WO2016165591A1 - 基于焦磷酸测序技术的mgmt基因启动子甲基化检测 - Google Patents

基于焦磷酸测序技术的mgmt基因启动子甲基化检测 Download PDF

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WO2016165591A1
WO2016165591A1 PCT/CN2016/078935 CN2016078935W WO2016165591A1 WO 2016165591 A1 WO2016165591 A1 WO 2016165591A1 CN 2016078935 W CN2016078935 W CN 2016078935W WO 2016165591 A1 WO2016165591 A1 WO 2016165591A1
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primer
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陈华
刘小青
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凯杰生物工程（深圳）有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof

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  • the present invention relates to MGMT gene promoter methylation detection based on pyrosequencing technology.
  • the invention relates to sequencing primers for detecting promoter methylation of MGMT genes based on pyrosequencing techniques.
  • the invention also relates to kits and microarrays comprising sequencing primers for detecting promoter methylation of MGMT genes based on pyrosequencing techniques.
  • the invention also relates to the use of sequencing primers in the preparation of kits and microarrays for detecting methylation of MGMT gene promoters based on pyrosequencing techniques.
  • the invention also relates to a method for detecting methylation of a MGMT gene promoter in a sample using sequencing primers.
  • Pyrosequencing technology is a sequence analysis technique based on the cascade reaction of enzymes and substrates.
  • the single-stranded PCR product was used as a template, the sequencing primers were annealed and the four dNTPs were added to the reaction system in a predetermined order. If the added dNTP is complementary to the base on the template strand, an equimolar amount of pyrophosphate group PPi is added and incorporated by the action of the DNA polymerase.
  • ATP adenosine triphosphate
  • ATP adenosine triphosphate
  • An optical signal occurs.
  • the light signal detected by the instrument is reflected in the fluorescence spectrum peak in the real-time sequencing map. If the added dNTP cannot bind to the template, it is directly degraded by the diphosphatase and the reaction continues to the next round.
  • the single-stranded PCR product is complementary and extended, and the base type is judged by the presence or absence of the signal peak at each base position, and the number of bases is judged by the height and area of the signal peak.
  • Pyrosequencing is suitable for the quantitative analysis of 10-100 bp length sequences. Its repeatability and precision are comparable to those of Sanger sequencing, and the detection speed is greatly improved.
  • Multiple pyrosequencing technology enhances the detection ability of sites above 100 bp while maintaining the advantages of pyrosequencing technology. Two or more sequencing reactions are performed simultaneously in the same well position, and a specific detection sequence is obtained by scientifically arranging the order of four dNTP additions, thereby realizing Simultaneous detection of multiple distant sites.
  • O6-methylguanine-DNA-methyltransferase is a ubiquitous and unique DNA repair protein that can methylate without any cofactor or protein. Transfer from the O6 position of the 6-methyl guanine to its own cysteine residue, so that the guanine is reduced, and temozolomide (TMZ) can transfer the methyl group to the DNA after hydrolysis in vivo.
  • the methylation site, 6-methylguanine is a key site for cytotoxicity of temozolomide.
  • MGMT repairs DNA alkylation damage and is the main cause of cell resistance to nitrosourea and temozolomide, which are commonly used in the treatment of malignant glioma.
  • MGMT-negative gliomas were significantly more effective in chemotherapy than chloroethylnitrosoure (CENU) than those with MGMT. Therefore, the repair effect of MGMT on alkylating agent damage can make the level of MGMT expression in tissue reflect the sensitivity difference of different individuals to alkylating agent drugs.
  • the degree of methylation in the promoter region of MGMT gene can indicate the expression level of MGMT in vivo, and then predict the survival of patients with malignant glioma receiving alkylating agent chemotherapy.
  • MGMT protein in glioma tissues is detected before the development of chemotherapy regimen.
  • the nitrosourea antitumor drugs and TMZ are not used in tumor patients with high expression of MGMT, which can avoid drug resistance and is expected to improve the efficacy of chemotherapy.
  • the US FDA also recommends MGMT promoter methylation analysis in patients taking temozolomide to guide patients to medication.
  • a variety of molecular biology detection techniques have been applied to the detection of MGMT gene promoter methylation sites, such as real-time quantitative PCR, LDR, RFLP, HRM, gene chip, direct sequencing and the like.
  • the non-sequencing method needs to rely on enzyme digestion or clustering to interpret the patient's genotype, and there is a large probability of typing errors, which is undoubtedly a clinical diagnosis.
  • the accuracy of direct sequencing is high, the DNA fragments generated by the sequencing reaction need to be separated by capillary electrophoresis, and then the detection signal is detected by the detection system, which takes a long time and has high detection cost.
  • Pyrosequencing can perform real-time quantitative analysis of sequences within a certain length. Its repeatability and accuracy are comparable to Sanger sequencing, and the detection speed is greatly improved. Significantly reduced. In addition, the detection sensitivity of pyrosequencing technology is much higher than that of Sanger sequencing. At present, the technology has been widely used in microbial identification and medical analysis, genetic analysis, and SNP detection. Therefore, pyrosequencing technology will have great value and potential in the application and promotion of MGMT gene promoter methylation detection.
  • the sequencing primer comprises the nucleotide sequence set forth in SEQ ID NO:3.
  • the sequencing primer has the nucleotide sequence set forth in SEQ ID NO: 3, or consists or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 3.
  • the target sequence to which the sequencing primer is directed is the sequence of 131, 265, 519 to 131, 265, 537 of the human genome chromosome 10 (SEQ ID NO: 6).
  • kits or microarray for detecting methylation of a MGMT gene promoter based on pyrosequencing technology.
  • the kit or microarray comprises a sequencing primer for pyrosequencing, the sequencing primer comprising the nucleotide sequence set forth in SEQ ID NO:3.
  • the sequencing primer has the nucleotide sequence set forth in SEQ ID NO: 3, or consists or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 3.
  • the target sequence to which the sequencing primer is directed is the sequence of 131, 265, 519 to 131, 265, 537 of the human genome chromosome 10 (SEQ ID NO: 6).
  • the kit or microarray further comprises a primer set comprising a primer having the nucleotide sequence set forth in SEQ ID NO: 1 and having the sequence shown by SEQ ID NO:
  • a primer for the nucleotide sequence consists of or consists essentially of a primer having the nucleotide sequence shown by SEQ ID NO: 1 and a primer having the nucleotide sequence shown by SEQ ID NO: 2.
  • kit or microarray further comprises instructions indicating that the analysis sequence is represented by SEQ ID NO: 4 and/or the order of assignment is set forth by SEQ ID NO: 5.
  • kits or microarrays for detecting MGMT gene promoter methylation in a sample based on pyrosequencing techniques.
  • the kit or microarray comprises a sequencing primer for pyrosequencing, the sequencing primer comprising the nucleotide sequence set forth in SEQ ID NO:3.
  • the sequencing primer has the nucleotide sequence set forth in SEQ ID NO: 3, or consists or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 3.
  • the target sequence to which the sequencing primer is directed is the sequence of 131, 265, 519 to 131, 265, 537 of the human genome chromosome 10 (SEQ ID NO: 6).
  • the kit or microarray further comprises a primer set comprising a primer having the nucleotide sequence set forth in SEQ ID NO: 1 and having the sequence shown by SEQ ID NO:
  • a primer for the nucleotide sequence consists of or consists essentially of a primer having the nucleotide sequence shown by SEQ ID NO: 1 and a primer having the nucleotide sequence shown by SEQ ID NO: 2.
  • the kit or microarray further comprises instructions for indicating that the analytical sequence used for pyrosequencing is represented by SEQ ID NO: 4 and/or the order of assignment is set forth by SEQ ID NO: 5.
  • the sample is a biological sample, preferably the sample is a body fluid sample or a tissue sample, and more preferably the sample is selected from the group consisting of a biopsy sample, a cell culture, whole blood, plasma, serum, saliva, Cerebral fluid, sweat, urine, feces, peritoneal fluid, secretions, and tears.
  • the invention also relates to a method for detecting methylation of a MGMT gene promoter in a sample based on pyrosequencing technology, the method comprising:
  • the pyrosequencing is performed using a sequencing primer comprising the nucleotide sequence set forth in SEQ ID NO:3.
  • the sequencing primer has the nucleotide sequence set forth in SEQ ID NO: 3, or consists or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 3.
  • the target sequence targeted by the sequencing primer is the sequence of 131, 265, 519 to 131, 265, 537 of the human genome chromosome 10 (SEQ ID NO: 6).
  • the amplification is PCR amplification, preferably the PCR amplification uses a primer set comprising a primer having the nucleotide sequence set forth in SEQ ID NO: 1 and having a primer for the nucleotide sequence shown by SEQ ID NO: 2, or consisting of or consisting essentially of a primer having the nucleotide sequence shown by SEQ ID NO: 1 and having the nucleotide represented by SEQ ID NO: Primer composition of the sequence.
  • the pyrosequencing also uses the analysis sequence set forth in SEQ ID NO: 4 and/or the assignment sequence set forth in SEQ ID NO: 5.
  • the sample is a biological sample, preferably the sample is a body fluid sample or a tissue sample, and more preferably the sample is selected from the group consisting of a biopsy sample, a cell culture, whole blood, plasma, serum, saliva, Cerebral fluid, sweat, urine, feces, peritoneal fluid, secretions, and tears.
  • Figure 1 is a schematic diagram of a technical process and main elements of pyrosequencing.
  • Figure 2 is a schematic diagram showing the methylation of the MGMT gene promoter region by pyrosequencing.
  • Figure 3 is a screenshot of the methylation test results of the MGMT gene promoter region, in which the abscissa represents the distribution sequence and the ordinate represents the fluorescence signal intensity collected by the instrument.
  • Figure 4 is a screenshot of the results of detection of promoter methylation of the MGMT gene using different sequencing primers, where the abscissa represents the distribution sequence and the ordinate represents the intensity of the fluorescent signal collected by the instrument.
  • Fig. 5 is a graph comparing the detected value of the methylation ratio detected by the sequencing system including the sequencing primer of the present invention with a theoretical value.
  • the present invention aims to solve the shortcomings of the prior art detection of the MGMT gene promoter methylation site, such as the probability of a large typing error, a long time-consuming or a high detection cost.
  • the present invention solves the above problems by using a pyrosequencing technique of the sequencing primer of the present invention.
  • sample includes any sample containing a nucleic acid molecule.
  • the sample may be derived from a biological source ("biological sample"), such as a tissue (eg, a biopsy sample), an extract, or a cell culture including cells (eg, tumor cells), cell lysates, and biological or physiological fluids, such as Whole blood, plasma, serum, saliva, brain pulp, sweat, urine, feces, secretions, milk, peritoneal fluid, etc. Samples obtained from the source or pretreated to improve the sample Samples after product characteristics (eg, plasma preparation from blood, diluted mucus, etc.) can be used directly.
  • the sample is a human physiological fluid, such as human serum.
  • the sample is a biopsy sample such as a tumor tissue or cell obtained by tissue examination.
  • the sample is a malignant or normal tissue sample.
  • Samples that can be analyzed and/or used in accordance with the present invention include polynucleotides of clinical origin, such as DNA or RNA.
  • DNA extraction can be performed, for example, with phenol and chloroform, or using commercially available DNA extraction reagents.
  • extraction can be carried out using a column kit (for example, GENERATION (registered trademark) Capture Column Kit Gentra).
  • nucleic acids can be purified by conventional purification methods in the art, for example using PrepSEQ TM kit and U.S. Patent No. 5,234,809 Method (from Applied Biosystems) and the like.
  • the present invention preferably uses a commercially available kit such as EpiTect Plus Lyse All Bisulfite Kit (QIAGEN) to extract and purify the nucleic acid. After extraction and purification of the nucleic acid, the nucleic acid, such as DNA, can be subjected to bisulfite treatment for the PCR reaction.
  • primer generally refers to a linear oligonucleotide that is complementary to and annealed to a target sequence.
  • the lower limit of primer length depends on the ability to hybridize, as very short primers (e.g., less than 5 nucleotides) do not form thermodynamically stable duplexes under most hybridization conditions. Primer lengths typically vary from 8 to 50 nucleotides. In certain embodiments, the primer is between about 15-25 nucleotides.
  • forward primer refers to an oligonucleotide that anneals to a particular strand of the target DNA.
  • reverse primer refers to an oligonucleotide that anneals to the opposite strand of the target DNA.
  • the forward and reverse primers are typically oriented on the target DNA sequence in a manner similar to PCR primers such that their 3' ends are closer to the target sequence than their 5' ends.
  • Naturally occurring nucleotides especially guanine, adenine, cytosine and thymine, hereinafter referred to as "G”, “A”, “C” and “T”
  • sequencing primer refers to an oligonucleotide primer for initiating a sequencing reaction on a nucleic acid.
  • amplification product refers to an amplified nucleic acid produced by nucleic acid amplification from a nucleic acid template.
  • template DNA or “template RNA” refers to a nucleic acid that is a desired target for amplification.
  • template RNA is reverse transcribed into cDNA, and the template cDNA is used to generate an amplification product.
  • nucleotide analog refers to a compound that is structurally similar to a naturally occurring nucleotide.
  • the nucleotide analog can have an altered phosphate backbone, a sugar moiety, a nucleobase, or a combination thereof.
  • Nucleotide analogs that typically have altered nucleobases in particular confer different base pairing and base stacking properties.
  • Nucleotide analogs with altered phospho-saccharide backbones e.g., peptide nucleic acids (PNA), locked nucleic acids (LNA) typically change chain characteristics, such as secondary structure formation, in particular.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • the nucleotide sequences of the PCR primers and sequencing primers of the present invention also include modified forms thereof as long as the amplification or sequencing effects of the primers are not significantly affected.
  • the modification may be, for example, the addition of one or more nucleotide residues in the nucleotide sequence or both ends, the deletion of one or more nucleotide residues in the nucleotide sequence, or the Multiple nucleotide residues are replaced with additional nucleotide residues, such as replacing A with T, replacing C with G, and the like.
  • the modified forms of the primers are also encompassed within the scope of the invention, particularly the claims.
  • the modified form of the nucleotide sequence of the PCR primers and sequencing primers is a chemically amplified primer as disclosed in CN103270174A.
  • Each nucleotide of the primer of the present invention can be chemically synthesized using, for example, a universal DNA synthesizer (for example, Model 394 manufactured by Applied Biosystems). Also available Any other method well known in the art to synthesize oligonucleotides, such as PCR primers and sequencing primers.
  • a universal DNA synthesizer for example, Model 394 manufactured by Applied Biosystems.
  • Amplification reactions include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCP), automated maintenance sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA) , Multiple Displacement Amplification (MDA) and Rolling Ring Amplification (RCA), which are disclosed in the following references, which are incorporated by reference: Mullis et al., U.S. Patent No. 4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al, U.S. Patent No.
  • the target nucleotide is amplified using a PCR method.
  • the PCR method itself is well known in the art.
  • the term "PCR” includes derived forms of the reaction including, but not limited to, reverse transcription PCR, real-time PCR, nested PCR, multiplex PCR, and real-time PCR.
  • the target nucleotide is quantitatively amplified using a real-time PCR method.
  • primers that hybridize to the sense strand and primers that hybridize to the antisense strand (forward primer) are used by annealing, extension, and denaturation steps.
  • the PCR is carried out by repeating about 30 to 50 times (for example, 45 times) in a cycle.
  • the PCR is real-time fluorescent quantitative PCR.
  • PCR uses the following primer set comprising a primer having the nucleotide sequence shown by SEQ ID NO: 1 and a primer having the nucleotide sequence shown by SEQ ID NO: 2. Or consisting of or consisting essentially of the primers.
  • amplification can be carried out using various conventional heat-resistant DNA polymerases including, but not limited to, FastStart Taq DNA polymerase (Roche), Ex Taq (Note)
  • the trademarks are Takara, Z-Taq, AccuPrime Taq DNA polymerase and HotStarTaq Plus DNA polymerase.
  • the fluorescent quantitative PCR reaction can be carried out using the following conditions: 95 ° C for 5 minutes, 95 ° C for 20 seconds, 60 ° C for 30 seconds, 72 ° C for 20 seconds, and cycle 45 times.
  • the reaction system can be 50 ⁇ L.
  • the PCR product can be processed to obtain a single-stranded PCR product that is complementary to the sequencing primer.
  • Production and purification of single-stranded PCR products can be carried out by methods well known in the art. Common methods for producing and purifying single-stranded PCR products include, but are not limited to, T7 reverse transcription (Hughes et al, Nat. Biotechnol., 2001, 19: 342-347), exonuclease (Higuchi and Ochman, Nucleic. Acids Res., 1989, 17: 5865), denaturing high-performance liquid chromatography (DHPLC) (Dickman and Hornby, Anal.
  • the invention obtains a single-stranded PCR product that binds complementary to a sequencing primer by a magnetic bead capture method.
  • the invention passes The PCR product was processed on a Q24 vacuum workstation according to the manufacturer's instructions to obtain a single-stranded PCR product that was complementary to the sequencing primer.
  • the single-stranded PCR product can be directly prepared using an asymmetric PCR method, thereby eliminating the need for additional processing after PCR.
  • Asymmetric PCR allows for the preparation of DNA single strands while PCR amplification.
  • Conventional asymmetric PCR uses two unequal amounts of primers for normal amplification in the initial cycle. As the cycle increases, fewer primers are depleted, and excess primers continue to linearly generate DNA single strands (Gyllensten and Erlich, Proc. Natl. Acad. Sci. USA, 1988, 85:7652- 7656).
  • the sequencing primer of the present invention can be used for pyrosequencing.
  • the sequencing primer may comprise a nucleotide sequence as set forth in SEQ ID NO:3.
  • the sequencing primer can have a nucleotide sequence as set forth in SEQ ID NO: 3, or consists essentially of or as set forth in SEQ ID NO: The nucleotide sequence consists of.
  • the target sequence to which the sequencing primer is directed is the sequence of 131, 265, 519 to 131, 265, 537 of the human genome chromosome 10 (SEQ ID NO: 6).
  • the sequencing primers used in the present invention can be as set forth in SEQ ID NO: 7, 10 or 13.
  • QIAGEN's PyroMark Gold Q24 Reagents kit and real-time quantitative pyrophosphate sequence analyzer are used (model: Q24MDx), pyrosequencing was performed according to the instructions. Pyrosequencing can be performed using the following hybridization conditions: heating at 80 °C for 2 min and annealing at room temperature for 20 minutes. In one embodiment, pyrosequencing also uses the analysis sequence set forth in SEQ ID NO: 4 and/or the order of assignment shown by SEQ ID NO: 5. The analysis sequence is used when the result of the sequencing experiment is completed, and the analyzer software compares the analysis sequence with the sequencing result.
  • the software will calculate the ratio of C/T to the site marked with Y; in practical applications, the scale value will change with the sample, and the ratio value indicates the degree of methylation.
  • the order of distribution is the sequence of the nucleotide substrate ejected by the instrument during the sequencing process; when sequencing, the instrument adds the nucleotide substrate to the reaction cell in order according to the order of distribution, and if a fluorescent signal is detected when A is injected, The sequencing result representing this site is A, and so on.
  • pyrosequencing can use analytical sequences and/or assignment sequences as shown in Table 3.
  • the present invention relates to a kit for detecting methylation of a MGMT gene promoter based on pyrosequencing technology, comprising a sequencing primer of the present invention or a combination of a sequencing primer and a primer set.
  • the invention also relates to the use of a sequencing primer of the invention or a combination of a sequencing primer and a primer set for the preparation of a kit for detecting methylation of a MGMT gene promoter based on pyrosequencing technology.
  • the sequencing primer may comprise a nucleotide sequence as set forth in SEQ ID NO:3.
  • the sequencing primer may have a nucleotide sequence as set forth in SEQ ID NO: 3, or consist of or consist essentially of the nucleotide sequence set forth in SEQ ID NO: 3.
  • the primer set comprises A primer having the nucleotide sequence shown by SEQ ID NO: 1 and a primer having the nucleotide sequence shown by SEQ ID NO: 2, or consisting of or consisting essentially of the primer.
  • Kits may comprise materials or reagents (including sequencing primers and primer sets) for use in practicing the methods of the invention.
  • Kits may include storage reagents (eg, primers, dNTPs, enzymes, etc. in suitable containers) and/or support materials (eg, buffers, instructions for performing assays, etc.).
  • the kit can include one or more containers (eg, boxes) containing corresponding reagents and/or support materials. Such contents can be delivered together or separately to a given recipient.
  • the first container may contain the enzyme for the assay
  • the second container contains the primer set
  • the third container contains the sequencing primer.
  • the kit may also contain a compartment suitable for holding the reagent or container.
  • the kit can contain sequencing primers, primer sets, PCR reaction buffers, instructions for use.
  • the kit may also contain a polymerase, dTNP, and the like.
  • the kit may also contain UNG, internal standards for quality control, positive and negative controls, and the like.
  • the kit may also contain reagents for preparing nucleic acids, such as DNA, from a sample.
  • the kit of the present invention may further comprise any sequencing primers and/or primer sets other than the sequencing primers and/or primer sets of the present invention, such as sequencing primers and/or primer sets capable of efficiently detecting methylation of the MGMT gene promoter. .
  • the above examples are not to be construed as limiting the kits and their contents suitable for use in the present invention.
  • the instructions in the kit indicate that the analytical sequence used for pyrosequencing is represented by SEQ ID NO: 4 and/or the order of assignment is set forth by SEQ ID NO: 5.
  • the present invention relates to a microarray for detecting promoter methylation of MGMT gene based on pyrosequencing technology, comprising a sequencing primer of the present invention or a combination of a sequencing primer and a primer set.
  • the invention also relates to the use of a sequencing primer of the invention or a combination of a sequencing primer and a primer set for the preparation of a microarray for detecting methylation of a MGMT gene promoter based on pyrosequencing technology.
  • the sequencing primer may comprise a nucleotide sequence as set forth in SEQ ID NO:3.
  • the sequencing primer may have a nucleotide sequence as set forth in SEQ ID NO: 3, or consist of or consist essentially of the nucleotide sequence set forth in SEQ ID NO: 3.
  • the primer set comprises a primer having the nucleotide sequence set forth in SEQ ID NO: 1 and having SEQ ID NO: The primer of the nucleotide sequence shown in 2 consists either of or consists essentially of the primer.
  • a microarray refers to a solid support having a flat surface having a nucleic acid array, each member of the array comprising the same copy of an oligonucleotide or polynucleotide immobilized at a spatially defined region or site, The region or site does not overlap with regions or sites of other members of the array; that is, the regions or sites are spatially discrete.
  • a spatially determined hybridization site can be "addressable" because its position and its immobilized oligonucleotide identity are known or predetermined (eg, known or prior to its use) definite).
  • the oligonucleotide or polynucleotide is single stranded and is typically covalently linked to the solid support by a 5'- or 3'-end.
  • the density of nucleic acids containing non-overlapping regions in the microarray is typically greater than 100/cm 2 , more preferably greater than 1000/cm 2 .
  • Microarray technology is disclosed, for example, in the following reference: Microarrays, edited by Schena: A Practical Approach (IRL Press, Oxford, 2000); Southern, Current Opin. Chem. Biol., 2: 404-410, 1998, the entire contents of which are The references are incorporated herein.
  • the human genomic DNA transformed and purified by bisulfite was first amplified on the Rotor-Gene Q platform using PCR Master Mix, HS Taq and two PCR primers.
  • the PCR Master Mix contained fluorescent dyes. It is capable of being embedded in an increasing number of PCR double stranded products during amplification. The entire product enrichment process can therefore be monitored in real time by Rotor-Gene Q software to ensure reliable PCR products for subsequent pyrosequencing analysis.
  • the PCR product was processed by a PyroMark Q24 vacuum workstation to finally obtain a single-stranded PCR product that was complementary to the sequencing primer.
  • the specific pyrosequencing program was then run on a PyroMark Q24 pyrosequencing instrument.
  • the instrument added four dNTPs in sequence according to the order of assignment shown in Table 2 below.
  • the dNTP was on the single-stranded product by a cascade of enzymes and substrates.
  • the effective extension is received by the instrument in the form of an optical signal and eventually appears in the software interface in real time in the form of a signal peak.
  • the results after the operation are automatically analyzed by the software, reducing the burden and error of manual analysis.
  • the PCR conditions were as follows: 95 ° C: 5 minutes; 95 ° C: 20 seconds, 60 ° C: 30 seconds, 72 ° C: 20 seconds for a total of 45 cycles.
  • Figure 2 is a schematic diagram showing the methylation of the MGMT gene promoter region by pyrosequencing.
  • the sequencing primer is the sequencing primer shown in SEQ ID NO: 3, and the sequencing conditions of the sequencing primer are: heating at 80 ° C for 2 min, annealing at room temperature for 20 minutes.
  • Example 2 The materials, instruments and methods and conditions used in this example were the same as in Example 1, but the combination of the sequencing primers shown in Table 3 below and the analysis sequence and the distribution sequence was used in the pyrosequencing process.
  • Sequencing primer 1 TTTAGAAAGTTTTGAGTTT (SEQ ID NO: 3)
  • Sequencing primer 2 TTTAGAAAGTTTTGAGTT (SEQ ID NO: 7)
  • Sequencing primer 3 GTTTTTAGAAAGTTTTG (SEQ ID NO: 10)
  • Sequencing primer 4 GATAGTTAGAGTTTTTAGA (SEQ ID NO: 13)
  • the PCR conditions were: 95 ° C: 5 minutes; 95 ° C: 20 seconds, 60 ° C: 30 seconds, 72 ° C: 20 seconds for a total of 45 cycles.
  • the hybridization conditions of the sequencing primers were as follows: heating at 80 ° C for 2 min, annealing at room temperature for 20 minutes.
  • Example 2 The experimental materials, instruments and experimental procedures and conditions used in the test are the same as in Example 1; the sequencer automatically obtains the percentage of C bases at each methylation site, which is the methylation ratio of the site. Detected value.
  • the results of the methylation ratio are shown in Table 4 below.
  • the results show that the detected value of the methylation ratio is close to the theoretical value, and the detection accuracy is high.
  • the standard deviation of each point is less than 2.2, and the average value is 0.81, which shows good.
  • the precision of the methylation ratio of the four sites showed a good linear relationship with the theoretical value, and the correlation coefficient of the linear fitting was greater than 0.99 (see Figure 5). Therefore, the sequencing primer of the present invention can be applied to quantitative detection with high detection accuracy.

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Abstract

一种基于焦磷酸测序技术的MGMT基因启动子甲基化检测。具体而言，涉及用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的测序引物，包含测序引物的用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的试剂盒和微阵列，测序引物在制备用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的试剂盒和微阵列中的用途，以及使用测序引物检测样品中MGMT基因启动子甲基化的方法。

Description

基于焦磷酸测序技术的MGMT基因启动子甲基化检测 技术领域
本发明涉及基于焦磷酸测序技术的MGMT基因启动子甲基化检测。具体而言，本发明涉及用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的测序引物。本发明还涉及包含测序引物的用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的试剂盒和微阵列。本发明还涉及测序引物在制备用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的试剂盒和微阵列中的用途。本发明还涉及使用测序引物检测样品中MGMT基因启动子甲基化的方法。
背景技术
焦磷酸测序技术是一种基于酶与底物的级联反应进行检测并定量的序列分析技术。在整个反应体系中，以单链的PCR产物作为模板，测序引物与其退火结合，四种dNTP按照既定的顺序加到反应体系中。如果加入的dNTP与模板链上的碱基互补结合，在DNA聚合酶的作用下释放与掺入量等摩尔数的焦磷酸基团PPi。硫酸化酶催化释放的PPi与腺苷-5'-磷酸硫酸酐形成等量的腺嘌呤核苷三磷酸(Adenosine Triphosphate,ATP)，ATP驱动荧光素酶介导的荧光素向氧化态转化，并发生光信号。仪器检测到的光信号则以荧光信号峰的方式反映在实时出现的测序图谱中。如加入的dNTP不能与模板互补结合，则直接被双磷酸酶降解，反应继续进入下一轮。通过上述过程的循环，单链的PCR产物得到互补并延伸，通过各碱基位置信号峰的有无判断碱基类型，通过信号峰的高度和面积判断碱基的数目。焦磷酸测序法适于对10-100bp长度序列进行定量分析，其重复性和精确性能与Sanger测序媲美，而检测速度则大大提高。多重焦磷酸测序技术在保持焦磷酸测序技术优势的同时，增强其对于相距100bp以上位点的检测能力。两个甚至多个测序反应在同一孔位中同时进行，通过科学的编排四种dNTP加入的顺序获得特异的检测序列，从而实现 多个相距较远的位点的同时检测。
O6-甲基鸟嘌呤-DNA-甲基转移酶(O6-methylguanine-DNA-methyltransferase,MGMT)是一种普遍存在的独特的DNA修复蛋白，能够在没有任何辅助因子或蛋白的参与下将甲基从6位甲基鸟嘌呤的O6位转移到自身的半胱氨酸残基上，使鸟嘌呤被还原，而替莫唑胺(temozolomide,TMZ)在体内经水解作用后可将甲基转移至DNA上的甲基化位点，6位甲基鸟嘌呤正是替莫唑胺细胞毒作用的关键位点。MGMT使DNA烷基化损伤得到修复，是细胞对治疗恶性脑胶质瘤的常用药物亚硝脲类及替莫唑胺产生耐药的主要原因。有研究发现，通过对MGMT基因启动子的甲基化致使MGMT失活是肿瘤对TMZ化疗敏感的一个重要因素。研究发现MGMT阴性胶质瘤者对氯乙基亚硝脲类(chloroethylnitrosoure,CENU)的化疗有效率明显高于MGMT阳性者。因此MGMT对烷化剂损伤的修复作用使得组织中MGMT表达水平的高低能够反映出不同个体对烷化剂药物的敏感性差异。MGMT基因启动子区甲基化程度可以指示体内MGMT表达水平，进而对恶性胶质瘤患者接受烷化剂化疗的生存期进行预测。故在制定化疗方案前检测脑胶质瘤组织中MGMT蛋白的表达，对MGMT高表达的肿瘤患者不选用亚硝脲类抗肿瘤药物及TMZ，可以避免耐药，有望提高化疗疗效。美国FDA亦推荐对使用替莫唑胺的患者进行MGMT启动子甲基化分析，以指导患者用药。
现已有多种分子生物学检测技术应用于MGMT基因启动子甲基化位点的检测，如实时定量PCR、LDR、RFLP、HRM、基因芯片、直接测序等。其中非测序方式需要依赖酶切或聚类的方式判读患者的基因型，存在较大的分型错误的几率，这无疑是临床诊断大忌。直接测序虽然准确度高，但测序反应产生的DNA片段需要经过毛细管电泳分离，其后由检测系统检测荧光信号，耗时较长且检测成本较高。
焦磷酸测序法可对一定长度内序列进行实时定量分析，其重复性和精确性能与Sanger测序媲美，而检测速度大大提高，检测成本明 显降低。此外，焦磷酸测序技术的检测灵敏度也远高于Sanger测序。目前该技术已广泛应用于微生物鉴定及分法医学鉴、遗传学分析、SNP检测等方面。因此，焦磷酸测序技术在MGMT基因启动子甲基化检测的应用与推广将具有极大价值与潜力。
然而，目前尚没有商业化实现焦磷酸测序技术在MGMT基因启动子甲基化检测的方案。
发明内容
本发明一方面涉及用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的测序引物。在一个实施方案中，所述测序引物包含如SEQ ID NO:3所示的核苷酸序列。在另一个实施方案中，所述测序引物具有如SEQ ID NO:3所示的核苷酸序列，或者由或基本由如SEQ ID NO:3所示的核苷酸序列组成。在一个实施方案中，所述测序引物针对的靶标序列为人基因组第10号染色体的131,265,519至131,265,537的序列(SEQ ID NO:6)。
本发明另一方面涉及一种用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的试剂盒或微阵列。在一个实施方案中，所述试剂盒或微阵列包含用于焦磷酸测序的测序引物，所述测序引物包含如SEQ ID NO:3所示的核苷酸序列。在另一个实施方案中，所述测序引物具有如SEQ ID NO:3所示的核苷酸序列，或者由或基本由如SEQ ID NO:3所示的核苷酸序列组成。在一个实施方案中，所述测序引物针对的靶标序列为人基因组第10号染色体的131,265,519至131,265,537的序列(SEQ ID NO:6)。
在一个实施方案中，所述试剂盒或微阵列还包含以下引物组，所述引物组包含具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO:2所示的核苷酸序列的引物，或者由或基本由具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO:2所示的核苷酸序列的引物组成。
在一个实施方案中，所述试剂盒或微阵列还包含表明分析序列由SEQ ID NO:4所示和/或分配顺序由SEQ ID NO:5所示的说明书。
本发明另一方面涉及测序引物在制备用于基于焦磷酸测序技术检测样品中的MGMT基因启动子甲基化的试剂盒或微阵列中的用途。在一个实施方案中，所述试剂盒或微阵列包含用于焦磷酸测序的测序引物，所述测序引物包含如SEQ ID NO:3所示的核苷酸序列。在另一个实施方案中，所述测序引物具有如SEQ ID NO:3所示的核苷酸序列，或者由或基本由如SEQ ID NO:3所示的核苷酸序列组成。在一个实施方案中，所述测序引物针对的靶标序列为人基因组第10号染色体的131,265,519至131,265,537的序列(SEQ ID NO:6)。
在一个实施方案中，所述试剂盒或微阵列还包含以下引物组，所述引物组包含具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO:2所示的核苷酸序列的引物，或者由或基本由具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO:2所示的核苷酸序列的引物组成。
在一个实施方案中，所述试剂盒或微阵列还包含表明焦磷酸测序所用的分析序列由SEQ ID NO:4所示和/或分配顺序由SEQ ID NO:5所示的说明书。
在一个实施方案中，所述样品为生物样品，优选所述样品为体液样品或组织样品，和更优选所述样品选自活组织检查样品、细胞培养物、全血、血浆、血清、唾液、脑髓液、汗液、尿液、粪便、腹膜液、分泌液和泪液。
本发明还涉及一种基于焦磷酸测序技术检测样品中的MGMT基因启动子甲基化的方法，所述方法包括：
(1)提取和任选纯化样品中的DNA，
(2)将提取和任选纯化的DNA进行扩增，从而获得扩增产物，
(3)对步骤(2)的扩增产物进行焦磷酸测序。
在一个实施方案中，所述焦磷酸测序使用以下测序引物进行，所述测序引物包含如SEQ ID NO:3所示的核苷酸序列。在另一个实施方案中，所述测序引物具有如SEQ ID NO:3所示的核苷酸序列，或者由或基本由如SEQ ID NO:3所示的核苷酸序列组成。在一个实施 方案中，所述测序引物针对的靶标序列为人基因组第10号染色体的131,265,519至131,265,537的序列(SEQ ID NO:6)。
在一个实施方案中，所述扩增为PCR扩增，优选所述PCR扩增使用以下引物组，所述引物组包含具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO:2所示的核苷酸序列的引物，或者由或基本由具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO:2所示的核苷酸序列的引物组成。
在一个实施方案中，所述焦磷酸测序还使用由SEQ ID NO:4所示的分析序列和/或由SEQ ID NO:5所示的分配顺序。
在一个实施方案中，所述样品为生物样品，优选所述样品为体液样品或组织样品，和更优选所述样品选自活组织检查样品、细胞培养物、全血、血浆、血清、唾液、脑髓液、汗液、尿液、粪便、腹膜液、分泌液和泪液。
附图说明
图1为焦磷酸测序的一个技术流程及各主要元素示意图。
图2为焦磷酸测序检测MGMT基因启动子区甲基化示意图。
图3为MGMT基因启动子区甲基化检测结果截图，其中横坐标代表分配序列，纵坐标代表仪器所采集到的荧光信号强度。
图4为使用不同测序引物检测MGMT基因启动子区甲基化的结果截图，其中横坐标代表分配序列，纵坐标代表仪器所采集到的荧光信号强度。
图5为使用包括本发明的测序引物在内的测序体系所检测到的甲基化比例的检测值与理论值相比较的曲线图。
具体实施方式
参考用于说明的示例应用在下文中描述本发明的数个方面。应当理解的是，陈述许多具体细节、关系和方法来提供对本发明的充分理解。然而，在相关领域的普通技术人员将容易地认识到，可在不含一个或多个具体细节的情况下实施本发明或者可用其他方法来实施本发明。
本发明目的在于解决现有技术中对MGMT基因启动子甲基化位点的检测的缺点，例如较大的分型错误的几率、耗时较长或检测成本较高。本发明通过使用本发明的测序引物的焦磷酸测序技术而解决了上述问题。
使用本发明的测序引物的焦磷酸测序技术具有以下优点：
1)仪器及试剂通过系统优化，可达到最优的检测效果，且易标准化；
2)检测结果自动由软件给出，避免结果判断主观性；
3)操作过程简便且省时；
4)在0％-100％甲基化范围内的线性度高，能用于定量检测。
除非另有说明，否则本文所用的所有科技术语具有本发明所属领域普通技术人员通常理解的含义。在细胞和分子生物学中的普通术语的定义可以参见：余龙等译的基因VIII，标准书号：ISBN:978-7-03-014597-0，科学出版社出版(2005)；张瑾峰等译的细胞与分子生物学，中信出版社出版(2004)，ISBN:978-7-50-860075-8；和王镜岩等编的生物化学，高等教育出版社出版(2002)，ISBN:978-7-04-011088-3；Kendrew,J.等人(编),The Encyclopedia of Molecular Biology,Blackwell Science Ltd.出版(1994),ISBN 0-632-02182-9；和Meyers,R.A.(编),Molecular Biology and Biotechnology:a Comprehensive Desk Reference,VCH Publishers,Inc.出版(1995),ISBN 1-56081-5698。虽然在实施本发明时可采用类似或等同于本文所述方法和材料的任何方法和材料，但是本文描述了具体的材料和方法。
样品
本文所用术语“样品”包括含有核酸分子的任何样品。样品可来源于生物来源(“生物样品”)，例如组织(例如活组织检查样品)、提取物或包括细胞(例如肿瘤细胞)、细胞裂解物在内的细胞培养物和生物或生理流体，例如全血、血浆、血清、唾液、脑髓液、汗液、尿液、粪便、分泌液、乳汁、腹膜液等。获自来源的样品或在预处理以改进样 品特征(例如从血液制备血浆、稀释黏液等)后的样品可直接使用。在本发明的某些方面，样品是人生理流体，例如人血清。在本发明的某些方面，样品是活组织检查样品例如经组织检查获得的肿瘤组织或细胞。在本发明的某些方面，样品是恶性或正常组织样品。
可按照本发明进行分析和/或使用的样品包括临床来源的多核苷酸，例如DNA或RNA。
从样品中提取核酸的方法是本领域众所周知的，可用例如苯酚和氯仿进行DNA提取，或者使用市售DNA提取试剂进行提取。例如，可使用柱试剂盒(例如GENERATION(注册商标)Capture Column Kit Gentra)进行提取。
应该理解的是，核酸可通过本领域众常规的纯化方法来纯化，例如使用PrepSEQTM试剂盒(来自Applied Biosystems)和美国专利号5,234,809中的方法等等。本发明优选采用市售试剂盒例如EpiTect Plus LyseAll Bisulfite Kit(QIAGEN公司)对核酸进行提取和纯化。在提取和纯化核酸后，可对核酸例如DNA进行亚硫酸氢盐处理以用于PCR反应。
引物
本文所用的“引物”通常指与靶序列互补和退火的线性寡核苷酸。引物长度的下限按杂交能力而定，因为非常短的引物(例如小于5个核苷酸)在大多数杂交条件下不形成热力学稳定的双链体。引物长度通常在8-50个核苷酸内变化。在某些实施方案中，引物介于大约15-25个核苷酸之间。本文使用的术语“正向引物”是指与靶DNA的一条特定链退火的寡核苷酸。本文使用的术语“反向引物”是指与靶DNA的相反链退火的寡核苷酸。总之，正向引物和反向引物通常以类似于PCR引物的方式定向在靶DNA序列上，使得其3'末端比其5'末端更接近靶序列。天然存在的核苷酸(尤其是鸟嘌呤、腺嘌呤、胞嘧啶和胸腺嘧啶，在下文称为“G”、“A”、“C”和“T”)以及核苷酸类似物，都可用于本发明的引物。本文使用的术语“测序引物”是指用于起始对核酸进行的测序反应的寡核苷酸引物。
本文使用的“扩增产物”是指自核酸模板，通过核酸扩增而产生的扩增的核酸。
本文使用的“模板DNA”或“模板RNA”是指作为用于扩增的所需靶标的核酸。例如，模板RNA被逆转录为cDNA，并且该模板cDNA用于产生扩增产物。
本文使用的术语“核苷酸类似物”指与天然存在的核苷酸在结构上相似的化合物。核苷酸类似物可以具有改变的磷酸骨架、糖部分、核碱基或其组合。通常具有改变的核碱基的核苷酸类似物尤其赋予不同的碱基配对和碱基堆积特性。具有改变的磷酸-糖骨架的核苷酸类似物(例如肽核酸(PNA)、锁核酸(LNA))通常尤其改变链特性，例如二级结构形成。
用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的PCR引物、测序引物及靶系列的实例见下表1。
表1.本发明所用的PCR引物、测序引物及相应的靶序列
Figure PCTCN2016078935-appb-000001
本发明的PCR引物和测序引物的核苷酸序列还包括其修饰形式，只要所述引物的扩增或测序效果不受到明显的影响即可。所述修饰可以为例如在核苷酸序列中或两端添加一个或多个核苷酸残基、在核苷酸序列中缺失一个或多个核苷酸残基、或者将序列中的一个或多个核苷酸残基替换成另外的核苷酸残基，例如将A替换成T，将C替换成G等。本领域技术人员清楚，所述修饰形式的引物也涵盖在本发明之内、特别是权利要求的保护范围之内。在一个实施方案中，PCR引物和测序引物的核苷酸序列的修饰形式为如CN103270174A中所公开的化学增强型引物。
可以使用例如通用DNA合成仪(例如由Applied Biosystems制造的394型)，经化学方法合成本发明引物中的各个核苷酸。还可采用 本领域众所周知的任何其它方法来合成寡核苷酸，例如PCR引物和测序引物。
使用从样品中提取的基因组DNA作为模板，并使用PCR引物对MGMT基因进行扩增反应，以获得扩增产物。扩增反应包括但不限于聚合酶链式反应(PCR)、连接酶链式反应(LCP)、自动维持序列复制(3SR)、基于核酸序列的扩增(NASBA)、链置换扩增(SDA)、多重置换扩增(MDA)和滚环扩增(RCA)，其公开于以下参考文献(在此引作参考)中：Mullis等，美国专利第4,683,195号；第4,965,188号；第4,683,202号；第4,800,159(PCR)号；Gelfand等，美国专利第5,210,015号(用“Taqman”或”Taq”[注册商标]探针进行的实时PCR)；Wittwer等，美国专利第6,174,670号；Kacian等，美国专利第5,399,491号(“NASBA”)；Lizardi,美国专利第5,854,033号；Aono等，日本专利公开第JP 4-262799号(滚环扩增)；等等。
优选使用PCR法对靶核苷酸进行扩增。PCR法本身是本领域众所周知的。术语“PCR”包括该反应的衍生形式，其包括但不限于反转录PCR、实时PCR、嵌套式PCR、多重PCR和荧光定量PCR等。优选使用荧光定量PCR法对靶核苷酸进行定量扩增。
在引物、模板DNA和耐热DNA聚合酶存在下，使用与有义链杂交的引物(反向引物)和与反义链杂交的引物(正向引物)，通过使退火、延伸和变性步骤的循环重复大约30次～50次(例如45次)来进行PCR。在一个实施方案中，PCR为实时荧光定量PCR。在一个实施方案中PCR使用了以下引物组，所述引物组包含具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO:2所示的核苷酸序列的引物，或者由或基本由所述引物组成。本领域技术人员能够理解的是，也可使用其它PCR法和引物组，只要可扩增出目标片段即可。本领域技术人员可根据需要常规地选择PCR法，并常规设计出所需的PCR引物。
在本发明的PCR中，可使用各种常规的耐热DNA聚合酶进行扩增，包括但不限于FastStart Taq DNA聚合酶(Roche)、Ex Taq(注 册商标,Takara)、Z-Taq、AccuPrime Taq DNA聚合酶和HotStarTaq Plus DNA聚合酶。
基于引物Tm值选择合适PCR反应条件的方法是本领域众所周知的，本领域普通技术人员可以根据引物长度、GC含量、目标特异性和灵敏度、所使用的聚合酶性质等，选出最佳条件。例如，可使用以下条件进行荧光定量PCR反应：95℃5分钟，95℃20秒，60℃30秒，72℃20秒，循环45次。反应体系可为50μL。
在获得PCR产物后，可对PCR产物进行处理，以获得与测序引物互补结合的单链PCR产物。单链PCR产物的产生和纯化可通过本领域周知的方法来进行。常见的产生和纯化单链PCR产物的方法包括但不限于T7逆转录法(Hughes等人,Nat.Biotechnol.,2001,19:342-347)、核酸外切酶法(Higuchi和Ochman,Nucleic.Acids Res.,1989,17:5865)、变性高效液相色谱法(denaturing high-performance liquid chromatography，DHPLC)(Dickman和Hornby,Anal.Biochem.,2000,284:164-167)和磁珠捕获法(Espelund等人,Nucleic.Acids Res.,1990,18:6157-6158)等。在一个实施方案中，本发明通过磁珠捕获法获得与测序引物互补结合的单链PCR产物。在另一个实施方案中，本发明通过
Figure PCTCN2016078935-appb-000002
 Q24真空工作站按照制造商的说明书对PCR产物进行处理，以获得与测序引物互补结合的单链PCR产物。
另外，也可使用不对称PCR方法直接制备单链PCR产物，从而省去了在PCR后进行额外的处理。不对称PCR可在PCR扩增的同时制备DNA单链。常规不对称PCR使用两条不等量的引物，在开始的循环里进行正常扩增。随着循环的增加，量少的引物被逐渐耗尽，而超量的引物可继续直线扩增生成DNA单链(Gyllensten和Erlich,Proc.Natl.Acad.Sci.U.S.A.,1988,85:7652-7656)。
在获得单链PCR产物后，可采用本发明的测序引物进行焦磷酸测序。在一个实施方案中，所述测序引物可包含如SEQ ID NO:3所示的核苷酸序列。在另一个实施方案中，所述测序引物可具有如SEQ ID NO:3所示的核苷酸序列，或者由或基本由如SEQ ID NO:3所示 的核苷酸序列组成。在一个实施方案中，所述测序引物针对的靶标序列为人基因组第10号染色体的131,265,519至131,265,537的序列(SEQ ID NO:6)。在一个实施方案中，用于本发明的测序引物可如SEQ ID NO:7、10或13所示。
在一个实施方案中，采用QIAGEN公司的PyroMark Gold Q24Reagents试剂盒和实时定量焦磷酸序列分析仪(型号：
Figure PCTCN2016078935-appb-000003
 Q24MDx)，按照说明书方法进行焦磷酸测序。可使用以下杂交条件进行焦磷酸测序：80℃加热2min，室温退火20分钟。在一个实施方案中，焦磷酸测序还使用由SEQ ID NO:4所示的分析序列和/或由SEQ ID NO:5所示的分配顺序。分析序列是测序实验完成后进行结果分析时使用的，分析仪的软件将此分析序列与测序结果进行比对。比如分析序列为YGAYGTTYGTAGGTTTTYG，软件就会对标注有Y的位点计算C/T的比例；在实际应用中，比例值会随着样本而变化，这个比例值则指示甲基化的程度。分配顺序是仪器在进行测序过程中喷出的核苷酸底物的顺序；测序时，仪器按照分配顺序按次序将核苷酸底物加入反应池中，如果喷入A时检测到荧光信号，就代表这个位点的测序结果为A，以此类推。本领域技术人员可根据需要常规地选择和设计分析序列和/或分配顺序，并在实际应用中根据具体的靶序列的情况而使用不同的分析序列和/或分配顺序。在一个实施方案中，焦磷酸测序可使用如表3所示的分析序列和/或分配顺序。
试剂盒
本发明涉及一种用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的试剂盒，其含有本发明的测序引物或者测序引物与引物组的组合。本发明还涉及本发明测序引物或者测序引物与引物组的组合在制备用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的试剂盒中的用途。在一个实施方案中，所述测序引物可包含如SEQ ID NO:3所示的核苷酸序列。在另一个实施方案中，所述测序引物可具有如SEQ ID NO:3所示的核苷酸序列，或者由或基本由如SEQ ID NO:3所示的核苷酸序列组成。在一个实施方案中，所述引物组包含 具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO:2所示的核苷酸序列的引物，或者由或基本由所述引物组成。
试剂盒可包含实施本发明方法所用的材料或试剂(包括测序引物和引物组)。试剂盒可以包括储存反应试剂(例如在合适容器中的引物、dNTP、酶等)和/或支持材料(例如缓冲液、实施检测的说明书等)。例如，试剂盒可以包括一个或多个含有相应反应试剂和/或支持材料的容器(例如盒子)。这样的内容物可一起或分开递送给既定的接受者。例如，第一个容器可含有用于测定的酶，第二个容器含有引物组、而第三个容器含有测序引物。所述试剂盒还可含有适合容纳所述试剂或容器的隔室。作为一个实例，试剂盒可含有测序引物、引物组、PCR反应缓冲液、使用说明书。试剂盒还可含有聚合酶和dTNP等。试剂盒还可含有UNG、用于质控的内标、阳性和阴性对照等。试剂盒还可包含用于从样品制备核酸例如DNA的试剂。本发明试剂盒还可包含除了本发明的测序引物和/或引物组之外的其它任何测序引物和/或引物组，例如能够有效检测MGMT基因启动子甲基化的测序引物和/或引物组。以上实例不能理解为限制适用于本发明的试剂盒及其内容物。
在一个实施方案中，试剂盒中的说明书表明了焦磷酸测序所用的分析序列由SEQ ID NO:4所示和/或分配顺序由SEQ ID NO:5所示。
微阵列
本发明涉及一种用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的微阵列，其含有本发明的测序引物或者测序引物与引物组的组合。本发明还涉及本发明测序引物或者测序引物与引物组的组合在制备用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的微阵列中的用途。在一个实施方案中，所述测序引物可包含如SEQ ID NO:3所示的核苷酸序列。在另一个实施方案中，所述测序引物可具有如SEQ ID NO:3所示的核苷酸序列，或者由或基本由如SEQ ID NO:3所示的核苷酸序列组成。在一个实施方案中，所述引物组包含具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO: 2所示的核苷酸序列的引物，或者由或基本由所述引物组成。
微阵列是指具有平坦表面的固相支持体，其具有核酸阵列，阵列中的各个成员包含固定在空间上确定的区域或位点上的寡核苷酸或多核苷酸的相同的拷贝，所述区域或位点不与阵列中的其它成员的区域或位点重叠；也就是说，所述区域或位点在空间上是离散的。此外，空间上确定的杂交位点可为“可寻址的”，因为其位置及其固定化的寡核苷酸的身份是已知或预先确定的(例如在其使用前是已知或预先确定的)。通常寡核苷酸或多核苷酸为单链，并通常由5'-端或3'-端与固相支持体共价连接。微阵列中含有非重叠区的核酸的密度通常大于100/cm2，更优选大于1000/cm2。微阵列技术公开于例如以下参考文献中：Schena编辑的Microarrays:A Practical Approach(IRL Press,Oxford,2000)；Southern,Current Opin.Chem.Biol.,2:404-410，1998，其全部内容通过引用结合到本文中。
虽然上文已描述了本发明的各种实施方案，但是应理解的是，其仅以实例的方式提供，而并非限制。对公开的实施方案的许多改变可依照本文的公开内容来进行，而不会背离本发明的精神或范围。因此，本发明的广度和范围不应受到任何上述的实施方案所限制。
本文提及的所有文献都通过引用结合到本文中。本申请引用的所有出版物和专利文件都为所有目的而通过引用结合，引用程度如同单独地指出各个出版物或专利文件一样。
实施例
除非另外说明，否则本文实施例所用的材料均市购获得，用于进行实验的各种具体实验方法均为本领域常规的实验方法(参见例如F.奥斯伯等主编的《精编分子生物学实验指南》(1999)，科学出版社，ISBN 7-03-006408-9和J.萨姆布鲁克等主编的《分子克隆实验指南(第三版)》(2002)，科学出版社，ISBN 7-03-010338-6)或者按照制造商所建议的步骤和条件，并能由本领域技术人员根据需要常规地确定。以下对某些材料和方法进行了详述。
实施例1：使用本发明测序引物进行的焦磷酸测序
材料：
Figure PCTCN2016078935-appb-000004
方法：
如图1所示，首先采用PCR Master Mix、HS Taq及两条PCR引物对亚硫酸氢盐转化并纯化的人类基因组DNA在Rotor-Gene Q平台上进行扩增，PCR Master Mix中包含荧光染料，在扩增过程中能够嵌入不断增加的PCR双链产物中。因此整个产物富集的过程能够通过Rotor-Gene Q的软件进行实时监测，从而确保质量可靠的PCR产物用于后续焦磷酸测序分析。通过PyroMark Q24真空工作站对PCR产物进行处理，最终获得与测序引物互补结合的单链PCR产物。之后在PyroMark Q24焦磷酸测序仪上运行特定的焦磷酸测序程序，仪器会根据下表2所示的分配顺序依次加入四种dNTP，通过酶与底物的级联反应，dNTP在单链产物上的有效延伸以光信号的形式被仪器接收，并最终以信号峰的形式实时出现在软件界面中。运行后的结果由软件进行自动分析，减少了人工分析的负担和误差。
表2.PyroMark Q24软件设置用的分析序列以及分配顺序
名称 序列(5'—>3') SEQ ID NO:
分析序列 YGAYGTTYGTAGGTTTTYG 4
分配顺序 GTCGTATCAGTCGTCATGTTCG 5
实验过程如下：
一.取一来自于临床医院的临床样本，用QIAGEN公司的EpiTect Plus LyseAll Bisulfite Kit按照制造商所建议的步骤和条件进行组织裂解、亚硫酸盐化转化、核酸纯化；
二.取纯化后的核酸用PCR引物1、2进行PCR扩增(采用QIAGEN公司的实时荧光定量PCR分析仪(型号：Rotor-Gene Q))；
PCR条件如下：95℃：5分钟；95℃：20秒，60℃：30秒，72℃：20秒，共45个循环。
三.焦磷酸测序
图2为焦磷酸测序检测MGMT基因启动子区甲基化示意图。
采用QIAGEN公司的PyroMark Gold Q24 Reagents试剂盒和实时定量焦磷酸序列分析仪(型号：
Figure PCTCN2016078935-appb-000005
 Q24MDx)，按照制造商说明书方法和条件进行操作，测序引物为SEQ ID NO:3所示测序引物，测序引物的杂交条件为：80℃加热2min，室温退火20分钟。
测序结果见图3。从图3可知，焦磷酸测序的结果与预期序列(即CGACGCCCGCAGGTCCTCG，人基因组第10号染色体的131,265,519至131,265,537的序列)一致，也与检测“金标准”Sanger测序结果一致，且测序背景低。
实施例2：不同测序引物的比较
本实施例中所用的材料、仪器与方法及条件均同实施例1，但在焦磷酸测序过程中使用了下表3所示的测序引物与分析序列和分配顺序的组合。
表3：不同的测序引物与分析序列和分配顺序的组合
Figure PCTCN2016078935-appb-000006
测序引物1：TTTAGAAAGTTTTGAGTTT(SEQ ID NO:3)
测序引物2：TTTAGAAAGTTTTGAGTT(SEQ ID NO:7)
测序引物3：GTTTTTAGAAAGTTTTG(SEQ ID NO:10)
测序引物4：GATAGTTAGAGTTTTTAGA(SEQ ID NO:13)
PCR条件均为：95℃：5分钟；95℃：20秒，60℃：30秒，72℃：20秒，共45个循环。
测序引物的杂交条件均为：80℃加热2min，室温退火20分钟。
实验结果见图4。结果显示测序引物1的结果最好，待分析区域显示为蓝色(表示结果通过)，与预期序列(即CGACGCCCGCAGGTCCTCG，人基因组第10号染色体的131,265,519-131,265,537的序列)一致，且测序背景低；测序引物2、3、4的待分析区域显示为黄色或红色(表示结果不确定或失败)，与预期序列不太一致，且测序背景高。因此选择测序引物1作为本发明的测序引物。
实施例3：本发明测序引物的准确性、精密度和线性性能评估
材料：
Figure PCTCN2016078935-appb-000007
方法：
选取已知甲基化比例分别为0％、25％、50％、75％、100％的标准临床样本各一份，采用包括本发明的测序引物(SEQ ID NO:3)在内的测序体系进行检测，每个样本重复检测8次，统计4个检测位点的甲基化比例的检测值，并与理论值进行比较。
检测所使用的实验材料、仪器以及实验过程和条件均同实施例1；测序仪自动得出每个甲基化位点上的C碱基的百分数，即为该位点的甲基化比例的检测值。
甲基化比例的检测结果如下表4所示，结果显示：甲基化比例的检测值与理论值接近，检测准确性较高；各位点的标准偏差小于2.2，平均值为0.81，显示出良好的精密度；4个位点的甲基化比例的检测值与理论值呈现良好的线性关系，线性拟合的相关系数均大于0.99(见图5)。因此，本发明的测序引物可应用于定量检测且检测准确性高。
表4：甲基化比例的理论值与检测值
Figure PCTCN2016078935-appb-000008

Claims (10)

  1. 一种用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的测序引物，所述测序引物包含如SEQ ID NO:3所示的核苷酸序列。
  2. 权利要求1的测序引物，其中所述测序引物具有如SEQ ID NO:3所示的核苷酸序列。
  3. 权利要求1或2的测序引物，其中所述测序引物针对的靶标序列为人基因组第10号染色体的131,265,519至131,265,537的序列：SEQ ID NO:6。
  4. 一种用于基于焦磷酸测序技术检测MGMT基因启动子甲基化的试剂盒或微阵列，其包含权利要求1-3中任一项的测序引物。
  5. 权利要求4的试剂盒或微阵列，其还包含以下引物组，所述引物组包含具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO:2所示的核苷酸序列的引物。
  6. 权利要求4或5的试剂盒或微阵列，其还包含表明分析序列由SEQ ID NO:4所示和/或分配顺序由SEQ ID NO:5所示的说明书。
  7. 权利要求1-3中任一项的测序引物在制备用于基于焦磷酸测序技术检测样品中的MGMT基因启动子甲基化的试剂盒或微阵列中的用途。
  8. 权利要求7的用途，其中所述试剂盒或微阵列还包含以下引物组，所述引物组包含具有由SEQ ID NO:1所示的核苷酸序列的引物和具有由SEQ ID NO:2所示的核苷酸序列的引物。
  9. 权利要求7或8的用途，其中所述试剂盒或微阵列还包含表明焦磷酸测序所用的分析序列由SEQ ID NO:4所示和/或分配顺序由SEQ ID NO:5所示的说明书。
  10. 权利要求7或8的用途，其中所述样品为生物样品，优选所述样品为体液样品或组织样品，和更优选所述样品选自活组织检查样品、细胞培养物、全血、血浆、血清、唾液、脑髓液、汗液、尿液、 粪便、腹膜液、分泌液和泪液。
PCT/CN2016/078935 2015-04-13 2016-04-11 基于焦磷酸测序技术的mgmt基因启动子甲基化检测 WO2016165591A1 (zh)

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CN105779402A (zh) * 2016-03-30 2016-07-20 苏州偲聚生物材料有限公司 用于检测血清中mgmt抗体的多肽，检测器件和检测试剂盒
CN108570504A (zh) * 2018-06-15 2018-09-25 上海润达榕嘉生物科技有限公司 一种mgmt启动子甲基化检测引物及其检测方法
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