Method for evaluating the efficiency of DNA methylation transformation

Abstract

The present application relates to the field of biotechnology, and particularly relates to a method for evaluating DNA methylation transformation efficiency. The present application can simply, quickly, directly and comprehensively evaluate DNA methylation transformation efficiency of the sample to be tested by detecting the methylation and non-methylation ΔCt values of four genes GNAS, GPR1, PAX6 and Actin respectively through conventional fluorescent PCR primer design, and calculating (2 ΔCtMSP +2 ΔCtUSP ) / (2 ΔCtMSP +2 ΔCtUSP +1). The method provided by the present application can be used for evaluation and quality control before carrying out methylation experiment or constructing methylation sequencing library, and the sample with low transformation efficiency can be detected in time to avoid carrying out subsequent experiment and causing resource waste such as experimental cost.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for evaluating DNA methylation conversion efficiency. Background Technology

[0002] The main methods for evaluating DNA methylation conversion efficiency include fluorescent PCR, digital PCR, and multiplex PCR. In 2015, Liu Yangyang et al. reported on "An Evaluation Method for Bisulfite-Treated DNA Transformation Efficiency in DNA Methylation Analysis" in the journal *Genetics*. They used TaqMan qPCR for quantitative detection, establishing standard curves for Ct values ​​and corresponding DNA copy numbers of transformed and untreated DNA standards at different concentration gradients of bisulfite treatment, and then calculating the conversion rate of the tested sample DNA. However, this method is relatively cumbersome, only selects housekeeping genes, has insufficient coverage for high methylation levels, and the standard curve established using exogenous DNA standards cannot accurately reflect the copy number of β-actin in the tested sample.

[0003] Currently, some authorized and published invention patents require the introduction of exogenous genes, some require the preparation of methylation positive controls, and some require the creation of standard curves. Furthermore, all of these methods only target a single gene for detection. For example, patent application number 201511008137.2 detects only one gene and requires the preparation of methylation positive control samples, making the operation complex, time-consuming, and inefficient. Patent application number 201911419949.4 also detects only one gene and requires dedicated ddPCR equipment, resulting in higher costs. No schemes were found that simultaneously assess methylation conversion efficiency for multiple endogenous genes with different methylation levels using ΔCt values ​​for methylation and unmethylation. Summary of the Invention

[0004] In view of this, the present invention provides a method for evaluating DNA methylation conversion efficiency. This method detects the ΔCt values ​​of methylated and unmethylated DNA in four genes: GNAS, GPR1, PAX6, and Actin, respectively, and then evaluates the efficiency by (2... ΔCtMSP +2 ΔCtUSP ) / (2 ΔCtMSP +2 ΔCtUSP +1) A simple, fast, direct and comprehensive way to evaluate the DNA methylation conversion efficiency of the sample to be tested.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] A method for evaluating DNA methylation conversion efficiency, comprising:

[0007] 1) Treat the DNA sample to be tested with bisulfite to obtain the transformed DNA sample to be tested;

[0008] 2) Using the transformed DNA of the test sample as a template, quantitative real-time PCR was performed to amplify the untransformed, methylated, and unmethylated sequences of the test gene in the DNA sample, respectively, and the Ct values ​​were recorded as Ct0, Ct1, and Ct2; the test gene includes at least two of GNAS, GPR1, PAX6, and Actin; the bisulfite conversion rate of each test gene was calculated according to the following formula:

[0009] Bisulfite conversion rate (%) = (2) ΔCtMSP +2 ΔCtUSP ) / (2 ΔCtMSP +2 ΔCtUSP +1)

[0010] Wherein, ΔCtMSP=Ct1-Ct0, ΔCtUSP=Ct2-Ct0;

[0011] 3) The transformation efficiency of the DNA sample to be tested is evaluated based on the amplification curve of the quantitative real-time PCR of the gene to be tested and the bisulfite conversion rate.

[0012] In this invention, the unconverted sequence is the original gene sequence, which has not undergone bisulfite conversion. The unmethylated sequence is one that has not undergone methylation modification and, after bisulfite treatment, transforms C to T. The methylated sequence is one where C on the CG island is methylated, while C at other positions remains unmethylated; after bisulfite treatment, C on the CG island remains unchanged, while C at other positions transforms to T (methylated C is usually limited to C on the CG island and does not include scattered C). The unconverted, methylated, and unmethylated sequences are located in the same region of the gene.

[0013] In some implementation schemes, the evaluation criteria are as follows:

[0014] a) If all amplification curves of the gene to be tested are S-shaped and the average bisulfite conversion efficiency is ≥98%, then the DNA sample to be tested is determined to have a high methylation conversion efficiency, and the DNA sample to be tested can be used for the construction and sequencing of DNA methylation libraries.

[0015] b) If the amplification curves of the genes to be tested are all S-shaped and the average conversion efficiency is less than 98%, then the DNA sample to be tested is determined to have low conversion efficiency and is not suitable for the construction and sequencing of DNA methylation libraries. The sample needs to be removed or the bisulfite conversion process needs to be further optimized until the standard of step a) is met.

[0016] c) If an S-type amplification curve appears in the gene to be tested, then the transformed DNA sample to be tested is prepared again according to step 1); then the experiment is carried out according to step 2) until a typical S-type amplification curve appears, and the results are evaluated according to the standards of a) to b).

[0017] In some embodiments, the target genes include GNAS, GPR1, PAX6, and Actin. Primers for amplifying the four target genes include primers for amplifying unconverted, methylated, and unmethylated sequences. For each gene, there are three pairs of primers amplifying its unconverted, methylated, and unmethylated sequences, for a total of 12 pairs of primers for the four genes, the sequences of which are shown in SEQ ID NO: 1–24.

[0018] Specifically:

[0019] Primers for amplifying the untransformed sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 1 and the downstream primer with the sequence shown in SEQ ID NO: 2;

[0020] Primers for amplifying the methylated sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 3 and the downstream primer with the sequence shown in SEQ ID NO: 4;

[0021] Primers for amplifying the unmethylated sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 5 and the downstream primer with the sequence shown in SEQ ID NO: 6;

[0022] Primers for amplifying the untransformed sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 1 and the downstream primer with the sequence shown in SEQ ID NO: 2;

[0023] Primers for amplifying the methylated sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 3 and the downstream primer with the sequence shown in SEQ ID NO: 4;

[0024] Primers for amplifying the unmethylated sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 5 and the downstream primer with the sequence shown in SEQ ID NO: 6;

[0025] Primers for amplifying the untransformed sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 1 and the downstream primer with the sequence shown in SEQ ID NO: 2;

[0026] Primers for amplifying the methylated sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 3 and the downstream primer with the sequence shown in SEQ ID NO: 4;

[0027] Primers for amplifying the unmethylated sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 5 and the downstream primer with the sequence shown in SEQ ID NO: 6;

[0028] Primers for amplifying the untransformed sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 1 and the downstream primer with the sequence shown in SEQ ID NO: 2;

[0029] Primers for amplifying the methylated sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 3 and the downstream primer with the sequence shown in SEQ ID NO: 4;

[0030] Primers for amplifying the unmethylated sequence of the target gene GNAS: the upstream primer with the sequence shown in SEQ ID NO: 5 and the downstream primer with the sequence shown in SEQ ID NO: 6.

[0031] In some implementation schemes, the bisulfite treatment time in step 1) is 2 to 16 hours; in some specific embodiments, the bisulfite treatment time is specifically 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours.

[0032] In some implementations, the concentration of the DNA sample to be tested in the quantitative PCR detection described in step 2) is 10 ng / μL.

[0033] In some implementation schemes, the system for the quantitative real-time PCR detection includes:

[0034] Add 5 μL of Master Mix, 1 μL each of upstream and downstream primers, 1 μL of template DNA, and water to a final volume of 10 μL.

[0035] In some implementations, the procedure for the quantitative PCR detection includes:

[0036] 98℃ for 5 minutes;

[0037] 95℃ for 30 seconds, 56℃ for 30 seconds, 72℃ for 45 seconds, for a total of 40 cycles.

[0038] This invention uses a conventional real-time PCR instrument to detect four endogenous genes with different methylation levels. Based on the ΔCt values ​​of genes treated with bisulfite and those not methylated, the results are obtained through (2... ΔCtMSP +2 ΔCtUSP ) / (2 ΔCtMSP +2 ΔCtUSP+1) Directly obtaining DNA methylation conversion rate allows for evaluation and quality control before conducting methylation experiments or constructing methylation sequencing libraries. It enables timely detection of samples with low conversion efficiency, thus avoiding the waste of resources such as experimental costs in subsequent experiments. Attached Figure Description

[0039] Figure 1 A flowchart illustrating the method for evaluating DNA methylation conversion efficiency of the present invention;

[0040] Figure 2 The effect of different conversion times of disulfite on conversion efficiency. Detailed Implementation

[0041] This invention provides a method for evaluating DNA methylation conversion efficiency. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments; those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0042] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0043] The present invention will be further illustrated below with reference to the embodiments:

[0044] Example 1

[0045] Primers used to assess DNA methylation conversion efficiency include:

[0046] The upstream primer sequence for the methylated region of the GNAS gene is SEQ ID NO:1, and the downstream primer sequence is SEQ ID NO:2; the upstream primer sequence for the unmethylated region is SEQ ID NO:3, and the downstream primer sequence is SEQ ID NO:4; the upstream primer sequence for the pre-bisulfite transformation sequence (i.e., the original genome sequence) is SEQ ID NO:5, and the downstream primer sequence is SEQ ID NO:6.

[0047] The upstream primer sequence for the methylated region of the GPR1 gene is SEQ ID NO:7, and the downstream primer sequence is SEQ ID NO:8; the upstream primer sequence for the unmethylated region of the sequence is SEQ ID NO:9, and the downstream primer sequence is SEQ ID NO:10; the upstream primer sequence for the pre-bisulfite transformation sequence (i.e., the original genome sequence) is SEQ ID NO:11, and the downstream primer sequence is SEQ ID NO:12.

[0048] The upstream primer sequence for the methylated region of the PAX6 gene is SEQ ID NO:13, and the downstream primer sequence is SEQ ID NO:14; the upstream primer sequence for the unmethylated region of the sequence is SEQ ID NO:15, and the downstream primer sequence is SEQ ID NO:16; the upstream primer sequence for the pre-bisulfite transformation sequence (i.e., the original genome sequence) is SEQ ID NO:17, and the downstream primer sequence is SEQ ID NO:18.

[0049] The upstream primer sequence for the methylated region of the Actin gene is SEQ ID NO:19, and the downstream primer sequence is SEQ ID NO:20; the upstream primer sequence for the unmethylated region is SEQ ID NO:21, and the downstream primer sequence is SEQ ID NO:22; the upstream primer sequence for the pre-bisulfite conversion region is SEQ ID NO:23, and the downstream primer sequence is SEQ ID NO:24.

[0050] The specific sequences are shown in Table 1:

[0051] Table 1

[0052]

[0053]

[0054] The primers consist of three pairs: each gene is methylated, unmethylated, and before transformation.

[0055] Example 2: Bisulfite Conversion of Genomic DNA

[0056] (I) Genomic DNA Extraction and Bisulfite Conversion

[0057] (1) Genomic DNA was extracted from blood and tissue samples using a genomic DNA extraction kit;

[0058] (2) Take 1 μg of genomic DNA and add water to 45 μL, then perform bisulfite conversion according to the operating procedure;

[0059] (3) Add 5 μL of M-Dilution Buffer to 45 μL of sample and mix well by pipetting.

[0060] (4) PCR at 42℃ for 30 min;

[0061] (5) Add 100 μL of the prepared CT Conversion Reagent to the corresponding sample and mix well;

[0062] (6) React on a PCR instrument. The program is: 95℃ for 30 seconds, 50℃ for 1 hour, 16 cycles, 4℃ for 10 minutes (or place on ice for 10 minutes).

[0063] (7) Add 400 μL of M-Binding Buffer to the EZ purification column. Add the sample processed in (6) above to the EZ purification column, mix by inversion, and then incubate at 13,000 rpm for 30 seconds.

[0064] (8) Discard the liquid in the collection tube, add 100 μL Wash Buffer, 13,000 rpm, 30 sec;

[0065] (9) Add 200 μL M-Desulphonation Buffer, incubate at room temperature for 15 min-20 min, 13,000 rpm, 30 sec;

[0066] (10) Add 200 μL Wash Buffer, 13,000 rpm, 30 sec;

[0067] (11) Add 200 μL Wash Buffer, 13,000 rpm, 30 sec;

[0068] (12) Discard the liquid in the collection tube, empty the tube, and run at 13,000 rpm for 1 min.

[0069] (13) Place the EZ purification column in a clean 1.5 mL centrifuge tube;

[0070] (14) Add 13 μL M-Elution Buffer, incubate at room temperature for 5 min, then incubate at 13,000 rpm for 1 min.

[0071] (15) The DNA after bisulfite treatment was quantified using Qubit and adjusted to 10 ng / μL.

[0072] (II) Quantitative Real-Time PCR Detection

[0073] (1) Thaw the PCR Master Mix, primers and samples on ice. Before use, shake continuously for 15 seconds to mix, then centrifuge for 10 seconds to prepare the corresponding number of PCR reaction tubes.

[0074] (2) Prepare PCR reaction system: Prepare PCR reaction system according to the table below.

[0075] Table 2

[0076] Components Volume (μL) Master Mix 5 primer mixture 2 <![CDATA[ddH2O]]> 2 Total volume 10

[0077] (3) After mixing the prepared PCR mixture by shaking for 15 seconds, centrifuge for 10 seconds and dispense 9 μL / tube into PCR reaction tubes.

[0078] (4) Add 1 μL (10 ng) of bisulfite-converted genomic DNA to each PCR reaction well, centrifuge at 4000 rpm for 15 s, and centrifuge the liquid on the tube wall to the bottom of the tube. If there are air bubbles, tap and centrifuge until the air bubbles are removed. Then perform PCR amplification according to the procedure in the table below.

[0079] Table 3

[0080]

[0081] (III) Analysis of test results.

[0082] First, the specificity of the melting curve must be checked:

[0083] If the S-type amplification curve is abnormal, the bisulfite-converted DNA sample needs to be prepared again and then tested again.

[0084] If all amplification curves of the gene to be tested are S-shaped and the average bisulfite conversion efficiency of all genes is ≥98%, then the DNA sample to be tested is determined to have a high methylation conversion efficiency, and the DNA sample to be tested can be used for the construction and sequencing of DNA methylation libraries.

[0085] If the amplification curves of the genes to be tested all show a typical S-shape, and the average bisulfite conversion efficiency of all genes is less than 98%, then the DNA sample to be tested is determined to have low conversion efficiency and is not suitable for the construction and sequencing of DNA methylation libraries. The sample to be tested should be removed or the bisulfite conversion process should be further optimized until the average bisulfite conversion efficiency of all genes is ≥98%.

[0086] Example 3: Effect of different conversion times on conversion efficiency of bisulfite

[0087] Peripheral blood, normal tissue, and tumor tissue samples were treated with bisulfite for 0.5 h, 2 h, 6 h, and 16 h overnight, respectively, and the transformation efficiency was assessed. Three technically replicate qPCR experiments were performed on each of the four samples for four genes (GNAS, GPR1, PAX6, and Actin), with each sample starting with 10 ng of bisulfite-transformed genomic DNA. This gradient experiment revealed that bisulfite treatment time is related to transformation efficiency. When treatment time was insufficient, the DNA methylation transformation efficiency of the four genes in different samples was mostly below 30%, indicating significant incomplete transformation. However, after 16 h of treatment, the bisulfite transformation efficiency reached over 99% in both blood and tissue samples, indicating complete transformation with excellent technical reproducibility.

[0088] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for evaluating DNA methylation conversion efficiency, characterized in that, include: 1) Treat the DNA sample to be tested with bisulfite for 16 hours to obtain the transformed DNA sample to be tested; 2) Using the transformed DNA of the test sample as a template, quantitative real-time PCR was performed to amplify the DNA and obtain the Ct values ​​of the untransformed, methylated, and unmethylated sequences of the test gene in the DNA sample, which were denoted as Ct, respectively. 0、 Ct 1、 Ct2; the test genes are GNAS, GPR1, PAX6, and Actin; the bisulfite conversion rate of each test gene is calculated according to the following formula: Bisulfite conversion rate (%) = (2) ΔCtMSP +2 ΔCtUSP ) / (2 ΔCtMSP +2 ΔCtUSP +1) Wherein, ΔCtMSP=Ct1-Ct0, ΔCtUSP=Ct2-Ct0; 3) The transformation efficiency of the DNA sample to be tested was evaluated based on the amplification curve of the quantitative real-time PCR of the gene to be tested and the bisulfite conversion rate. The evaluation criteria are as follows: a) If all amplification curves of the gene to be tested are S-shaped and the average bisulfite conversion efficiency is ≥98%, then the DNA sample to be tested can be used for the construction and sequencing of DNA methylation libraries; b) If the amplification curves of the genes to be tested are all S-shaped and the average bisulfite conversion efficiency of the genes to be tested is less than 98%, then the genes are not suitable for the construction and sequencing of DNA methylation libraries. The sample should be removed or the bisulfite conversion process should be further optimized until the standard of step a) is met. c) If no S-type amplification curve appears in the gene to be tested, then the transformed DNA sample to be tested is prepared again according to step 1); then the experiment is carried out according to step 2) until an S-type amplification curve appears, and the results are evaluated according to the standards of a)~b).

2. The evaluation method according to claim 1, characterized in that, The primers used to amplify the unconverted, methylated, and unmethylated sequences of the gene to be tested are shown in SEQ ID NO: 1~24.

3. The evaluation method according to claim 1, characterized in that, In step 2), the concentration of the DNA sample to be tested in the quantitative PCR detection is 10 ng / μL.

4. The evaluation method according to claim 1, characterized in that, The system for the quantitative real-time PCR detection includes: 5 μL of PCR Master Mix, 1 μL each of upstream and downstream primers, 1 μL of template DNA, and water to a final volume of 10 μL.

5. The evaluation method according to claim 1, characterized in that, The procedure for the quantitative real-time PCR detection includes: 95℃ for 5 minutes; 95℃ for 30 seconds, 56℃ for 30 seconds, 72℃ for 45 seconds, for a total of 40 cycles.

Images