WO2020019700A1 - Réactif de détection, kit et procédé de méthylation de l'adn - Google Patents

Réactif de détection, kit et procédé de méthylation de l'adn Download PDF

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WO2020019700A1
WO2020019700A1 PCT/CN2019/074716 CN2019074716W WO2020019700A1 WO 2020019700 A1 WO2020019700 A1 WO 2020019700A1 CN 2019074716 W CN2019074716 W CN 2019074716W WO 2020019700 A1 WO2020019700 A1 WO 2020019700A1
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methylation
disease
cpg
dna
probe
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Chinese (zh)
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禹汇川
骆衍新
白亮亮
唐冠楠
王小琳
李英杰
黄增鸿
黄美近
汪建平
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Sixth Affiliated Hospital of Sun Yat Sen University
Sun Yat Sen University
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Sixth Affiliated Hospital of Sun Yat Sen University
Sun Yat Sen University
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the invention belongs to the technical field of analysis methods, and relates to a DNA methylation detection reagent, a kit and a method, in particular to a DNA methylation detection reagent, a kit and a method for a flanking sequence without a CpG site.
  • Cytosine-5 DNA methylation present in cancer tissues is considered to be an apparent DNA modification with potential clinical value [1].
  • m5C occurs mainly in CpG dinucleotides. It has been confirmed in a variety of tumors that abnormal methylation of the CpG island (CGI) of the oncogene promoter leads to transcription inactivation [2].
  • CGI in the promoter represents only a small part of methylation, and CpG, which is mainly located in the genome, represents the most conserved DNA methylation target in eukaryotes, but the methylated region in this region The function is unclear.
  • genomic methylation may be a potential therapeutic target in cancer [3].
  • genomic methylation may be a potential mechanism for RNA alternative splicing regulation [4], and can limit transcription initiation, thereby preventing abnormal transcription [5].
  • DMPs differential methylation positions
  • Methylation-specific PCR is an endpoint analysis technique [6,7]; after that, the second generation of PCR-based technology-MethyLight (quantitative MSP) was used for quantitative detection [8] -10].
  • MethyLight technology based on the AluC4 control reaction has been widely used to detect CGI methylation in tissue samples [11,12].
  • the region outside the CpG island in the genome includes regions such as open sea and CpG shore, which are mainly located in the gene body and intergenic regions. There are fewer CG sites in this region, and there are few adjacent CG sites. Design primers and probes required for qMSP technology.
  • the absolute quantification of the methylation level depends on the methylation standard. If you want to obtain the absolute value of the methylation level at a certain site, you must set the 100% methylation standard at the same time, and calculate it by the ratio of the sample and the standard. Percentage of methylation (PM). If the methylation standard production batches are different or there are unavoidable quality defects, such as the CG site is not 100% methylated, the spontaneous deamination of m5C is converted to C, and the DNA is degraded, the absolute value of methylation level will be quantified Serious errors.
  • the current methylation detection is for the detection of methylation sites on CpG islands. Such sites can only be detected unless sequencing (bisulfite pyrosequencing) is used, but it is not used in large sample cohort verification. Cost-effectiveness.
  • the purpose of the present invention is to provide a methylation detection reagent, a kit and a detection method.
  • Another object of the present invention is to provide a DNA methylation detection reagent, a kit and a method for any CpG site on the entire gene sequence.
  • Another object of the present invention is to provide a DNA methylation detection reagent, a kit and a method for a flanking sequence without a CpG site.
  • Another object of the present invention is to provide a DNA methylation detection reagent, a kit and a method that do not require a methylation standard.
  • Another object of the present invention is to provide a DNA methylation detection reagent, a kit and a method with high accuracy, cheapness, convenience, and speed.
  • the present invention provides a method for detecting DNA methylation, the method comprising the following steps:
  • oligonucleotide probe binds to a small groove Binder (minor groove binder, MGB);
  • the method detects that the CpG site is a CpG site of a heteromethylated region, or an isolated CpG site flanked by CpG, or a CpG site of a co-methylated region, or a non-co-methylated region CpG site.
  • the CpG site detected by the method of the present invention is an isolated CpG site flanked by CpG.
  • the CpG site is a CpG site outside the CpG island, or a CpG site within the CpG island.
  • the CpG site is located in a genome, an intergenic region or a promoter.
  • the CpG site is located in the CpG open sea, CpG shore, and CpG shelf of the genome or intergenic region.
  • CpG islands CGI
  • MethyLight methylation-specific quantitative PCR
  • CpG islands CGI
  • Co-methylation is common in adjacent CpG sites.
  • the traditional PCR-based methylation detection technology is based on the assumption that all CG sites in the CpG island are methylated simultaneously or not at the same time, which is called a co-methylated region.
  • the designed primers and probes cover multiple CG sites in this area, and the ratio of simultaneous methylation of these CG sites is detected, not a high-resolution detection technique for a single CG site.
  • CGI co-methylation in the promoter represents only a small part of methylation, and there are still many non-co-methylation
  • the region of methylation, in which some adjacent CG sites are methylated, and some are unmethylated, are called hybrid methylated regions.
  • Heteromethylated regions mainly exist in CpG open sea, shore, shelf, and some CpG islands.
  • isolated CpG sites that are not located in the CGI, but are located in the flanking sequences of the genome and intergenic regions without CpG.
  • existing PCR-based technologies cannot detect such hybrid methylated CpG sites or isolated CpG sites without CpG flanking sequences.
  • the isolated CpG sites without CpG flanks are generally CpG sites located outside the CpG island, such as CpG opensea, CpG shore, and CpG shelf at the genome or intergenic region.
  • step a all unmethylated cytosine bases of the DNA sample are converted into uracil, while the methylated cytosine bases remain unchanged, and the unmethylated Cytosine bases are converted to uracil.
  • the conversion agent is not particularly limited, and all the agents reported in the prior art that can achieve the conversion of cytosine to uracil may be, such as hydrazine, bisulfite, and bisulfite (such as sodium metabisulfite, Potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.).
  • the converting agent is selected from bisulfite.
  • Methylation occurs when a methyl group is added to cytosine. After treatment with a conversion agent such as bisulfite or bisulfite or hydrazine, unmethylated cytosine will become uracil because When performing amplification, uracil is similar to thymine and will be recognized as thymine. In the amplified sequence, cytosine that has not been methylated becomes thymine (C becomes T), and methylated cells Pyrimidine (C) does not change.
  • a conversion agent such as bisulfite or bisulfite or hydrazine
  • the methylated CpG remains CpG (CG), while the unmethylated CpG is deaminated to TpG (TG) after conversion, and then a CG / TG-specific probe is used Binding CpG site.
  • the length of the primer amplification is 50-200 bp.
  • the primer should avoid the CG site: this technique is particularly suitable for CG sites in the genome and intergenic regions, open seas and other regions, there are fewer adjacent CG sites, and the probe covers the CG site to be tested Primers should avoid other CG sites to avoid the effect of hybrid methylation on the detection results.
  • one of the pair of oligonucleotide probes covering the CpG site to be tested specifically binds the CG sequence and the other specifically binds the TG sequence, that is, the pair of probes.
  • the pair of probes one for binding methylated CpG sites and one for binding unmethylated CpG sites.
  • the pair of oligonucleotide probes covering the CpG site to be detected is a pair of Taqman probes.
  • the 5 'end of each Taqman probe is connected to a fluorophore, and the 3' end is connected to a quencher and MGB group.
  • the fluorophores attached to the 5 'ends of the two Taqman probes have different emission light wavelengths.
  • the probe structure is destroyed, and the fluorescence is detected.
  • one of the probes is a TG-Taqman-MGB probe directed to an unmethylated CpG site, and the other is a CG-Taqman-MGB probe directed to a methylated CpG site.
  • the fluorescent group is not particularly limited, and may be selected from the fluorescent groups for probes in the prior art.
  • each probe it may be selected from, for example, FAM, VIC, ROX, TAMRA, One of SYTO9, JOE / TET / HEX, Texas Red, NED / BODIPY / TMR-X, etc., but it is necessary to maintain a pair of probes, each of which has a different fluorophore with a different emission The wavelength of light so that they can be distinguished during fluorescence detection.
  • FAM and SYTO9 have the same emission light wavelength
  • VIC and JOE / TET / HEX have the same emission light wavelength
  • ROX and Texas Red have the same emission light wavelength
  • TAMRA and NED / BODIPY / TMR-X With the same emitted light wavelength, the selection of the above-mentioned fluorophores with the same emitted light wavelength should be avoided. Amplification is shown by the increase and decrease of fluorescence, and the increase and decrease of fluorescence is also directly used for analysis, and the methylation status of the DNA under study can be inferred from the fluorescence signal.
  • the quencher is not particularly limited, and the quencher of the fluorescent group in the prior art may be used, such as one of NFQ, BHQ1, and BHQ2.
  • the MGB group connected at the 3 'end can increase the annealing temperature of the probe.
  • MGB genotyping of DNA polymorphisms.
  • MGB is introduced into the probe in the methylation detection, and the characteristics of the single base mismatch close to MGB, which makes the annealing temperature up to 10 ° C or more, such as 17 ° C, can be fully utilized in a simple way.
  • the methylated and non-methylated CG sites of the location are designed in the region of the probe near the MGB, that is, the detection of non-CpG island methylation sites in the prior art has been overcome for many years.
  • the present invention finds that if the MGB is not connected and the probe annealing temperature reaches 70 ° C, the probe must be very long, especially in non-CpG island areas, where the CG content is low and may grow to 40bp. In such a long probe, the only CG / TG mismatch difference has almost no effect on the annealing temperature, so CG / TG cannot be identified.
  • the number of probe bases is reduced as much as possible: the probe annealing temperature should be 10 degrees higher than the primer annealing temperature to ensure that the binding of the probe to the DNA template occurs before the primer annealing extension.
  • the shorter the probe the more specific the probe is to identify methylated and unmethylated CG sites.
  • the length of the probe should be controlled between 10 and 20 bp. As a more preferred embodiment, the length of the probe should be controlled between 12 and 18 bp.
  • the CpG site to be detected should be located as far as possible in the region 3 'to the middle of the probe.
  • the CpG site is located in a third region near the 3 ′ end of the probe.
  • the single base mismatch in the 3 ′ half region (MGB region) of the probe has an annealing temperature difference of up to 17 ° C, while the single base mismatch in the 5 ′ end region can only produce 2 ⁇ 10 ° C annealing temperature difference. Therefore, the probe at the 3 'end of the CG site to be detected has higher specificity.
  • the 5 'end of the probe should be close to the 3' end of the forward primer, but should not overlap with the forward primer, and can be spaced more than 1 base from the 3 'end of the forward primer.
  • the probe may overlap the reverse primer.
  • the 5 'end of the probe should be as close as possible to the forward primer, which can ensure that the fluorescent group coupled to the 5' end of the probe can extend to the maximum It is cleaved by DNA polymerase and releases fluorescence to ensure the accuracy of detection.
  • the probe bound to the template strand can be efficiently cleaved to avoid premature termination of DNA extension and inaccurate detection.
  • the invention ingeniously designs primers and probes, and adopts MGB-binding probes.
  • genomic DNA is treated with a transforming agent, unmethylated cytosine is converted to uracil (UpG), and methylated cytosine residues Unaffected (mCpG), which results in methylation-dependent sequence differences in genomic DNA.
  • UpG uracil
  • mCpG methylated cytosine residues Unaffected
  • fluorescently labeled TaqMan MGB probes with different emission wavelengths were used to distinguish single-base differences from transformant treatment.
  • the same pair of primers are used to amplify methylated and unmethylated alleles. Sequence discrimination only occurs during the process of hybridization of fluorescent probes. This distinction is based on the complete sequence pairing and error The matching annealing temperature difference ( Figure 1).
  • the present invention uses a fluorescently labeled MGB probe that emits light wavelengths to specifically bind methylated allele sequences, and uses another fluorescently labeled MGB probe that emits light wavelengths to specifically bind unmethylated alleles sequence.
  • the single base mismatch in the 3 'half region (MGB region) of the probe has an annealing temperature difference as high as 17 ° C compared to the perfect pairing. This allows us to design probes to cover only a single CpG dinucleotide, which can measure the methylation level of isolated CpG.
  • the method of the present invention can simultaneously detect methylation of multiple isolated CpG sites.
  • a pair of primers and a pair of oligonucleotide probes covering the CpG site to be tested need to be designed.
  • the primer amplification regions of each CpG site to be tested do not overlap to avoid competition in the multiplex PCR process, and the fluorophore attached to the 5 'end of each probe has a different emission light wavelength.
  • the compatibility of multiple qMSP primers and probe combinations needs to be analyzed: After the primer probe design is completed, the secondary structure that may exist between all primer probes in the multiple qMSP system should be theoretically excluded first.
  • step c) the methylation state in the DNA sample to be tested is analyzed by the methylation percentage parameter PM.
  • the calculation method of the methylation percentage parameter PM is: (methylated fluorescence value / internal reference fluorescence value) test sample / (methylated fluorescence value / internal reference fluorescence value) fully methylated standard
  • the methylation percentage parameter PM is:
  • the present invention more directly expresses the methylation ratio of the sample to be measured, and does not need to use an internal reference reaction and a fully methylated standard, which is more convenient. At the same time, errors caused by the use of internal reference reactions and fully methylated standards are also avoided.
  • the present invention also provides a reagent for detecting DNA methylation, the reagent contains at least one pair of oligonucleotide probes, the probes cover a CpG site, and the probes It is a probe connected to MGB.
  • the reagents of the invention do not contain fully methylated standards.
  • one pair of oligonucleotide probes specifically binds the CG sequence and the other specifically binds the TG sequence.
  • a pair of oligonucleotide probes in the reagent is a pair of Taqman probes, and a 5 'end of each Taqman probe is connected to a fluorophore, and a 3' end is connected to a quencher and an MGB group. And the fluorophores connected at the 5 'ends of the two Taqman probes have different emission wavelengths.
  • the fluorophore may be selected from any two of FAM (or SYTO9), VIC (or JOE / TET / HEX), ROX (or Texas Red), TAMRA (or NED / BODIPY / TMR-X) Species.
  • the quencher is selected from one of NFQ, BHQ1, BHQ2, and the like.
  • the length of the probe is between 10 and 20 bp; more preferably, the length of the probe is between 12 and 18 bp.
  • the CpG site to be tested is located in a region near the 3 'end of the probe; preferably, the CpG site is located in a third region near the 3' end of the probe.
  • the reagent further includes at least a pair of primers; the pair of primers corresponds to a pair of probes.
  • a pair of primers includes a forward primer and a reverse primer
  • a pair of probes includes a CG-binding probe and a TG-binding probe.
  • the pair of probes must be located in the amplicon region of the pair of primers. Consider keeping the probe as close as possible to the forward primer, but not overlapping it.
  • This technology can be applied to multiple PCR systems, using two or more pairs of primers and two or more pairs of probes corresponding to them respectively, to simultaneously detect two or more test sites in a reaction system. Methylation level.
  • the length of the primer amplification is 50-200 bp.
  • the 5 'end of the probe is close to the 3' end of the forward primer.
  • the reagent further contains a transforming agent, and the transforming agent is selected from one or more of hydrazine, bisulfite, and bisulfite; preferably, the transforming agent is selected Since bisulfite.
  • the reagent further contains one or more of a DNA polymerase, dNTPs, Mg 2+ ions, and a buffer solution; preferably, the reagent contains a DNA polymerase, dNTPs, Mg 2+ ions And buffer.
  • the amplification regions of each pair of primers do not overlap, and between the primers and the primers, between the probes and the probes, and between the probes and the Primers should be compatible with each other without secondary structures such as dimers and hairpin-like structures to ensure the efficiency and specificity of multiplex PCR amplification.
  • the present invention also provides a kit for detecting DNA methylation, the kit containing the above-mentioned reagent for detecting DNA methylation.
  • the kit of the present invention includes one or more containers divided into receiving reagents therein.
  • a first container containing a probe that specifically binds to a CpG site
  • a second container containing an amplification primer
  • a third container containing a conversion agent that sensitively converts unmethylated cytosine, and the like.
  • the invention also provides a system for detecting DNA methylation, the system comprising:
  • the DNA methylation detecting member contains the above-mentioned reagent or kit.
  • the output component is used for outputting methylation ratio or methylation percentage parameter of methylation, PM.
  • the present invention also provides applications of the above-mentioned DNA methylation detection method, reagent, kit, and system in disease diagnosis.
  • the disease is a methylation-related disease.
  • the diseases include but are not limited to: tumors; CNS dysfunction and injury; brain injury; mental disorders; dementia; cardiovascular diseases; gastrointestinal diseases; respiratory diseases; inflammation, infection, immunity; skin, muscle, Connective tissue or bone disease; endocrine and metabolic diseases; headache or sexual dysfunction, etc. It has been reported in the prior art that the occurrence of DNA methylation may be related to various diseases [18-36].
  • the tumor includes, but is not limited to, colorectal cancer, breast cancer, lung cancer, prostate cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, nasopharyngeal cancer, thyroid cancer, kidney cancer, bladder cancer, cervical cancer, ovarian cancer, Nervous system tumors, lymphomas, and leukemias.
  • Promoter methylation of tumor-related genes is an important indicator of cancer and can therefore be used in many applications, including cancer diagnosis and early detection, estimation of cancer development risk, cancer prognosis, follow-up examination after treatment, and anti-cancer Estimate of treatment response. Recently, active attempts have been made to examine the methylation of promoters of tumor-related genes in blood, sputum, saliva, stool or urine, and the results have been used in the diagnosis and treatment of various cancers.
  • the method, reagent and kit for detecting DNA methylation of the present invention can also be used for non-disease diagnostic applications, such as, but not limited to, cell line authentication and organization using species methylation characteristics as markers Source identification, prenatal diagnosis, microbial identification, germline identification of new species, etc.
  • non-disease diagnostic applications such as, but not limited to, cell line authentication and organization using species methylation characteristics as markers Source identification, prenatal diagnosis, microbial identification, germline identification of new species, etc.
  • the application of methylation as a marker has been reported in the prior art [37-46].
  • the present invention also provides a method for diagnosing a disease.
  • the method is to detect the DNA methylation status of a sample to be tested by using the methods, reagents, kits, and systems described above, and determine whether the DNA methylation status is determined based on the DNA methylation status. Sickness, risk, probability of illness, course of illness, type of illness, etc.
  • the present invention also provides a disease diagnosis system, which includes the above-mentioned system for detecting DNA methylation.
  • the disease diagnosis system further includes a result judgment component.
  • the result judging component is used to compare the methylation results of the sample to be tested with the normal sample, so as to analyze whether the disease, the risk, the probability of the disease, the course of the disease, the type of the disease, etc. One or more. Further, the result judgment component compares the methylation status of the test sample and the normal sample, and outputs whether or not the disease is present, the risk of the disease, and the probability of the disease according to the difference or difference between the test sample and the normal sample. The course of the disease, the type of disease, etc.
  • the present invention proposes a novel QASM (quantitative analysis of single-CpG methylation) technology for the first time, which uses a probe combined with a minor groove binder (MGB) and uses a real-time quantitative PCR
  • MGB minor groove binder
  • the detection design of this technology is simpler and does not require an independent input reference response, so the inevitable deviations and errors from the input reference response will not accumulate and will not affect the final result.
  • the standard deviation of the two independent measurements is significantly smaller than the standard deviation using the Alu-C4 response as the input reference.
  • the PM of this technology is determined by the ratio of methylation / (methylation + unmethylation), it is not necessary to calculate fully methylated DNA as a reference. If the reference DNA is not fully methylated, the PM determined by the ratio of the test sample to the reference DNA is obviously incorrect, which also explains why the results of this technique and pyrosequencing are almost identical, while traditional MethyLight The results of phosphate sequencing are linearly related, but there is a certain bias in PM.
  • the present invention is a high-resolution detection technique for a single CG site, which is much higher than the low resolution of 3 to 10 CG sites in the conventional technique, the present invention can effectively detect the presence of heterogeneous methyl groups in the methylation group.
  • Pyrosequencing is the gold standard technique for detecting DNA methylation levels and can also be used to detect methylation levels of isolated CG sites targeted by this technology.
  • pyrosequencing requires a special pyrosequencer, which is difficult to obtain (generally not available in laboratories), expensive sequencing (about 300 / sample), and long sequencing cycle (about 1 month); this technique requires only one routine
  • the real-time PCR instrument is cheap, and the data can be obtained immediately.
  • the traditional MethyLight technology is based on the assumption that all CpGs of CGI are methylated or unmethylated at the same time, and the percentage of co-methylation of all CpG sites covered by the primer probe is determined. However, if the CpGs covered by the primers and probes in the test site and flanking sequences are not co-methylated, the original technology will not be able to accurately check the methylation status of the test zone. Because this technique shows a high degree of sensitivity, specificity and accuracy for a single CpG measurement.
  • the method of the present invention is used to determine the methylation level of a single CpG dinucleotide, but it is not limited to open seas with flanking sequences lacking CpG. The method of the invention can more accurately detect the methylation level of the CGI test area.
  • Figure 1 is the detection principle of the present invention.
  • Figure 3 is a quantitative comparison of methylation quantification accuracy of FAT3, FHIT and KIAA1026 with bisulfite pyrosequencing;
  • Figure 4 shows the results of screening EPIC microarrays in 45 tumor samples using this technique.
  • FIG. 5 compares the accuracy of the PM calculation method (A) of the present invention with the traditional PM calculation method (B).
  • CpG site of a heteromethylated region and "CpG site of a non-co-methylated region” mean one.
  • the epigenetics community usually assumes that these 10 CpGs are methylated or unmethylated at the same time, which is called a "co-methylated region”.
  • the genome Many regions are such "co-methylated regions”.
  • the traditional technology is based on such a high probability "co-methylated” phenomenon. Primers and probes are designed to cover multiple CpG sites in a 100 bp region. What is actually detected is The proportion of cells with multiple CpG sites simultaneously methylated in this region.
  • CpG sites in hybrid methylated regions are called "CpG sites in non-co-methylated regions” [47-48].
  • Genome A gene is all the nucleotide sequences required to produce a polypeptide chain or functional RNA.
  • a gene body is the main part of a gene. It usually refers to a gene that removes its promoter region (usually refers to the upstream and 2000bp region downstream).
  • the intergenic region refers to the spacer sequence between genes and is a segment in the genome that does not have a genetic effect and does not belong to the genetic structure.
  • CpG islands The distribution of CpG dinucleotides in the human genome is very uneven. In certain sections of the genome, CpG remains at or above the normal frequency. CpG islands are mainly located in the promoter and exon regions of the gene, and are some regions rich in CpG dinucleotides, with a length of 300-3000bp. It is usually defined as GC content exceeding 55%, and the ratio of actual to expected CpG dinucleotide numbers is greater than 65%.
  • the expected CpG dinucleotide number calculation method is (C number * G number) / sequence length.
  • CpG shore In the areas immediately adjacent to the CpG island, the length is about 100-3000 bp.
  • the frequency of CpG dinucleotides does not meet the requirements of the CpG island definition, but it is higher than other regions of the genome.
  • These immediate flanking regions are called "shore"
  • CpG islands of CpG islands In regions of the genome that are far away from CpG islands, the frequency of CpG dinucleotides is much lower than that of CpG islands. Compared to CpG islands, these genomes are the most extensive Area is called "open sea"
  • CpG shelf In the areas on both sides of CpG shore, the frequency of CpG dinucleotides is lower than shore, but higher than open sea. Compared to CpG islands, “shores” and the broad “high seas”, these are far from CpG islands and The area closer to the "shore” is called the CpG "shelf"
  • Flanking sequences do not have isolated CpG sites for CpG: In the CpG open sea and shore regions, CpG dinucleotides appear less frequently, and CG sites are often isolated, often lacking in 100-200bp PCR amplified sequences on their sides. Other CpG sites.
  • Tissue DNA methylation level has potential clinical application value.
  • There are various methods for detecting DNA methylation such as using MethyLight (methylation-specific quantitative PCR) to check the methylation of CpG islands (CGI).
  • CGI CpG islands
  • the CGI in the promoter represents only a small part of the methylation group, and it is now gradually found that CpG, which is mainly located in the genome, plays an important role in the occurrence and development of disease and abnormal intracellular molecular events.
  • existing PCR-based techniques cannot detect such isolated CpG sites without CpG on both sides of the sequence. Therefore, the present invention provides a new QASM analysis method, which uses a Taqman probe bound to a minor groove binder (MGB).
  • MGB minor groove binder
  • the present invention utilizes the 5 'exonuclease activity of a DNA polymerase to cleave a dual-labeled probe, and hybridizes with the CG / TG sequence of the DNA treated by the transforming agent. Cleavage of the 5 'exonuclease separates the 5'-fluorophore from the 3'-quenching agent, releasing the fluorophore and generating a detectable fluorescent signal.
  • Minor groove binding (MGB) binding to 3'-quencher allows the use of shorter probes, and has high sensitivity and specificity for single base mismatches.
  • Methylated CpGs remain CpG during the conversion agent treatment, while unmethylene CpG is deaminated to TpG after conversion by the conversion agent. Therefore, using a CG / TG specific probe carrying two different fluorophores can When performing a one-step methylation detection, the single-base mismatch in the 3 'half region (MGB region) of the probe has an annealing temperature difference of up to 17 ° C compared to the perfect pairing. This allows us to design probes to cover only a single CpG dinucleotide, which can measure the methylation level of isolated CpG. PM is determined by the CT threshold ratio of the two fluorescent probes in the amplification cycle.
  • the ratio between the signals from the methylated probe and the unmethylated probe can achieve accurate quantification of the methylation level of the test site. And no need to amplify control genes, such as Alu-C4 and ACTB, to reflect and correct the amount and integrity of the starting DNA template; in addition, it is no longer necessary to use CpGenome fully methylated DNA samples as a reference for calculating each sample PM samples.
  • control genes such as Alu-C4 and ACTB
  • the QASM detection method of the present invention is different from the traditional MethyLight in that it has a high resolution for detecting the methylation status in the genome and can detect the methylation level of a single CpG.
  • This detection based on the ratio of methylated probes to non-methylated probes can improve the sample normalization method in PCR-based detection technology, because it does not need to use a positive control to calculate PM (fully methylated DNA),
  • PM fully methylated DNA
  • the total amount of control-corrected DNA template (Alu-C4PCR reaction) needs to be entered. Therefore, it is not susceptible to cancer-related copy number variation. This advantage is even more pronounced in complex clinical tissues with large samples.
  • This technology is not only very specific and sensitive, but also easy to operate. It does not require control reactions and fully methylated DNA standards as references, which can overcome their shortcomings. In addition, it has higher repeatability and accuracy than existing MethyLight technology. We used this technique to determine three isolated CpG loci located in CpG open sea in 45 colorectal cancer samples, and found that this method can be used to quantitatively detect methylation in clinically complex samples.
  • the QASM detection method is technically sufficient to reliably detect methylation levels in isolated CpG sites. If suitable primers and probes are designed, they can also be used to more accurately detect the methylation level of CGI.
  • Fresh frozen tumor tissue samples were obtained from 45 patients with primary colorectal adenocarcinoma. The patients included 32 men and 13 women. Of these patients, 17 were stage I tumors and 28 were stage II tumors; 18 patients had recurrence at follow-up and 27 patients had no recurrence at follow-up. See Table 1 for details.
  • genomic DNA was amplified by real-time quantitative PCR.
  • bisulfite-converted genomic DNA was amplified using primers and a pair of oligonucleotide probes covering the CpG site to be tested, with a fluorescent report attached to the 5 'end of each oligonucleotide probe
  • the dye 6FAM or VIC specifically binds the CG sequence and the TG sequence, respectively
  • MGB-NFQ quencher-MGB group
  • Taq DNA polymerase will cleave the probe and release the reporter gene during 5 'to 3' exonuclease activity during DNA extension.
  • the initial DNA template concentration can be obtained from the CT (cycle threshold) value of the fluorescent signal [15].
  • the fully methylated and completely unmethylated standard samples were mixed at a certain ratio and then tested by QASM to draw a standard curve.
  • We use a 20uL reaction system which includes 500nM primers, 150nM probes, 200nM each of dATP, dCTP, dGTP, and dTTP, 2.25mM MgCl 2 , 0.75U HotStar Taq enzyme, 1X PCR buffer.
  • the reaction conditions were: DNA after bisulfite conversion at 95 ° C for 15 minutes, then 50 cycles of 94 ° C for 30 seconds, 56-60 ° C for 1 minute, and 72 ° C for 1 minute.
  • the QASM test data of the CpG site to be tested is expressed in PM, but the calculation method is different from the previously reported method [8].
  • the PM of each sample is equal to methylation / (methylation + non-methylation) ⁇ 100.
  • PM 100 / (1 + 1/2 - ⁇ CT )
  • ⁇ CT CT FAM -CT VIC
  • Tables 4 to 6 show the detection results of FAT3, FHIT, and KIAA1026 in 10 clinical colorectal cancer tissues, respectively.
  • DMPs differentially Methylated Positions
  • MethyLight a high-throughput assay to DNA measure methylation .Nucleic Acids Res. 2000.28 (8): E32.).
  • PM standard deviation of two independent measurements is higher than the PM standard deviation of the methylated / unmethylated signal ratio used in this technique to correct the input difference ( Figure 2E).
  • the higher standard deviations observed in traditional MethyLight may come from random PCR at low template concentrations [16] and the accumulation of bias in two independent reactions. However, this technique is less likely to be affected by these factors because the signals from methylated and unmethylated probes are generated using the same primers in the same reaction system.
  • EPIC array is the second-generation product of Illumina HM450microarray. Its detection probe coverage in CpG open sea has increased significantly, which provides a valuable tool for screening biologically and clinically significant isolated CpGs [17] . Forty-five colorectal adenocarcinoma tissues were used for this experiment (Table 1).
  • EPIC microarray (EPIC microarray technology refers to Pidsley, R, Zotenko, Peters, TJ, et al. Critical evaluation of the Illumina Methylation EPIC BeadChip microarray for whole-genome DNA methylation, profiling.
  • Embodiment 5 Accuracy Comparison between the PM Calculation Method of the Present Invention and the Traditional PM Calculation Method

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Abstract

La présente invention concerne un réactif de détection, un kit et un procédé de méthylation de l'ADN, en particulier un réactif de détection, un kit et un procédé de méthylation de l'ADN sans sites CpG au niveau de séquences flanquantes. Le réactif, le kit et le procédé utilisent une sonde Taqman se liant à un lieur de sillon mineur (MGB) et peuvent déterminer la méthylation d'un îlot CpG (CGI), et peuvent déterminer des sites CpG isolés dans une pleine mer CpG et équivalents dans un génosome sans CpG au niveau de deux séquences flanquantes. Le réactif, le kit et le procédé sont spécifiques et sensibles, simples à utiliser, et ne nécessitent pas de réaction de commande ou de substance d'ADN standard entièrement méthylée en tant que référence, ce qui permet de surmonter les défauts résultant de la réaction de commande et de la substance d'ADN étalon entièrement méthylée. De plus, le réactif, le kit et le procédé ont une répétabilité et une précision supérieures à la technologie MethyLight existante.
PCT/CN2019/074716 2018-07-27 2019-02-03 Réactif de détection, kit et procédé de méthylation de l'adn Ceased WO2020019700A1 (fr)

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CN111269963B (zh) * 2019-12-31 2021-07-13 广东凯普生物科技股份有限公司 一步法核酸提取转化试剂盒及其使用方法
CN112375822B (zh) * 2020-06-01 2021-11-02 广州市基准医疗有限责任公司 用于检测乳腺癌的甲基化生物标记物及其应用
CN113817822B (zh) * 2020-06-19 2024-02-13 中国医学科学院肿瘤医院 一种基于甲基化检测的肿瘤诊断试剂盒及其应用
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