites

(2) Obtaining DNA samples

(3) Bisulfite treating DNA to convert unmethylated C in the sequence to U

(4) Amplifying target fragment by using primer designed in step 1

(5) Digesting the product in the step 4 by SAP to remove the unconsumed dNTP

(6) Single base extension of the product of step 5 with ddNTP and UEP primers

(7) Desalting and purifying the product, and performing flight mass spectrometry detection

(8) And analyzing and interpreting the result.

2. The method for the quantitative detection of the time-of-flight mass spectrometry multiple-target DNA methylation sites according to claim 1, wherein the detection method is used for simultaneously detecting CpG sites in a plurality of different regions.

3. The method for the quantitative detection of multiple target DNA methylation sites according to claim 1, wherein the specific primers in the step 1 include but are not limited to: a specific forward primer and a reverse primer designed according to the sequence of the specific CpG locus to be detected or a single base extension primer designed according to the specific CpG locus to be detected.

4. The method for quantitative detection of multiple target DNA methylation sites according to claim 1, wherein the step 4 specifically comprises performing a first round of PCR using an upstream primer-F and a downstream primer-R of a specific CpG site to amplify the template DNA to obtain a fragment containing the specific CpG site.

5. The method for the quantitative detection of the multiple target DNA methylation sites by time-of-flight mass spectrometry according to claim 4, wherein the reaction conditions of the first round of PCR in the step 4 are as follows: pre-denaturation at 95 deg.C (5 min); amplification: 95 deg.C (20sec), 56 deg.C (30sec), 72 deg.C (1min) for 45 cycles; extension: 72 deg.C (3min), 4 deg.C (∞).

6. The method for the quantitative detection of the multiple target DNA methylation sites by the time-of-flight mass spectrometry of claim 1, wherein in the step 5, the fragment obtained after the step 4 is subjected to dephosphorylation treatment to obtain a dephosphorylated fragment.

7. The method for the quantitative detection of the multiple target DNA methylation sites by time-of-flight mass spectrometry according to claim 6, wherein the dephosphorylation reaction in the step 5 is performed under the following conditions: dephosphorizing at 37 deg.c for 40 min; inactivation of SAP enzyme: 85 deg.C (5min), 4 deg.C (∞).

8. The method for the quantitative detection of the methylation sites of the DNA with multiple targets by the time-of-flight mass spectrometry of claim 1, wherein the step 6 specifically comprises a step of performing single base extension on the dephosphorylated fragment obtained after the step 5 by using-UEP of a specific CpG site, so as to detect the methylation degree.

9. The method for the quantitative detection of multiple target DNA methylation sites by time-of-flight mass spectrometry according to claim 8, wherein the reaction conditions for single base extension in step 6 are as follows: pre-denaturation at 94 deg.C (30 sec); amplification inner circulation, namely 52 ℃ (5sec) and 80 ℃ (5sec), and 5 circulations are performed; amplification cycles of 94 deg.C (5sec), 52 deg.C (5sec), and 80 deg.C (5sec) for 40 cycles; extension: 72 deg.C (3min), 4 deg.C (∞).

10. The method for the quantitative detection of the multiple target DNA methylation sites by time-of-flight mass spectrometry according to claim 1, wherein the step 7 specifically comprises the following steps:

s1 spreading clean resin on the resin plate with 6mg resin per hole, air dryingThe method comprises the following steps of (1) taking minutes;

s2, adding the sample to be tested into the sample space of the sample plate, and adding the sample into each sample holeddH20, sealed with a sealing film, and subjected to instantaneous centrifugation;

s3, opening the sealing film covering the sample holes, reversely buckling the sample plate on the resin plate for fixation, integrally turning over the fixed sample plate and the resin plate, tapping the resin plate to enable the resin in the resin plate to completely fall into the corresponding sample holes on the sample plate, sealing the sample holes by the sealing film, and performing instant centrifugation;

and S4, shaking the sample resin mixture, slowly rotating the sample resin mixture on a rotator for 15min, centrifuging the sample resin mixture for 5min at 4000rpm, spotting the centrifuged sample on a computer, and detecting the sample by using a mass spectrometer.

11. The method for the quantitative detection of multiple target DNA methylation sites according to claim 1, wherein in the step 8, the original result obtained in the step 7 is interpreted; and calculating according to the signal-to-noise ratios (SNR-1 and SNR-2) in the generated data table to obtain the methylation degree of the sample methylation site. The calculation formula is as follows: SNR-C/(SNR-C + SNR-T).

Technical Field

The invention belongs to the technical field of biology, and particularly discloses a flight time mass spectrum multiple-target DNA methylation site quantitative detection method.

Technical Field

Methylation of DNA, an important chemical modification of DNA, affects the genetic appearance of DNA without altering the DNA sequence. In vertebrates, the vast majority of DNA methylation is modified to 5 methylcytosine (5mc), and in the human genome, approximately 70% to 80% of CpG dinucleotides are involved in the methylation of this modified DNA and can be involved in a number of biological processes, including genomic imprinting, silencing of transposable elements, stem cell differentiation, embryonic development, and the like. Methylation of DNA is one of the important research contents of epigenetics as an important epigenetic phenomenon.

The rapid development of DNA methylation detection methods, high-throughput sequencing technology and chip technology for whole genome methylation detection and quantitative methylation mass spectrometry detection are increasingly applied to methylation research. As researchers continue to study methylation, the need for quantitative detection of methylation sites is increasing.

The principle of the existing methylation quantitative detection technology based on the MassARRAY platform is that sodium bisulfite is used for treating DNA under the catalysis of hydroquinone under the alkaline condition, so that unmethylated cytosine is subjected to deamination reaction and is converted into uracil (U), and methylated cytosine cannot be subjected to deamination reaction and still remains as cytosine. In the first round of amplification, transformed U pairs with A and is further converted to T in the second round of amplification. Finally, the difference between methylated C and unmethylated C becomes the difference between C and T. PCR amplification is carried out on a DNA sample treated by CT to obtain an amplification product corresponding to a target fragment, SAP digestion is carried out on the amplification product, then reverse transcription and enzyme digestion are carried out on the product synchronously by using T7 RNA, DNA Polymerase and RNase A enzyme to obtain a small fragment containing CpG site, the molecular weight of the methylated fragment and the non-methylated fragment is different, and the fragments can be distinguished and the methylation ratio can be calculated through time-of-flight mass spectrometry. The detection method has the disadvantages that CpG sites which are not in the same range cannot be simultaneously detected in the same reaction well, and the CpG sites in the amplified fragment cannot be detected certainly or the result of single detection cannot be ensured due to the technical limitation.

Disclosure of Invention

Aiming at the problems, the invention provides a flight time mass spectrum multiple-target DNA methylation site quantitative detection method, which can realize the detection of the methylation degree of specific CpG sites at a plurality of different positions simultaneously based on a MassARRAY nucleic acid mass spectrum platform, can obtain the result of the individual methylation degree of the CpG sites to be detected, and reduces the experiment cost.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a flight time mass spectrum multiple target DNA methylation site quantitative detection method comprises the following steps:

(1) designing specific primers for specific sites

(2) Obtaining DNA samples

(3) Bisulfite treating DNA to convert unmethylated C in the sequence to U

(4) Amplifying target fragment by using primer designed in step 1

(5) Digesting the product in the step 4 by SAP to remove the unconsumed dNTP

(6) Single base extension of the product of step 5 with ddNTP and UEP primers

(7) Desalting and purifying the product, and performing flight mass spectrometry detection

(8) And analyzing and interpreting the result.

Further, the method for the quantitative detection of the multiple target DNA methylation sites by the time-of-flight mass spectrometry is used for simultaneously detecting the CpG sites in a plurality of different regions.

Further, in the method for the quantitative detection of multiple target DNA methylation sites by time-of-flight mass spectrometry, the specific primers in step 1 include, but are not limited to: a specific forward primer and a reverse primer designed according to the sequence of the specific CpG locus to be detected or a single base extension primer designed according to the specific CpG locus to be detected.

Further, in the method for quantitatively detecting multiple target DNA methylation sites by time-of-flight mass spectrometry, the step 4 specifically includes performing a first round of PCR by using an upstream primer-F and a downstream primer-R of a specific CpG site, and amplifying the template DNA to obtain a fragment containing the specific CpG site.

Further, in the method for the quantitative detection of the multiple target DNA methylation sites by time-of-flight mass spectrometry, the reaction conditions of the first round of PCR in the step 4 are as follows: pre-denaturation at 95 deg.C (5 min); amplification: 95 deg.C (20sec), 56 deg.C (30sec), 72 deg.C (1min) for 45 cycles; extension: 72 deg.C (3min), 4 deg.C (∞).

Further, in the method for quantitatively detecting the multiple target DNA methylation sites by using the time-of-flight mass spectrometry, in the step 5, the fragment obtained in the step 4 is subjected to dephosphorylation treatment to obtain a dephosphorylated fragment.

Further, in the method for the quantitative detection of the multiple target DNA methylation sites by time-of-flight mass spectrometry, the dephosphorylation reaction conditions in the step 5 are as follows: dephosphorizing at 37 deg.c for 40 min; inactivation of SAP enzyme: 85 deg.C (5min), 4 deg.C (∞).

Further, in the method for quantitatively detecting the methylation sites of the multiple target DNAs by time-of-flight mass spectrometry, step 6 specifically includes performing single base extension on the dephosphorylated fragment obtained after step 5 by using-UEP of a specific CpG site, so as to detect the methylation degree.

Further, in the method for the quantitative detection of the multiple target DNA methylation sites by time-of-flight mass spectrometry, the reaction conditions for single base extension in the step 6 are as follows: pre-denaturation at 94 deg.C (30 sec); amplification inner circulation, namely 52 ℃ (5sec) and 80 ℃ (5sec), and 5 circulations are performed; amplification cycles of 94 deg.C (5sec), 52 deg.C (5sec), and 80 deg.C (5sec) for 40 cycles; extension: 72 deg.C (3min), 4 deg.C (∞).

Further, in the method for quantitatively detecting multiple target DNA methylation sites by time-of-flight mass spectrometry, the step 7 specifically comprises the following steps:

s1 spreading clean resin on the resin plate with 6mg resin per hole, air dryingThe method comprises the following steps of (1) taking minutes;

s2, adding the sample to be tested into the sample space of the sample plate, and adding the sample into each sample holeddH20, sealed with a sealing film, and subjected to instantaneous centrifugation;

s3, opening the sealing film covering the sample holes, reversely buckling the sample plate on the resin plate for fixation, integrally turning over the fixed sample plate and the resin plate, tapping the resin plate to enable the resin in the resin plate to completely fall into the corresponding sample holes on the sample plate, sealing the sample holes by the sealing film, and performing instant centrifugation;

and S4, shaking the sample resin mixture, slowly rotating the sample resin mixture on a rotator for 15min, centrifuging the sample resin mixture for 5min at 4000rpm, spotting the centrifuged sample on a computer, and detecting the sample by using a mass spectrometer.

Further, in the method for quantitatively detecting multiple target DNA methylation sites by time-of-flight mass spectrometry, in the step 8, the original result obtained in the step 7 is read; and calculating according to the signal-to-noise ratios (SNR-1 and SNR-2) in the generated data table to obtain the methylation degree of the sample methylation site. The calculation formula is as follows: SNR-C/(SNR-C + SNR-T).

Compared with the prior art, the invention has the following advantages: by the method, the methylation degree of the specific CpG sites at different positions can be detected simultaneously based on a MassARRAY platform, and the result of the individual methylation degree of the CpG sites to be detected can be obtained, so that the result is more accurate, and the experiment cost is greatly reduced.

Drawings

FIG. 1: a brief experimental flow and a schematic diagram of a MassARRAY iPLEX detection method;

FIG. 2: a peak image of an original result shows that the signal-to-noise ratio of C in the CpG sites to be detected is higher, and the methylation degree of the result is higher after calculation;

FIG. 3: a peak image of an original result shows that the signal-to-noise ratio of T in the CpG sites to be detected is higher, and the methylation degree of the result is lower after calculation;

FIG. 4: the results are derived from the original, where the signal-to-noise ratio is calculated to yield the corresponding degree of methylation at the site.

Examples

1. In the following technical procedures, all general detection methods are used unless otherwise specified. For convenience of explanation, we illustrate by way of example.

2. Materials, reagents and the like used in the following techniques are commercially available from normal sources unless otherwise specified.

3. In the embodiment, the sample is human, and the experimental DNA is obtained after blood is extracted by a hundred-tach magnetic bead whole blood genome extraction kit (model AU 18016).

4. Selection of CpG sites to be detected: cg03915055, cg15989608, cg19226017, cg19554255, cg25114611, cg01616956, cg09994445, cg16779463 designed specific probes for the chosen sites:

5. searching the gene position of the selected CpG site on a UCSC website, and expanding 200bp on the upstream and downstream respectively to obtain a sequence corresponding to the site to be detected. Specific PCR primers and single base extension primers are designed aiming at the selected CpG sites to be detected, the specific sequence composition is shown in the following table 1, and the sequence numbers are shown in SEQ ID NO: 1, starting:

TABLE 1 primer sequence Listing

Detailed Description

The experimental process is shown in figure 1.

For convenience, the reagents required for the experiments are summarized below: EZ DNA Methylation Gold kit (ZYMO), ddH₂O、PCR Buffer、MgCl₂、dNTPs、Taq Polymerase、SAP Buffer、Shrimp Alkaline Phosphatase Enzyme、iPLEX Buffer Plus、iPLEX Termination Mix、iPLEX Enzyme。

(1) The detection method has wide sample type sources, such as blood, tissues, cells and the like, can efficiently extract and prepare the genome DNA, and is used for quantitative methylation detection.

(2) CT treatment of DNA:

I. reaction system

Reagent	Volume for CT Conversion Reagent
		ddH2O	9mL
M-Dilution Buffer	3mL
		M-Dissolving Buffer	500μL

II. Reaction procedure

Temperature of	Time	Number of cycles
			98℃	10min	1
64℃	30min	5
			4℃	∞	1

(3) Amplifying a target fragment:

I. primer primers for reaction System primers primer pairs sequences in Table 1

Components	5ul reaction System addition (ul)
		ddH2O	1.8
10×PCR Buffer	0.5
		2.5mM MgCl2	0.4
10mM dNTPs	0.1
		Taq Polymerase(5U/ul)	0.2
Primer mix	1
		DNA template	1

II. Reaction procedure

(4) SAP digestion

I. Reaction system

II. Reaction procedure

Temperature of	Time	Number of cycles
			37℃	40min	1
85℃	5min	1
			4℃	∞	1

(5) Single base extension

I. Primer for reaction System primer in Table 1 Single base extension primer

Components	5ul reaction System addition (ul)
		ddH2O	0.5949
10×iPLEX Buffer Plus	0.2
		iPLEX Termination Mix	0.22
Primer mix	0.94
		iPLEX Enzyme	0.0451

II. Reaction procedure

(6) Flight mass spectrometry detection

I. Spreading clean resin on the resin plate for later use, 6mg resin per hole, and air dryingThe method comprises the following steps of (1) taking minutes;

II. Adding the sample to be tested into the sample space of the sample plate, and adding the sample into each sample holeddH20, sealed with a sealing film, and subjected to instantaneous centrifugation;

III, opening a sealing film covering the sample hole, reversely buckling the sample plate on the resin plate for fixing, integrally overturning the fixed sample plate and the resin plate, tapping the resin plate to enable the resin in the resin plate to completely fall into the corresponding sample hole in the sample plate, sealing by using the sealing film, and carrying out instantaneous centrifugation;

and IV, shaking the sample resin mixture, slowly rotating the sample resin mixture on a rotator for 15min, centrifuging the sample resin mixture for 5min at 4000rpm, spotting the sample on a computer after centrifugation, and detecting the sample by using a mass spectrometer.

(7) Results analysis and interpretation

And V, obtaining a quantitative methylation result of the relevant sites through calculation according to the signal-to-noise ratio of the C site and the signal-to-noise ratio of the T site, as shown in the figure 2-4.

The results of the above embodiments show that the method for quantitatively detecting multiple target DNA methylation sites by time-of-flight mass spectrometry disclosed by the invention can realize detection of methylation degrees of specific CpG sites at multiple different positions simultaneously based on a MassARRAY platform, and can obtain a result of individual methylation degrees of the CpG sites to be detected, thereby reducing experiment cost.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.

SEQUENCE LISTING

<110> Bo vast Biotechnology (Beijing) Ltd

<120> flight time mass spectrum multiple target DNA methylation site quantitative detection method

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