WO2017143866A1 - 用于定量检测rprm基因dna甲基化试剂盒及方法 - Google Patents

用于定量检测rprm基因dna甲基化试剂盒及方法 Download PDF

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WO2017143866A1
WO2017143866A1 PCT/CN2016/113680 CN2016113680W WO2017143866A1 WO 2017143866 A1 WO2017143866 A1 WO 2017143866A1 CN 2016113680 W CN2016113680 W CN 2016113680W WO 2017143866 A1 WO2017143866 A1 WO 2017143866A1
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dna
rprm
methylation
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陈骐
王瀚泽
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福建师范大学
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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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a DNA methylation kit and method for quantitatively detecting RPRM gene.
  • DNA methylation is one of the most common molecular molecular changes in tumors, especially in tumor suppressor genes and mismatch repair genes, which can lead to mutations in gene coding regions and genes. Inactivation is beneficial to the development of tumors and is one of the important indicators of cancer.
  • the establishment of methylation pattern maps of different types of cancer genes in humans is an important part of research in related fields. Abnormal methylation of genes has been found in lung cancer, gastric cancer, head and neck cancer, bladder cancer, prostate cancer, leukemia, lymphoma, and gallbladder cancer.
  • RPRM gene reprimo, TP53dependent G2arrest mediator candidate, is a p53 tumor suppressor-inducible gene located at chromosome 2q23. This allele imbalance has been demonstrated with lung cancer, breast cancer and colon cancer. Associated. Abnormal methylation of RPRM gene has been detected in many cancer and tumor cell lines, including gastric cancer, prostate cancer, pancreatic cancer, lung cancer, colorectal cancer, esophageal cancer, gallbladder cancer, lymphoma, leukemia, breast cancer patients. .
  • RPRM methylation is potential as a biomarker for early serological biopsy of gastric cancer.
  • the detection of RPRM methylation in gastric cancer patients combined with gastric mucosal atrophy markers and Helicobacter pylori detection may establish an early detection index for gastric cancer.
  • RPRM methylation is one of the important indicators of gastric cancer patients, it may be used for prognosis detection of gastric cancer.
  • the current RPRM methylation assay has been approved for clinical trials in the United States. Therefore, methylation of RPRM gene, as an important serum molecular marker in cancer diagnosis and treatment, can be used as an important reference for diagnosis and treatment.
  • methylation mainly occurs in C (ie, cytosine) at the CpG site.
  • C ie, cytosine
  • cytosine will be converted into uracil, while methylated cytosine will remain unchanged.
  • Cellulite and uridine are distinguished by methods such as DNA sequencing analysis.
  • methylation or non-methylation of cytosine can be distinguished by bisulfite conversion.
  • the invention improves the method for bisulfite conversion, and optimizes the bisulfite conversion PCR method to establish a DNA methyl group by establishing a methylated modified and non-modified internal reference plasmid, a real-time PCR method and a threshold interception method.
  • the sensitive melting curve analysis achieves the purpose of quantitative detection of DNA methylation.
  • This method can be used not only to quantitatively detect DNA methylation of RPRM gene, but also to quantitatively detect DNA methylation of other genes, and eliminate the steps of DNA sequencing. Moreover, the degree of methylation can be quantitatively analyzed, the operation is relatively simple, and the cost is low, which is very suitable for high-throughput analysis of a large number of samples in the future.
  • the object of the present invention is to provide a DNA methylation kit and a method for quantitatively detecting RPRM gene, which can be used not only for quantitative detection of DNA methylation of RPRM gene, but also for quantitative detection of DNA methylation of other genes, and is exempted from The steps of DNA sequencing, and the degree of methylation can be quantitatively analyzed, the operation is simple and the cost is low, which is very suitable for high-throughput analysis of a large number of samples in the future.
  • the present invention adopts the following technical solutions:
  • a DNA methylation kit for quantitative detection of RPRM gene comprising: a DNA conversion reagent component (heavy sulfite conversion reaction solution), a DNA purification reagent component (a glass fiber membrane spin column, a binding buffer, a rinse buffer, Desulfurization buffer, elution buffer), methylation detection reagent component (2 ⁇ SYBR GREEN I premixed reaction solution, primer RPRM-F and RPRM-R, methylation positive control plasmid S M and unmethylated Positive control plasmid S U ).
  • a DNA conversion reagent component heavy sulfite conversion reaction solution
  • a DNA purification reagent component a glass fiber membrane spin column, a binding buffer, a rinse buffer, Desulfurization buffer, elution buffer
  • methylation detection reagent component 2 ⁇ SYBR GREEN I premixed reaction solution, primer RPRM-F and RPRM-R, methylation positive control plasmid S M and unmethylated Positive control
  • a method for quantitatively detecting DNA methylation of a RPRM gene comprising the following:
  • the melting curve method in step (3) uses two standard plasmids as a reference: the M-seq of the methylated RPRM gene sequence after bisulfite conversion, the sequence is SEQ ID NO. 2, but not methylation After the sequence is transformed, U-seq, the sequence is SEQ ID NO. 3, and then the two sequences are separately cloned into a vector as positive control standard samples S M and S U , and the real-time PCR amplification described in the above (3) is performed. Melting curve analysis.
  • a DNA methylation kit for quantitative detection of RPRM gene comprising: a DNA conversion reagent component (heavy sulfite conversion reaction solution), a DNA purification reagent component (a glass fiber membrane spin column, a binding buffer, a rinse buffer, Desulfurization buffer, elution buffer), methylation detection reagent component (2 ⁇ SYBR GREEN I premixed reaction solution, primers RPRM-F and RPRM-R, methylation positive control plasmid SM and non-methylation positive Control plasmid SU).
  • a DNA conversion reagent component heavy sulfite conversion reaction solution
  • a DNA purification reagent component a glass fiber membrane spin column, a binding buffer, a rinse buffer, Desulfurization buffer, elution buffer
  • methylation detection reagent component 2 ⁇ SYBR GREEN I premixed reaction solution, primers RPRM-F and RPRM-R, methylation positive control plasmid SM and non-methylation
  • the bisulfite conversion reaction solution contains 5 mol/L NaHSO3, 12.5 wt.% ethylene glycol dimethyl ether (DME), pH 5.0.
  • the binding buffer contains 6 mol/L guanidine hydrochloride; the rinsing buffer contains 10 mmol/L Tris-Cl, pH 6.8, volume fraction 80% ethanol; and the desulfurization buffer contains 200 mmol/ NaOH, 100 mmol/L NaCl, volume fraction 30% ethanol, volume fraction 5% glycerol; elution buffer containing 10 mmol/L Tris-Cl, 1 mmol/L EDTA, pH 8.0.
  • the primer RPRM-F is: 5'-GTTTTAGAAGAGTTTAGTTGTTG-3'; RPRM-R is 5'-CTACTATTAACCAAAAACAAAC-3'.
  • the 2 ⁇ SYBR GREEN I premixed reaction solution contains 2 mmol/L MgCl2, 100 mmol/L Tris-Cl, 1 enzyme activity unit/ ⁇ L Taq DNA polymerase, 5 mmol/L dNTPs, 2 ⁇ SYBR GREEN I dye and 2 ⁇ ROX I calibration dye.
  • 2 ⁇ SYBR GREEN I dye and 2 ⁇ ROX I calibration dye were purchased from Beijing Bolingke Biotechnology Co., Ltd.
  • a method for quantitatively detecting DNA methylation of RPRM gene including the following:
  • a 0.2 ml PCR tube add 20 ⁇ l of DNA aqueous solution containing 1 ng to 1 ⁇ g of DNA, and then mix with 80 ⁇ l of the conversion reaction solution; place the PCR tube in a thermocycler for the following reaction: 95 ° C Pre-denaturation for 3 minutes; [95 ° C for 30 seconds, 70 ° C for 10 minutes] 12 cycles.
  • the PCR tube was placed in a fluorescence quantitative PCR machine to carry out the following reaction: amplification stage: 95 ° C for 30 seconds pre-denaturation; [95 ° C 5 seconds, 56 ° C 15 seconds, 72 ° C 30 seconds] a total of 40 cycles; melting curve stage : 95 ° C 15 seconds pre-denaturation, then read the fluorescence value from 72 ° C -88 ° C every 0.1 ° C increase, the results obtained by melting curve analysis to determine the RPRM gene promoter specific DNA sequence methylation in the sample Degree.
  • the invention has the advantages that: by constructing a positive control standard plasmid, a real-time PCR method and a melting curve analysis, the bisulfite conversion PCR method is optimized, and the methylation-sensitive melting curve analysis is performed to reach the DNA. Methylation is used for quantitative detection purposes.
  • the method can be used not only for quantitative detection of DNA methylation of RPRM gene, but also for quantitative detection of DNA methylation of other genes, and eliminates the steps of DNA sequencing, and the degree of methylation can be quantitatively analyzed, and the operation is relatively simple. The lower cost is ideal for high-throughput analysis of a large number of samples in the future.
  • Figure 2 uses S M as a standard DNA sample, which is sequentially diluted to 1 ⁇ 10 -1 nanomoles / liter, 1 ⁇ 10 -2 nanomoles / liter, 1 ⁇ 10 -3 nanomoles / liter, 1 ⁇ 10 -4 nanomolar / l, 1 ⁇ 10 -5 nmol / l concentration by qPCR amplification Ct value in the ordinate, a standard curve generated for the value of the concentration as abscissa.
  • Figure 3 shows the conversion and degradation of S M DNA by different bisulfites.
  • A conversion of S M with mixed sulfite, NaHSO 3 , and Na 2 S 2 O 5 for 2 hours and 6 hours;
  • B mixed sulfite, NaHSO 3 , and Na 2 S 2 O 5 was converted to S M and the rate of DNA degradation after 6 hours of reaction.
  • Figure 4 Effect of high temperature heating conditions on S M DNA transformation.
  • A the effect of different high temperature heating conditions on DNA conversion rate;
  • B the effect of different high temperature heating conditions on DNA degradation rate.
  • the reaction conditions were: a, [95 ° C for 5 minutes, 60 ° C for 30 minutes] 6 cycles; b, 95 ° C for 3 minutes, [95 ° C for 30 seconds, 60 ° C for 10 minutes] 18 cycles; c, [95 ° C10 Seconds, 60 ° C for 10 minutes] 18 cycles; d, 95 ° C for 3 minutes, 60 ° C for 3 hours.
  • Figure 5 Effect of transformation reaction temperature on S M DNA transformation.
  • A the effect of different transformation temperatures on DNA conversion rate
  • B the effect of different transformation temperatures on DNA degradation rate.
  • the reaction conditions were: e, 95 ° C for 3 minutes, [95 ° C for 30 seconds, 60 ° C for 10 minutes] 18 cycles; f, 95 ° C for 3 minutes, [95 ° C for 30 seconds, 70 ° C for 10 minutes] 18 cycles; , 95 ° C for 3 minutes, [95 ° C 30 seconds, 80 ° C for 10 minutes] 18 cycles; h, 95 ° C for 3 minutes, [95 ° C 30 seconds, 90 ° C for 10 minutes] 18 cycles.
  • FIG. 1 Effect of denaturant on S M DNA transformation.
  • A the effect of different denaturants on DNA conversion;
  • B the effect of different denaturants on DNA degradation rate.
  • FIG. 7 Effect of denaturant on genomic DNA transformation of AGS cell lines.
  • A the promotion of AGS DNA transformation by different denaturants within 2 hours of reaction;
  • B the effect of different denaturants on the final copy number of the target sequence within 2 hours of the reaction.
  • Figure 8 Effect of denaturant on PBMC genomic DNA transformation.
  • A the promotion of PBMC DNA transformation by different denaturants within 2 hours of reaction;
  • B the effect of different denaturants on the final obtained copy number of the target sequence within 2 hours of the reaction.
  • FIG. 1 Comparison of optimized formulations, Qiagen kits and traditional transformation methods for S M DNA transformation.
  • A DME's bisulfite solution, Qiagen kit and traditional conversion method for S M DNA conversion
  • B DME's bisulfite solution, Qiagen kit and traditional transformation method for degradation of S M DNA rate.
  • Figure 10 compares the optimized formulation, Qiagen kit and traditional transformation methods for BGC-823 DNA transformation.
  • A the addition of DME bisulfite solution, Qiagen kit and traditional transformation method for the conversion of BGC-823 DNA
  • B the addition of DME bisulfite solution, Qiagen kit and traditional transformation method to convert 1 microgram BGC-823DNA The target sequence copy number obtained.
  • Figure 11 compares the optimized formulation, Qiagen kit and traditional transformation methods for PBMC DNA transformation.
  • A the addition of DME bisulfite solution, Qiagen kit and traditional transformation method for PBMC DNA transformation;
  • B DME addition of bisulfite solution, Qiagen kit and traditional transformation method to convert 1 microgram of PBMC DNA obtained Target sequence copy number.
  • Figure 12 Peak shape of RPRM gene melting curve in 20 PBMC samples.
  • Figure 13 is a standard curve showing the relationship between methylated amplification products of mixed samples and initial methylated DNA content.
  • Figure 14 is an example of RPRM methylation analysis of plasma DNA samples in normal and gastric cancer patients.
  • A 5 normal human plasma DNA samples RPRM methylation test results;
  • B 5 gastric cancer patients plasma DNA samples RPRM methylation test results.
  • Quantitative detection of RPRM gene DNA methylation kit including glass fiber membrane spin column, conversion reaction solution (5 mol/L NaHSO3, 12.5 wt.% DME, pH 5.0), binding buffer (6 mol/L guanidine hydrochloride) ), rinse buffer (10 mmol/L Tris-Cl pH 6.8, 80% ethanol) desulfurization buffer (200 mmol/L NaOH, 100 mmol/L NaCl, 30% ethanol, 5% glycerol), elution Buffer (10 mmol/L Tris-Cl, 1 mmol/L EDTA, pH 8.0), 2 ⁇ SYBR GREEN I premixed reaction, primer RPRM-F (10 ⁇ mol/L, sequence GTTTTAGAAGAGTTTAGTTGTTG) and RPRM -R (10 micromol/L, sequence CTACTATTAACCAAAAACAAAC), methylation positive control plasmid S M and unmethylated positive control plasmid S U .
  • conversion reaction solution 5
  • a method for quantitatively detecting DNA methylation of a RPRM gene comprising the following:
  • the rinse buffer was then transferred to a new 1.5 ml Eppendorf tube; 30 ⁇ l of elution buffer was added, allowed to stand at room temperature for 2 minutes, and then centrifuged at 12,000 rpm for 1 minute to collect the purified DNA.
  • the primer RPRM-F is: 5'-GTTTTAGAAGAGTTTAGTTGTTG-3'; RPRM-R is 5'-CTACTATTAACCAAAAACAAAC-3'.
  • the melting curve method in step (3) uses two standard plasmids as a reference: the M-seq of the methylated RPRM gene sequence after bisulfite conversion, the sequence is SEQ ID NO. 2, but not methylation After the sequence is transformed, U-seq, the sequence is SEQ ID NO. 3, and then the two sequences are separately cloned into a vector as positive control standard samples S M and S U , and the real-time PCR amplification described in the above (3) is performed. Melting curve analysis.
  • the binding buffer is 6 mol/L guanidine hydrochloride; the rinsing buffer is 10 mmol/L Tris-Cl pH 6.8, 80% ethanol; the desulfurization buffer is 200 mmol/L NaOH, 100 m. Molar/liter NaCl, 30% ethanol, 5% glycerol; elution buffer containing 10 mmol/L Tris-Cl, 1 mmol/L EDTA, pH 8.0.
  • Conversion reaction solution formulation 5 mol/L NaHSO 3 , 12.5% DME, pH 5.0.
  • the standard S M can also be used as a DNA template for bisulfite conversion.
  • There are 30 cytosine sites in the target sequence of S M which are gradually converted to uracil after treatment with bisulfite.
  • the peak shape of S M will gradually transition to the peak shape of S U ; when the conversion rate reaches 100%, the peak shape of MS-MCA will completely coincide with the peak shape of S U .
  • the peak shape of S M and S U , and the peak shape (S X ) of S M in the transformed state are shown in Fig. 1. Therefore, the MS-MCA can be used to detect the product state of S M after bisulfite conversion, thereby selecting the formulation with the best conversion rate and the transformation conditions.
  • the conversion rate is assumed to be an average melting temperature of the conversion product of Tm (X).
  • control group (0.1 ng S M , 0.5 mmol/L hydroquinone, pH 5.0, 100 ⁇ l);
  • the catalytic reaction conditions of bisulfite to cytosine mainly include catalytic reaction temperature (generally 55-60 ° C) and high temperature heating of the solution at different time intervals (to maintain the single-stranded state of DNA).
  • the template was validated by genomic DNA of gastric cancer AGS cell line, gastric cancer BGC-823 cell line and normal human peripheral blood mononuclear cell (PBMC) as template. Since the sequence of genomic DNA can only be recognized by primers after bisulfite conversion, the CT value obtained by qPCR amplification is a comprehensive reflection of the degradation rate and conversion rate of the template. The higher the conversion rate, the lower the degradation rate. The higher the copy number of the final target sequence.
  • PBMC peripheral blood mononuclear cell
  • Control group (0.1 ng S M , 100 ⁇ l);
  • Non-denaturing agent group (0.1 ng S M , 4 mol/L NaHSO 3 , pH 5.0, 100 ⁇ l);
  • Guanidine hydrochloride group (0.1 ng S M , 4 mol/L NaHSO 3 , 150, 300 or 600 mmol/L guanidine hydrochloride, pH 5.0, 100 ⁇ l);
  • DME group (0.1 ng S M , 4 mol/L NaHSO 3 , 5%, 10% or 20% DME, pH 5.0, 100 ⁇ l);
  • reaction conditions were: 95 ° C for 3 minutes, [95 ° C for 30 seconds, 70 ° C for 10 minutes] 18 cycles;
  • Non-denaturing agent group (1 ⁇ g AGS DNA, 4 mol/L NaHSO 3 , pH 5.0, 100 ⁇ l);
  • Guanidine hydrochloride group (1 ⁇ g AGS DNA, 4 mol/L NaHSO 3 , 300 mmol/L guanidine hydrochloride, pH 5.0, 100 ⁇ m
  • reaction conditions were: 95 ° C for 3 minutes, [95 ° C for 30 seconds, 70 ° C for 10 minutes] 12 cycles;
  • Non-denaturing agent group (1 ⁇ g PBMC DNA, 4 mol/L NaHSO 3 pH 5.0, 100 ⁇ l);
  • Guanidine hydrochloride group (1 ⁇ g PBMC DNA, 300 mmol/L guanidine hydrochloride, pH 5.0, 100 ⁇ l);
  • DME group (1 ⁇ g PBMC DNA, 4 mol/L NaHSO 3, 10% DME, pH 5.0, 100 ⁇ l);
  • reaction conditions were: 95 ° C for 3 minutes, [95 ° C for 30 seconds, 70 ° C for 10 minutes] 12 cycles;
  • optimization group The optimized formula and conditions of the laboratory (hereinafter referred to as "optimization group”) Plus Bisulfite Conversion Kit (Qiagen, Germany) (hereinafter referred to as “Qiagen”) commercialization kit, and traditional bisulfite conversion method (hereinafter referred to as "traditional method”) for comparison, testing various conversion methods for S M , BGC -823 and PBMC genomic DNA transformation efficiency.
  • Control group (0.1 ng S M , 100 ⁇ l);
  • c.Qiagen (0.1 ng S M , Bisulfite Mix and DNA Protect Buffer total 140 ⁇ l 0.1 ng);
  • group d Conventional method (0.1 ng S M , 2.6 mol/L NaHSO 3 , 0.5 mmol/L hydroquinone, 100 ⁇ l 0.1 ng); wherein group a does not react, group b reaction conditions are: 95 ° C for 3 minutes, [95 ° C for 30 seconds, 70 ° C for 10 minutes] 12 cycles ( ⁇ 2 hours); Group c reaction conditions according to the kit instructions: 95 ° C for 5 minutes, 60 ° C for 25 minutes, 95 ° C for 5 minutes, 60 ° C 85 minutes, 95 ° C for 5 minutes, 60 ° C for 175 minutes ( ⁇ 5 hours); group d DNA was first denatured at 0.3 mole / liter of NaOH, 37 ° C for 10 minutes, then added traditional formula bisulfite solution to 100 microliters system The reaction was carried out at 50 ° C for 16 hours.
  • the reaction conditions of group a are: 95 ° C for 3 minutes, [95 ° C for 30 seconds, 70 ° C for 10 minutes] 12 cycles ( ⁇ 2 hours); group b reaction conditions according to the kit instructions: 95 ° C for 5 minutes, 60 ° C for 25 minutes , 95 ° C for 5 minutes, 60 ° C for 85 minutes, 95 ° C for 5 minutes, 60 ° C for 175 minutes ( ⁇ 5 hours); Group C DNA was first denatured at 0.3 mole / liter of NaOH, 37 ° C for 10 minutes, and then added to the traditional formula The sulfate solution was passed to a 100 microliter system and reacted at 50 ° C for 16 hours.
  • the results show that the conversion results of the optimized bisulfite reaction solution to the genomic DNA of the BGC-823 cell line in the 2-hour reaction time are comparable to those of the Qiagen kit at 5 hours and the conventional method at 16 hours. This indicates that this formulation can complete the conversion of methylated DNA samples in a short time. And the target sequence copy number obtained by transforming 1 microgram of BGC-823 DNA by this method is higher than that of the Qiagen kit.
  • reaction conditions of group a are: 95 ° C for 3 minutes, [95 ° C for 30 seconds, 70 ° C for 10 minutes] 12 cycles ( ⁇ 2 hours); Group b reaction conditions according to the kit instructions: 95 ° C for 5 minutes, 60 ° C for 25 minutes, 95 ° C for 5 minutes, 60 ° C for 85 minutes, 95 ° C for 5 minutes, 60 ° C for 175 minutes ( ⁇ 5 hours); Group c DNA first at 0.3 Mole / liter of NaOH, denaturation at 37 ° C for 10 minutes, then add the traditional formulation of bisulfite solution to 100 microliters of system, reaction at 50 ° C for 16 hours. The results (Fig.
  • MS-MCA Real-time quantitative PCR amplification and melting curve analysis of methylation sensitivity
  • the PCR tube was placed in a fluorescence quantitative PCR machine to carry out the following reaction: amplification stage: 95 ° C for 30 seconds pre-denaturation; [95 ° C 5 seconds, 56 ° C 15 seconds, 72 ° C 30 seconds] a total of 40 cycles; melting curve stage Pre-denaturation at 95 ° C for 15 seconds, then read the fluorescence value from 0.1 ° C per 72 ° C - 88 ° C, and the results obtained were analyzed by melting curve method to determine the specificity of the RPRM gene promoter in the plasma sample. The degree of methylation of the DNA sequence.
  • PBMC DNA sample can represent unmethylated DNA sample.
  • Analysis of 20 PBMC DNA samples revealed that the unmethylated peaks were between 77 ° C and 81 ° C ( Figure 12).
  • the peak shape of some samples is close to but not completely consistent with the S U control, which may be due to the hypomethylation or single nucleotide polymorphism between different individuals, or the level of amplification products, which may lead to melting curves. A slight drift.
  • the sample is determined to be an unmethylated sample; when the peak shape partially or completely exceeds the non-methyl group When the interval is normalized, it is determined that the sample has a certain degree of methylation.
  • the melting curve has only one peak type, so it is easy to judge whether the sample has Methylation.
  • mixed DNA sample types such as surgically resected tumor tissue samples (mixed normal type cells and tumor cells), tumor patient plasma DNA samples (mixed normal blood cells and tumor cell-derived DNA)
  • the melting curve may appear two or even More peak types. In most cases, the signals of multiple peak types are more obvious, and the sample can be judged to be a mixed sample of methylated and unmethylated. However, in some cases, the signal of the methylated peak is not significant. In this case, an indicator is needed to discriminate between these samples.
  • the fluorescence signal of DNA is proportional to the abundance of PCR amplification products. As the temperature increases, the DNA double strands unwind and the fluorescence signal decreases. However, the rate of decline of the fluorescent signal of the amplified product of the methylated and unmethylated samples is different. Fluorescence intensity represents the total amount of DNA that remains in a double-stranded state at a certain temperature. Therefore, if we determine the fluorescence signal of the unmethylated amplification product at 77 ° C to 81 ° C, the non-methylated amplification product can be relatively quantified by integrating the fluorescence signal. Likewise, the product of methylated DNA can be quantified by temperature from 81 ° C to 86 ° C.
  • FIGS 14A and 14B are representative of 5 normal human plasma DNA samples (RPRM unmethylated) and 5 gastric cancer patient plasma DNA samples (RPRM methylation), respectively.

Abstract

本发明提供了用于定量检测RPRM基因DNA甲基化试剂盒及方法，所述试剂盒包含DNA转化试剂组分，DNA纯化试剂组分和甲基化检测试剂组分。

Description

用于定量检测RPRM基因DNA甲基化试剂盒及方法 技术领域
本发明的属于生物技术领域，具体涉及用于定量检测RPRM基因DNA甲基化试剂盒及方法。
背景技术
DNA甲基化作为最基本的表观遗传学调控机制，是肿瘤中最常见的基因分子改变之一，尤其表现在肿瘤抑制基因与错配修复基因上，能导致基因编码区的突变和使基因失活而有利于肿瘤的发展，是癌症发生的重要指标之一。人类不同类型的癌症基因甲基化模式图谱的建立是相关领域研究的一项重要内容。目前已在肺癌、胃癌、头部和颈部癌症、膀胱癌、前列腺癌、白血病、淋巴瘤、和胆囊癌等中发现基因的异常甲基化。
RPRM基因的全称：reprimo,TP53dependent G2arrest mediator candidate，是一种p53肿瘤抑制因子诱导表达的基因，位于染色体2q23位点上，该位点等位基因失衡已被证明与肺癌、乳腺癌和结肠癌等相关联。在许多癌症和肿瘤细胞株的检测中，都发现了RPRM基因的异常甲基化，包括胃癌、前列腺癌、胰腺癌、肺癌、大肠癌、食道癌、胆囊癌、淋巴瘤、白血病、乳腺癌患者。特别是在早期胃癌的血浆样品检测中，只有RPRM有高甲基化频率(>70％)，而正常的对照血浆无明显的经常性甲基化，说明发现于癌症的RPRM甲基化，很少发生在非恶性的组织中。因此，RPRM甲基化作为胃癌早期血清学活检的生物标志物是有潜力的。另一方面，胃癌患者RPRM甲基化检测并与胃黏膜萎缩标记和幽门螺杆菌检测相结合有可能建立起胃癌早期检测的指标。此外，由于RPRM甲基化是胃癌患者的重要指标之一，有可能用于胃癌的预后检测中。目前RPRM甲基化检测已在美国被批准进入临床试验阶段。因此，RPRM基因甲基化，作为癌症诊治中重要的血清分子标志物，可以作为诊断和治疗中的一项重要参考依据。
基因甲基化主要发生于CpG位点的C(即胞嘧啶)上，在重亚硫酸盐的作用下，胞嘧啶将转变成尿嘧啶，而甲基化的胞嘧啶则保持不变，再进一步通过DNA测序分析等方法，区别胞密啶及尿密啶。因此，通过重亚硫酸盐转化可以区别胞嘧啶的甲基化或者非甲基化。本发明，改进了重亚硫酸盐转化的方法，并通过建立甲基化修饰及非修饰内参质粒，配合实时PCR的方法及阈值截取，将重亚硫酸盐转化PCR方法加以优化，进行DNA甲基化敏感的熔解曲线分析，达到对DNA甲基化进行定量检测的目的。该方法不仅可用于定量检测RPRM基因DNA甲基化，也可用于定量检测其他基因DNA甲基化，且免除了DNA测序的步骤， 而且对甲基化的程度可以加以定量分析，操作较为简便，成本较低，非常适合于未来大量样本的高通量分析。
发明内容
本发明的目的在于提供用于定量检测RPRM基因DNA甲基化试剂盒及方法，该方法不仅可用于定量检测RPRM基因DNA甲基化，也可用于定量检测其他基因DNA甲基化，且免除了DNA测序的步骤，而且对甲基化的程度可以加以定量分析，操作较为简便，成本较低，非常适合于未来大量样本的高通量分析。
为实现上述目的，本发明采用如下技术方案：
用于定量检测RPRM基因DNA甲基化试剂盒，包含：DNA转化试剂组分(重亚硫酸盐转化反应液)，DNA纯化试剂组分(玻璃纤维膜离心柱，结合缓冲液，漂洗缓冲液、脱硫缓冲液，洗脱缓冲液)，甲基化检测试剂组分(2×SYBR GREEN I预混反应液，引物RPRM-F与RPRM-R，甲基化阳性对照质粒SM及非甲基化阳性对照质粒SU)。
用于定量检测RPRM基因DNA甲基化的方法，所述方法包括如下：
(1)在0.2毫升的PCR管中，加入含有1纳克至1微克DNA的20微升DNA水溶液，然后加入80微升重亚硫酸盐转化反应液，进行DNA转化反应；将上述转化反应后100微升的DNA溶液，进行如下纯化处理。
(2)将上述转化反应后100微升溶液，与500微升结合缓冲液进行混合，通过玻璃纤维膜离心柱收集转化后的DNA，并用漂洗缓冲液进行清洗；离心弃去漂洗缓冲液，加入脱硫缓冲液进行脱硫处理，用漂洗缓冲液再清洗两次；离心弃去漂洗缓冲液，然后用30微升洗脱缓冲液收集玻璃纤维膜离心柱上经处理后的DNA。
(3)取上述纯化后的DNA溶液1微升转移到0.1毫升的PCR管中，在含1×SYBR GREEN I预混反应液，0.25微摩尔/升RPRM-F、RPRM-R引物的反应液中进行实时聚合酶链式反应扩增；条件如下：扩增阶段：95℃、30秒；[95℃、5秒，56℃、15秒，72℃、30秒]40个循环；熔解曲线阶段：95℃、15秒，然后从72℃-88℃每升高0.1℃读取一次荧光值，将得到的结果进行熔解曲线法分析，以确定样本中RPRM基因启动子特异DNA序列甲基化的程度。
步骤(3)中的熔解曲线法分析使用两种标准质粒作为参照：甲基化的RPRM基因序列经重亚硫酸盐转化后的M-seq，序列为SEQ ID NO.2，而非甲基化序列转化后U-seq，序列为SEQ ID NO.3，然后分别将这两种序列克隆至载体上作为阳性对照标准样品SM和SU，进行上述(3)所描述的实时PCR扩增及熔解曲线分析。
具体为：
用于定量检测RPRM基因DNA甲基化试剂盒，包含：DNA转化试剂组分(重亚硫酸盐转化反应液)，DNA纯化试剂组分(玻璃纤维膜离心柱，结合缓冲液，漂洗缓冲液、脱硫缓冲液，洗脱缓冲液)，甲基化检测试剂组分(2×SYBR GREEN I预混反应液，引物RPRM-F与RPRM-R，甲基化阳性对照质粒SM及非甲基化阳性对照质粒SU)。
所述DNA转化试剂组分中，重亚硫酸盐转化反应液含5摩尔/升NaHSO3，12.5wt.％乙二醇二甲醚(DME)，pH 5.0。
所述DNA纯化试剂组分中，结合缓冲液含6摩尔/升盐酸胍；漂洗缓冲液含10毫摩尔/升Tris-Cl，pH 6.8，体积分数80％乙醇；脱硫缓冲液含200毫摩尔/升NaOH，100毫摩尔/升NaCl，体积分数30％乙醇，体积分数5％甘油；洗脱缓冲液含10毫摩尔/升Tris-Cl，1毫摩尔/升EDTA，pH 8.0。
所述的引物RPRM-F为：5’-GTTTTAGAAGAGTTTAGTTGTTG-3’；RPRM-R为5’-CTACTATTAACCAAAAACAAAC-3’。
所述的2×SYBR GREEN I预混反应液含有2毫摩尔/升MgCl2、100毫摩尔/升Tris-Cl，1个酶活力单位/微升Taq DNA聚合酶，5毫摩尔/升dNTPs，2×SYBR GREEN I染料和2×ROX I校准染料。其中2×SYBR GREEN I染料、2×ROX I校准染料购买自北京博凌科为生物科技有限公司。
用于定量检测RPRM基因DNA甲基化的方法，包括如下：
(1)DNA重亚硫酸盐转化反应：
在0.2毫升的PCR管中，加入含有1纳克至1微克DNA的20微升DNA水溶液，然后与80微升转化反应液混匀；将PCR管置于热循环仪中进行如下反应：95℃3分钟预变性；[95℃30秒，70℃10分钟]12个循环。
(2)DNA纯化处理：
将上述转化反应后100微升溶液，与500微升结合缓冲液进行混合，然后全部转移至玻璃纤维膜离心柱中，12000转/秒转速离心1分钟；弃去流出液，加入500微升漂洗缓冲液，12000转/秒转速离心1分钟；弃去流出液，加入250微升脱硫缓冲液，室温放置10分钟，12000转/秒转速离心1分钟；弃去流出液，加入500微升漂洗缓冲液，12000转/秒转速离心1分钟，然后再重复此步骤一次；12000转/秒转速离心2分钟彻底甩去残留的漂洗缓冲液，然后将离心柱转移至新的1.5毫升Eppendorf管中；加30微升洗脱缓冲液，室温放置2分钟，然后12000转/秒转速离心1分钟收集纯化后的DNA。
(3)PCR反应及MS-MCA(Methylation-sensitive Melt Curve Analysis)分析：
在0.1毫升的荧光定量PCR管中，加入8微升超纯水，10微升2×SYBR GREEN I预混反应液，浓度为10微摩尔/升RPRM-F和RPRM-R引物各0.5微升，最后加入1微升上述纯化的DNA，混匀。然后将PCR管置于荧光定量PCR仪中进行如下反应：扩增阶段：95℃30秒预变性；[95℃5秒，56℃15秒，72℃30秒]共40个循环；熔解曲线阶段：95℃15秒预变性，然后从72℃-88℃每升高0.1℃读取一次荧光值，将得到的结果进行熔解曲线法分析，以确定样本中RPRM基因启动子特异DNA序列甲基化的程度。
(4)阳性对照：
在上述(3)的分析中，需要在另外的荧光定量PCR管中使用两种标准质粒SM和SU作为参照。人基因组RPRM靶基因序列(SEQ ID No.1,Ref-seq)，完全甲基化的基因组DNA经重亚硫酸盐转化后相应的序列(SEQ ID No.2,M-seq)，以及非甲基化的基因组转化后相应的序列(SEQ ID No.3,U-seq)如下：
SEQ ID No.1,Ref-seq：
Figure PCTCN2016113680-appb-000001
SEQ ID No.2,M-seq，SM：
Figure PCTCN2016113680-appb-000002
SEQ ID No.3,U-seq，SU：
Figure PCTCN2016113680-appb-000003
将上述M-seq和U-seq两种序列分别克隆至
Figure PCTCN2016113680-appb-000004
载体(Promega，USA)上作为阳性对照标准样品SM和SU，与样品进行上述(3)所描述的实时PCR扩增及熔解曲线分析。引物RPRM-F与RPRM-R可对SM和SU进行无差异的扩增，但在熔解曲线阶段将区分为完全甲基化和非甲基化的标准峰型。
本发明的优点在于：通过构建阳性对照标准质粒，配合实时PCR的方法及熔解曲线分析，将重亚硫酸盐转化PCR方法加以优化，同时通过甲基化敏感的熔解曲线分析，达到对DNA 甲基化进行定量检测的目的。该方法不仅可用于定量检测RPRM基因DNA甲基化，也可用于定量检测其他基因DNA甲基化，且免除了DNA测序的步骤，而且对甲基化的程度可以加以定量分析，操作较为简便，成本较低，非常适合于未来大量样本的高通量分析。
附图说明
图1SM的转化态峰型示例。其中，SM质粒经重亚硫酸盐的转化以及MS-MCA所得到的峰型如图中带符号标记曲线所示(SX)。左右两个实线曲线分别为SU和SM阳性对照质粒产生的峰型。
图2以SM作为标准DNA样品，依次稀释为1×10-1纳摩尔/升，1×10-2纳摩尔/升，1×10-3纳摩尔/升，1×10-4纳摩尔/升，1×10-5纳摩尔/升浓度，经qPCR扩增，以Ct值为纵坐标，浓度的对数值为横坐标生成标准曲线。
图3不同重亚硫酸盐对SM DNA的转化及降解情况。A，用混合亚硫酸盐，NaHSO3，以及Na2S2O5对SM进行转化，反应2小时及6小时的转化率；B，用混合亚硫酸盐，NaHSO3，以及Na2S2O5对SM进行转化，反应6小时后的DNA降解率。
图4高温加热条件对SM DNA转化影响。A，不同的高温加热条件对DNA转化率的影响；B，不同的高温加热条件对DNA降解率的影响。其中，反应条件分别为：a，[95℃5分钟，60℃30分钟]6循环；b，95℃3分钟，[95℃30秒，60℃10分钟]18循环；c，[95℃10秒，60℃10分钟]18循环；d，95℃3分钟，60℃3小时。
图5转化反应温度对SM DNA转化影响。A，不同的转化温度对DNA转化率的影响；B，不同的转化温度对DNA降解率的影响。其中，反应条件分别为：e，95℃3分钟，[95℃30秒，60℃10分钟]18循环；f，95℃3分钟，[95℃30秒，70℃10分钟]18循环；g，95℃3分钟，[95℃30秒，80℃10分钟]18循环；h，95℃3分钟，[95℃30秒，90℃10分钟]18循环。
图6变性剂对SM DNA转化影响。A，不同的变性剂对DNA转化率的影响；B，不同的变性剂对DNA降解率的影响。
图7变性剂对AGS细胞系基因组DNA转化影响。A，反应2小时内不同的变性剂对AGS DNA转化的促进作用；B，反应2小时内不同的变性剂对最终获得的靶序列拷贝数的影响。
图8变性剂对PBMC基因组DNA转化影响。A，反应2小时内不同的变性剂对PBMC DNA转化的促进作用；B，反应2小时内不同的变性剂对最终获得的靶序列拷贝数的影响。
图9优化配方，Qiagen试剂盒及传统转化方法对于SM DNA转化的比较。A，加入DME的重亚硫酸盐溶液，Qiagen试剂盒及传统转化方法对SM DNA的转化率；B，加入DME的重亚 硫酸盐溶液，Qiagen试剂盒及传统转化方法对SM DNA的降解率。
图10优化配方，Qiagen试剂盒及传统转化方法对于BGC-823DNA转化的比较。A，加入DME的重亚硫酸盐溶液，Qiagen试剂盒及传统转化方法对BGC-823DNA的转化情况；B，加入DME的重亚硫酸盐溶液，Qiagen试剂盒及传统转化方法转化1微克BGC-823DNA获得的靶序列拷贝数。
图11优化配方，Qiagen试剂盒及传统转化方法对于PBMC DNA转化的比较。A，加入DME的重亚硫酸盐溶液，Qiagen试剂盒及传统转化方法对PBMC DNA的转化情况；B，加入DME的重亚硫酸盐溶液，Qiagen试剂盒及传统转化方法转化1微克PBMC DNA获得的靶序列拷贝数。
图12 20例PBMC样本RPRM基因熔解曲线峰型。
图13混合样本的甲基化扩增产物与初始甲基化DNA含量关系的标准曲线。
图14正常人及胃癌患者血浆DNA样本RPRM甲基化分析示例。A，5例正常人血浆DNA样本RPRM甲基化检测结果；B，5例胃癌患者血浆DNA样本RPRM甲基化检测结果。
具体实施方式
用于定量检测RPRM基因DNA甲基化试剂盒，包含玻璃纤维膜离心柱，转化反应液(5摩尔/升NaHSO3、12.5wt.％DME，pH 5.0)，结合缓冲液(6摩尔/升盐酸胍)，漂洗缓冲液(10毫摩尔/升Tris-Cl pH 6.8，80％乙醇)脱硫缓冲液(200毫摩尔/升NaOH、100毫摩尔/升NaCl、30％乙醇、5％甘油)，洗脱缓冲液(10毫摩尔/升Tris-Cl、1毫摩尔/升EDTA，pH 8.0)，2×SYBR GREEN I预混反应液，引物RPRM-F(10微摩尔/升，序列为GTTTTAGAAGAGTTTAGTTGTTG)与RPRM-R(10微摩尔/升，序列为CTACTATTAACCAAAAACAAAC)，甲基化阳性对照质粒SM及非甲基化阳性对照质粒SU。
用于定量检测RPRM基因DNA甲基化的方法，所述方法包括如下：
(1)在0.2毫升的PCR管中，加入含有1纳克至1微克DNA的20微升DNA水溶液，然后加入80微升含5摩尔/升NaHSO3、12.5％DME，pH 5.0的转化反应液，混匀，使转化反应液终浓度达到4摩尔/升NaHSO3、10％DME，pH 5.0的条件；反应条件为：95℃3分钟；接着，95℃30秒，70℃10分钟，共12个循环；将上述转化反应后100微升的DNA溶液，进行如下纯化处理。
(2)将上述转化反应后100微升溶液，与500微升结合缓冲液进行混合，然后全部转移至玻璃纤维膜离心柱中，12000转/秒转速离心1分钟；弃去流出液，加入500微升漂洗缓冲液，12000转/秒转速离心1分钟；弃去流出液，加入250微升脱硫缓冲液，室温放置10分钟， 12000转/秒转速离心1分钟；弃去流出液，加入500微升漂洗缓冲液，12000转/秒转速离心1分钟，然后再重复此步骤一次；12000转/秒转速离心2分钟彻底甩去残留的漂洗缓冲液，然后将离心柱转移至新的1.5毫升Eppendorf管中；加30微升洗脱缓冲液，室温放置2分钟，然后12000转/秒转速离心1分钟收集纯化后的DNA。
(3)取上述纯化后的DNA溶液1微升转移到0.1毫升的PCR管中，加入10微升2×SYBR GREEN I预混反应液(2毫摩尔/升MgCl2、100毫摩尔/升Tris-Cl，1个酶活力单位/微升Taq DNA聚合酶，5毫摩尔/升dNTPs，2×SYBR GREEN I染料和2×ROX I校准染料)，10微摩尔/升引物RPRM-F、RPRM-R各0.5微升，补足超纯水至20微升，然后在PCR仪器上进行实时聚合酶链式反应扩增；条件如下：扩增阶段：95℃、30秒；[95℃、5秒，56℃、15秒，72℃、30秒]40个循环；熔解曲线阶段：95℃、15秒，然后从72℃-88℃每升高0.1℃读取一次荧光值，将得到的结果进行熔解曲线法分析，以确定样本中RPRM基因启动子特异DNA序列甲基化的程度。
所述的引物RPRM-F为：5’-GTTTTAGAAGAGTTTAGTTGTTG-3’；RPRM-R为5’-CTACTATTAACCAAAAACAAAC-3’。
步骤(3)中的熔解曲线法分析使用两种标准质粒作为参照：甲基化的RPRM基因序列经重亚硫酸盐转化后的M-seq，序列为SEQ ID NO.2，而非甲基化序列转化后U-seq，序列为SEQ ID NO.3，然后分别将这两种序列克隆至载体上作为阳性对照标准样品SM和SU，进行上述(3)所描述的实时PCR扩增及熔解曲线分析。
所述的结合缓冲液为6摩尔/升盐酸胍；所述漂洗缓冲液为10毫摩尔/升Tris-Cl pH 6.8、80％乙醇；所述脱硫缓冲液为200毫摩尔/升NaOH、100毫摩尔/升NaCl、30％乙醇、5％甘油；洗脱缓冲液含10毫摩尔/升Tris-Cl、1毫摩尔/升EDTA，pH 8.0。
实施例1
1.转化反应液配方：5摩尔/升NaHSO3、12.5％DME，pH 5.0。
2.转化反应条件：95℃3min，[95℃30秒，70℃10分钟]12循环。
3.DNA模板
标准品SM除了可以作为MS-MCA时甲基化峰型的阳性对照外，还可作为进行重亚硫酸盐转化的DNA模板。SM的靶序列中具有30个胞嘧啶位点，经过重亚硫酸盐的处理这些胞嘧啶将逐渐转化成尿嘧啶。随着转化的进行，SM的峰型将逐渐向SU的峰型进行过渡；当转化率达100％时，其MS-MCA的峰型将完全与SU的峰型重合。SM和SU的峰型，以及处于转化态的SM的峰型(SX)如图1所示。因此可以通过MS-MCA对SM经重亚硫酸盐转化后的产 物状态进行检测，从而挑选出转化率最优的配方及转化条件。
4.C-T转化率计算
SM和SU的熔解温度分别由10次独立的qPCR扩增及MS-MCA确定，分别为Tm(SM)＝84.21±0.09℃和Tm(SU)＝79.12±0.10℃。当以SM为模板进行转化时，假设转化产物的平均熔解温度为Tm(X)，则转化率
Figure PCTCN2016113680-appb-000005
5.模板降解率计算
因为重亚硫酸盐的转化将不可避免的造成DNA的断裂及降解，因此DNA的降解情况也是衡量重亚硫酸盐溶液配方及转化条件优劣的重要指标。以SM作为标准DNA样品，依次稀释为1×10-1纳摩尔/升、1×10-2纳摩尔/升、1×10-3纳摩尔/升、1×10-4纳摩尔/升、1×10-5纳摩尔/升浓度，经qPCR扩增，以Cycle Threshold(Ct)值为纵坐标，浓度的对数值为横坐标生成标准曲线(图2)。当以SM为模板进行转化时，经过qPCR分别得到转化前后的Ct值，通过标准曲线可分别求得转化前以及转化后靶序列的拷贝数C1和C2，则降解率
Figure PCTCN2016113680-appb-000006
6.改进的条件
1)加入变性剂DME促进转化；
2)减小反应体系至100微升；
3)反应时间缩短至2小时。
7.与其它方法的对比优势：
  优化方法 QIAGEN试剂盒 传统方法
添加剂 10％DME DNA protect buffer 0.5毫摩尔/升对苯二酚
反应体系 100微升 140微升 500微升
转化时间 2小时 5小时 16–18小时
转化率 高 中 低
DNA降解 中 中 高
实施例2不同重亚硫酸盐的比较
比较不同的重亚硫酸盐，包括NaHSO3，Na2S2O5，以及NaHSO3与Na2S2O5不同比例的混合亚硫酸盐之间的转化效率。
实验设计：对照组(0.1纳克SM，0.5毫摩尔/升对苯二酚，pH 5.0，100微升)；
混合亚硫酸盐(0.1纳克SM，0.052克混合亚硫酸盐、0.5毫摩尔/升对苯二酚，pH 5.0，100微升)；
NaHSO3(0.1纳克SM，4摩尔/升NaHSO3、0.5毫摩尔/升对苯二酚，pH 5.0，100微升)；
Na2S2O5(0.1纳克SM，2摩尔/升Na2S2O5、0.5毫摩尔/升对苯二酚，pH 5.0，100微升)；
上述转化反应条件均为：[95℃5分钟，58℃30分钟]4个循环或者12个循环。
不同重亚硫酸盐对SM的转化及降解情况见图3A及3B。其中，纯NaHSO3在相同条件下对DNA的转化效率较高，对DNA造成的降解程度稍低。
实施例3不同温度反应条件对转化效率的影响
重亚硫酸盐对胞嘧啶的催化反应条件主要包括催化反应温度(一般为55～60℃)及以不同时间间隔对溶液进行高温加热(以维持DNA的单链状态)两个方面。
1)高温加热条件的影响
实验设计：0.1纳克SM，4摩尔/升NaHSO3，pH 5.0，100微升。
取4组上述溶液分别进行如下反应(～3小时)：
a.[95℃5分钟，60℃30分钟]6循环；
b.95℃3分钟，[95℃30秒，60℃10分钟]18循环；
c.[95℃10秒，60℃10分钟]18循环；
d.95℃3分钟，60℃3小时。
结果(图4)显示高温加热时间越长，转化率相对提高，但同时DNA的降解程度也越高。
2)转化反应温度的影响
实验设计：0.1纳克SM，4摩尔/升NaHSO3，pH 5.0，100微升。
取4组上述溶液分别进行如下反应(～3小时)：
e.95℃3分钟，[95℃30秒，60℃10分钟]18循环；
f.95℃3分钟，[95℃30秒，70℃10分钟]18循环；
g.95℃3分钟，[95℃30秒，80℃10分钟]18循环；
h.95℃3分钟，[95℃30秒，90℃10分钟]18循环；
结果(图5)显示转化反应温度越高，转化率相对提高，但降解率也相对增加。
实施例4、变性剂DME和盐酸胍对反应的影响
模板除SM外，还通过胃癌AGS细胞系，胃癌BGC-823细胞系和正常人外周血单核细胞(Peripheral Blood Mononuclear Cell,PBMC)的基因组DNA为模板验证不同方法的有效性。由于基因组DNA只有经重亚硫酸盐转化后序列才能被引物所识别，因此其经qPCR扩增后获 得的CT值是模板的降解率与转化率的综合反映，转化率越高，降解率越低，则最终靶序列的拷贝数则越高。
1)实验设计(以SM为模板)：
对照组(0.1纳克SM，100微升)；
无变性剂组(0.1纳克SM，4摩尔/升NaHSO3，pH 5.0，100微升)；
盐酸胍组(0.1纳克SM，4摩尔/升NaHSO3，150、300或600毫摩尔/升盐酸胍，pH 5.0，100微升)；
DME组(0.1纳克SM，4摩尔/升NaHSO3，5％、10％或20％DME，pH 5.0，100微升)；
反应条件为：95℃3分钟，[95℃30秒，70℃10分钟]18循环；
结果(图6)显示盐酸胍在较宽浓度范围无论对DNA的转化率或是降解率方面均无明显作用。而DME能相对提高转化率，虽然降解率也相对增加。
2)实验设计(以AGS细胞系基因组DNA为模板)；
无变性剂组(1微克AGS DNA，4摩尔/升NaHSO3，pH 5.0，100微升)；
盐酸胍组(1微克AGS DNA，4摩尔/升NaHSO3，300毫摩尔/升盐酸胍，pH 5.0，100微
升)；DME组(1微克AGS DNA，4摩尔/升NaHSO3，10％DME，pH 5.0，100微升)；
反应条件为：95℃3分钟，[95℃30秒，70℃10分钟]12循环；
结果(图7)显示在反应时间约2小时内盐酸胍无论对DNA的转化率或是降解率方面均无明显作用。而DME能明显的促进转化的进行。
3)实验设计(正常人PBMC细胞基因组DNA为模板)：
无变性剂组(1微克PBMC DNA，4摩尔/升NaHSO3pH 5.0，100微升)；
盐酸胍组(1微克PBMC DNA，300毫摩尔/升盐酸胍，pH 5.0，100微升)；
DME组(1微克PBMC DNA，4摩尔/升NaHSO3，10％DME，pH 5.0，100微升)；
反应条件为：95℃3分钟，[95℃30秒，70℃10分钟]12循环；
结果(图8)显示在反应时间约2小时内各组反应均能转化PBMC DNA，可能是由于重亚硫酸盐对于非甲基化的DNA样本转化能力较强。
实施例5、与商业化试剂盒及传统方法的比较
本实验室优化的配方及条件(以下简称“优化组”)与
Figure PCTCN2016113680-appb-000007
Plus Bisulfite Conversion Kit(Qiagen，Germany)(以下简称“Qiagen”)商业化试剂盒，以及传统重亚硫酸盐转化方法(以下简称“传统方法”)进行比较，测试各种转化方法对SM、BGC-823及PBMC基因组DNA的转化效率。
1)实验设计(以SM为模板)：
a.对照组(0.1纳克SM，100微升)；
b.优化组(0.1纳克SM，4摩尔/升NaHSO3，10％DME，pH 5.0，100微升)；
c.Qiagen(0.1纳克SM，Bisulfite Mix及DNA Protect Buffer共140微升0.1纳克)；
d.传统方法(0.1纳克SM，2.6摩尔/升NaHSO3，0.5毫摩尔/升对苯二酚，100微升0.1纳克)；其中，a组不进行反应，b组反应条件为：95℃3分钟，[95℃30秒，70℃10分钟]12循环(～2小时)；c组反应条件根据试剂盒说明书：95℃5分钟，60℃25分钟，95℃5分钟，60℃85分钟，95℃5分钟，60℃175分钟(～5小时)；d组DNA首先在0.3摩尔/升NaOH，37℃下变性10分钟，然后加入传统配方重亚硫酸盐溶液至100微升体系，50℃下反应16小时。
结果(图9)显示优化的重亚硫酸盐反应液在2小时反应时间内对SM DNA的转化率高于Qiagen试剂盒在5小时反应的转化率，也高于传统方法16小时的转化率。并且加入Trolox的配方对DNA造成的降解率与Qiagen试剂盒或传统方法的降解情况相近。
2)实验设计(以BGC-823细胞系基因组DNA为模板)
a.优化组(1微克BGC-823DNA，4摩尔/升NaHSO3，10％DME，pH 5.0，100微升)；
b.Qiagen(1微克BGC-823DNA，Bisulfite Mix及DNA Protect Buffer共140微升)；
c.传统方法(1微克BGC-823DNA，2.6摩尔/升NaHSO3，0.5毫摩尔/升对苯二酚，100微升)；
其中，a组反应条件为：95℃3分钟，[95℃30秒，70℃10分钟]12循环(～2小时)；b组反应条件根据试剂盒说明书：95℃5分钟，60℃25分钟，95℃5分钟，60℃85分钟，95℃5分钟，60℃175分钟(～5小时)；c组DNA首先在0.3摩尔/升NaOH，37℃下变性10分钟，然后加入传统配方重亚硫酸盐溶液至100微升体系，50℃下反应16小时。
结果(图10)显示优化的重亚硫酸盐反应液在2小时反应时间内对BGC-823细胞系基因组DNA的转化结果与Qiagen试剂盒在5小时和传统方法在16小时反应的转化结果相当，表明此配方能在较短时间内完成对甲基化DNA样本的转化。并且通过此方法转化1微克BGC-823DNA获得的靶序列拷贝数高于Qiagen试剂盒。
3)实验设计(PBMC基因组DNA为模板)
a.优化组(1微克PBMC DNA，4摩尔/升NaHSO3，10％DME，pH 5.0，100微升)；
b.Qiagen(1微克PBMC DNA，Bisulfite Mix及DNA Protect Buffer共140微升)；
c.传统方法(1微克PBMC DNA，2.6摩尔/升NaHSO3，0.5毫摩尔/升对苯二酚，100微升)；
其中，a组反应条件为：95℃3分钟，[95℃30秒，70℃10分钟]12循环(～2小时)； b组反应条件根据试剂盒说明书：95℃5分钟，60℃25分钟，95℃5分钟，60℃85分钟，95℃5分钟，60℃175分钟(～5小时)；c组DNA首先在0.3摩尔/升NaOH，37℃下变性10分钟，然后加入传统配方重亚硫酸盐溶液至100微升体系，50℃下反应16小时。结果(图11)显示优化的重亚硫酸盐反应液在2小时反应时间内对正常人PBMC基因组DNA的转化结果与Qiagen试剂盒在5小时反应和传统方法在16小时的转化结果相当，表明此配方能在较短时间内完成对非甲基化DNA样本的转化。并且通过此方法转化1微克PBMC DNA获得的靶序列拷贝数高于Qiagen试剂盒。
实施例6对血浆游离核酸中RPRM基因DNA甲基化的分析
(一)重亚硫酸盐转化
1.取血清或血浆1毫升，通过酚氯仿抽提方法或商业化试剂盒对DNA进行提取。
2.在0.2毫升的PCR管中，加入20微升提取的DNA溶液，然后加入80微升重亚硫酸盐转化反应液，混匀。
3.将PCR管置于热循环仪中进行如下反应：95℃3分钟预变性；[95℃30秒，70℃10分钟]12个循环。
4.将上述转化反应后100微升溶液，与500微升结合缓冲液进行混合，然后将溶液全部转移至玻璃纤维膜离心柱中，12000转/秒转速离心1分钟。
5.弃去流出液，加入500微升漂洗缓冲液，12000转/秒转速离心1分钟。
6.弃去流出液，加入250微升脱硫缓冲液，室温放置10分钟，然后12000转/秒转速离心1分钟，弃去流出液。
7.加入500微升漂洗缓冲液，12000转/秒转速离心1分钟，弃去流出液。
8.重复上述步骤1次。
9.12000转/秒转速离心2分钟，彻底甩去残留的漂洗缓冲液，然后将离心柱转移至新的1.5毫升EP管中
10.往膜中央滴加30微升洗脱缓冲液，室温放置2分钟，然后12000转/秒转速离心1分钟收集纯化后的DNA。
(二)实时荧光定量PCR扩增及甲基化敏感性的熔解曲线分析(MS-MCA)
1.实时荧光定量PCR扩增
在0.1毫升的荧光定量PCR管中，加入8微升超纯水，10微升2×SYBR GREEN I预混反应液，10微摩尔/升RPRM-F和RPRM-R引物各0.5微升，最后加入1微升上述纯化的DNA，混匀。除待测DNA样本外，还需要使用两种标准质粒SM和SU作为阳性对照，在另 外的PCR管中按相同的方法进行配制。然后将PCR管置于荧光定量PCR仪中进行如下反应：扩增阶段：95℃30秒预变性；[95℃5秒，56℃15秒，72℃30秒]共40个循环；熔解曲线阶段：95℃15秒预变性，然后从72℃-88℃每升高0.1℃读取一次荧光值，将得到的结果按下述方法进行熔解曲线法分析，以确定血浆样本中RPRM基因启动子特异DNA序列甲基化的程度。
2.非甲基化及甲基化峰型区间的确定
我们已经通过重亚硫酸盐测序证实正常人的PBMC中RPRM基因极少发生甲基化，并且其熔解曲线峰型与几乎阳性对照质粒的峰型一致，因此PBMC DNA样本可代表非甲基化的DNA样本。对20例PBMC DNA样本进行分析可发现其非甲基化峰型均介于77℃到81℃温度之间(图12)。有些样本的峰型与SU对照接近但不完全吻合，这可能是因为不同个体之间低甲基化或单核苷酸多态性，或是扩增产物含量高低，都可能会导致熔解曲线轻微的漂移。因此，我们将77-81℃区间作为非甲基化区间，而81-86℃则作为甲基化区间。依此原则，对于单一的DNA样本类型，当其熔解曲线峰型完全被包含于非甲基化区间时，则确定该样本为非甲基化样本；当其峰型部分或完全超出非甲基化区间时，则确定该样本存在一定程度的甲基化。
4.混合样本中甲基化与非甲基化DNA的相对定量分析
对于单一的DNA样本类型，如PBMC基因组DNA(一致非甲基化状态)，细胞系基因组DNA(程度均一的甲基化状态)等，其熔解曲线只有一个峰型，因此很容易判断样本是否具有甲基化。对于混合的DNA样本类型，如手术切除的肿瘤组织样本(混杂正常类型细胞及肿瘤细胞)，肿瘤病人血浆DNA样本(混杂正常血液细胞及肿瘤细胞来源的DNA)，其熔解曲线可能出现两个甚至更多的峰型。大多数情况下多个峰型的信号都较为明显，可以判断样本为甲基化与非甲基化的混合样本。然而，在某些情况下，甲基化峰型的信号不显著。在这种情形下，需要有一种指标来对这些样本进行判别。
DNA的荧光信号与PCR扩增产物的丰度成正比，随着温度的升高，DNA双链解旋，荧光信号下降。但甲基化与非甲基化样本的扩增产物荧光信号的下降速率不同。荧光强度代表着在一定温度下仍然维持双链状态的DNA总量。所以如果我们将非甲基化扩增产物的荧光信号确定在77℃到81℃，通过对荧光信号的积分就可对非甲基化扩增产物进行相对的定量。同样，甲基化DNA的产物可以通过从81℃到86℃的温度进行量化计算。
为了计算混合样品中非甲基化和甲基化DNA各自含量，首先将SM和SU以不同比例混合，扩增产生多组熔解曲线，然后对各组非甲基化与甲基化扩增产物的比例分别进行计算，从而 绘制甲基化扩增产物与初始甲基化DNA含量关系的标准曲线，如图13所示。通过标准曲线，可以计算混合DNA样品中甲基化DNA的含量。从中也可以发现，相同的引物对于甲基化DNA模板有一定的偏好性。这可能是这种方法的另一个优点，即使在DNA池中甲基化DNA极低的水平(甲基化DNA/总DNA＝1/1000)时，其甲基化DNA扩增信号也能被检测到。
5.血浆DNA样品甲基化阈值的确定
正常人血浆DNA中甲基化的DNA含量大致在0.21％到0.35％左右。我们将阈值设定为0.64％。当血浆样品中甲基化的DNA含量高于此阈值时，该样本将被视为甲基化的样本。图14A和14B分别为具有代表性的5例正常人血浆DNA样本(RPRM非甲基化)和5例胃癌患者血浆DNA样本(RPRM甲基化)。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
Figure PCTCN2016113680-appb-000008
Figure PCTCN2016113680-appb-000009
Figure PCTCN2016113680-appb-000010

Claims (6)

  1. 用于定量检测RPRM基因DNA甲基化试剂盒，其特征在于：所述试剂盒包含DNA转化试剂组分：重亚硫酸盐转化反应液，DNA纯化试剂组分：玻璃纤维膜离心柱、结合缓冲液、漂洗缓冲液、脱硫缓冲液、洗脱缓冲液，甲基化检测试剂组分：2×SYBR GREEN I预混反应液、引物RPRM-F与RPRM-R、甲基化阳性对照质粒SM及非甲基化阳性对照质粒SU。
  2. 根据权利要求1所述的用于定量检测RPRM基因DNA甲基化试剂盒，其特征在于：所述DNA转化试剂组分中，重亚硫酸盐转化反应液含5摩尔/升NaHSO3、12.5wt.％乙二醇二甲醚，pH 5.0。
  3. 根据权利要求1所述的用于定量检测RPRM基因DNA甲基化试剂盒，其特征在于：所述DNA纯化试剂组分中，结合缓冲液含6摩尔/升盐酸胍；漂洗缓冲液含10毫摩尔/升Tris-Cl，pH 6.8，体积分数80％乙醇；脱硫缓冲液含200毫摩尔/升NaOH，100毫摩尔/升NaCl，体积分数30％乙醇，体积分数5％甘油；洗脱缓冲液含10毫摩尔/升Tris-Cl，1毫摩尔/升EDTA，pH 8.0。
  4. 根据权利要求1所述的用于定量检测RPRM基因DNA甲基化试剂盒，其特征在于：所述的引物RPRM-F为：5’-GTTTTAGAAGAGTTTAGTTGTTG–3’；RPRM-R为5’-CTACTATTAACCAAAAACAAAC-3’。
  5. 用于定量检测RPRM基因DNA甲基化的方法，其特征在于：所述方法包括如下：
    (1)在0.2毫升的PCR管中，加入含有1纳克至1微克DNA的20微升DNA水溶液，然后加入80微升重亚硫酸盐转化反应液，混匀，使转化反应液终浓度达到4摩尔/升NaHSO3、10wt.％DME，pH 5.0的条件；反应条件为：95℃3分钟；接着，95℃30秒，70℃10分钟，共12个循环；接着将上述转化反应后100微升的DNA溶液，进行如下纯化处理；
    (2)将上述转化反应后100微升溶液，与500微升结合缓冲液进行混合，然后全部转移至玻璃纤维膜离心柱中，12000转/秒转速离心1分钟；弃去流出液，加入500微升漂洗缓冲液，12000转/秒转速离心1分钟；弃去流出液，加入250微升脱硫缓冲液，室温放置10分钟，12000转/秒转速离心1分钟；弃去流出液，加入500微升漂洗缓冲液，12000转/秒转速离心1分钟，然后再重复此步骤一次；12000转/秒转速离心2分钟彻底甩去残留的漂洗缓冲液，然后将离心柱转移至新的1.5毫升Eppendorf管中；加30微升洗脱缓冲液，室温放置2分钟，然后12000转/秒转速离心1分钟收集纯化后的DNA；
    (3)取上述纯化后的DNA溶液1微升转移到0.1毫升的PCR管中，加入10微升2×SYBR GREEN I预混反应液，浓度为10微摩尔/升引物RPRM-F、RPRM-R各0.5微升，补足超纯水至20微升，然后在PCR仪器上进行实时聚合酶链式反应扩增；条件如下：扩增阶段：95℃、 30秒；[95℃、5秒，56℃、15秒，72℃、30秒]40个循环；熔解曲线阶段：95℃、15秒，然后从72℃-88℃每升高0.1℃读取一次荧光值，将得到的结果进行熔解曲线法分析，以确定样本中RPRM基因启动子特异DNA序列甲基化的程度。
  6. 根据权利要求5所述的用于定量检测RPRM基因DNA甲基化的方法，其特征在于：步骤(3)中的熔解曲线法分析使用两种标准质粒作为参照：甲基化的RPRM基因序列经重亚硫酸盐转化后的M-seq，序列为SEQ ID NO.2，而非甲基化序列转化后U-seq，序列为SEQ ID NO.3，然后分别将这两种序列克隆至载体上作为阳性对照标准样品SM和SU，进行上述(3)所描述的实时PCR扩增及熔解曲线分析。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116445583A (zh) * 2023-06-15 2023-07-18 北京起源聚禾生物科技有限公司 一种用于细胞甲基化检测样本前处理试剂盒及使用方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105567850B (zh) * 2016-02-26 2019-03-12 福建师范大学 用于定量检测rprm基因dna甲基化试剂盒及方法
CN108130359A (zh) * 2016-11-29 2018-06-08 生工生物工程（上海）股份有限公司 一种dna甲基化检测试剂盒及其应用
CN107904313A (zh) * 2017-12-29 2018-04-13 韩林志 用于胃癌相关基因Reprimo、RNF180甲基化检测的引物对、试剂盒及方法
CN108103197B (zh) * 2018-02-08 2022-03-04 江苏为真生物医药技术股份有限公司 用于检测rprm基因和prdm5基因甲基化的引物组、试剂、试剂盒及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101285096A (zh) * 2008-06-03 2008-10-15 中国人民解放军第二军医大学 一种快速检测dna甲基化的方法
CN101353695A (zh) * 2007-07-23 2009-01-28 上海市肿瘤研究所 尿沉淀dna甲基化谱式分析诊断膀胱癌的方法和试剂盒
CN105567850A (zh) * 2016-02-26 2016-05-11 福建师范大学 用于定量检测rprm基因dna甲基化试剂盒及方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102021233B (zh) * 2009-09-22 2014-06-04 复旦大学附属华山医院 一种定量检测akap12甲基化水平的方法及其应用
CN102399862B (zh) * 2010-09-16 2016-03-30 苏州承美生物科技有限公司 一种基于解链曲线的甲基化dna检测方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101353695A (zh) * 2007-07-23 2009-01-28 上海市肿瘤研究所 尿沉淀dna甲基化谱式分析诊断膀胱癌的方法和试剂盒
CN101285096A (zh) * 2008-06-03 2008-10-15 中国人民解放军第二军医大学 一种快速检测dna甲基化的方法
CN105567850A (zh) * 2016-02-26 2016-05-11 福建师范大学 用于定量检测rprm基因dna甲基化试剂盒及方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116445583A (zh) * 2023-06-15 2023-07-18 北京起源聚禾生物科技有限公司 一种用于细胞甲基化检测样本前处理试剂盒及使用方法

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