A sample enrichment method for high sensitivity detection of idh1 gene r132h mutation

Abstract

The present application relates to the field of in vitro detection of gene mutation. Specifically, the present application provides a sample enrichment method for high-sensitivity detection of gene mutation, particularly for detection of IDH1 gene R132H mutation.

Description

Technical Field

[0001] This application relates to the field of in vitro detection of gene mutations. Specifically, it relates to a sample enrichment method for improving the sensitivity of gene mutation detection. Specifically, this application provides a method for detecting the R132H mutation in the IDH1 gene associated with acute myeloid leukemia (AML). Background Technology

[0002] Acute myeloid leukemia (AML) is a significant disease threatening national health. AML is a malignant blood disorder originating from myeloid hematopoietic cells and is the most common type of leukemia in adults. The pathogenesis of AML is highly complex and heterogeneous, involving the synergistic effects of multiple gene mutations, chromosomal abnormalities, and epigenetic abnormalities, leading to the accumulation of a large number of abnormally differentiated "primitive cells" in the patient's bone marrow. These primitive cells severely hinder the body's normal hematopoietic process and can also infiltrate various tissues and organs, damaging the patient's lung function and central nervous system.

[0003] AML has a rapid and severe onset; without timely intervention, a patient's life expectancy can only be extended to a few weeks. Currently, the main clinical treatment for AML is standardized therapy, namely daunorubicin combined with cytarabine, also known as the "3+7" therapy. However, due to the high heterogeneity of AML patients, the efficacy of standardized therapy varies among patients. Therefore, appropriate subtyping of AML patients is particularly important.

[0004] Currently, AML classification relies heavily on morphology and immunophenotype, employing multicolor staining and histochemical reactions to clarify the morphological characteristics of blood smears, and multichannel flow cytometry to detect cell surface expressed molecules. With advancements in molecular biology research and a deeper understanding of the pathogenesis of AML, gene mutations are receiving increasing attention in AML classification and treatment planning. Gene mutation detection has become a crucial basis for selecting AML treatment methods, assessing prognosis, and evaluating treatment efficacy. Some gene mutations, such as TP53, are clearly poor prognostic markers, significantly contributing to patient prognosis analysis; while others, such as FLT3 and IDH1 / 2, are gain-of-function (GOF) mutations, for which corresponding targeted drugs are available clinically. Detection of these gene mutations can effectively guide clinical targeted drug use, bringing AML treatment into the realm of precision medicine.

[0005] Although the treatment of AML has made great progress with the continuous improvement of chemotherapy regimens and the development of molecularly targeted drugs, minimal residual disease (MRD) remains a major challenge affecting the prognosis of AML. Minimal residual disease (MRD) refers to the state in which a small number of leukemia cells remain in the body after complete remission following treatment. Multichannel flow cytometry is currently the main method for detecting MRD, but the immunophenotype of AML cells overlaps with some normal hematopoietic stem and progenitor cells, interfering with MRD detection. Leukemia-specific gene mutations can also be used as a method for detecting MRD, but the sensitivity requirements for detecting gene mutations are high, which is difficult to meet with currently used clinical methods.

[0006] Twenty percent of AML patients carry IDH1 / 2 mutations, which promote the production of 2-hydroxyglutarate (2-HG), leading to DNA and histone hypermethylation, resulting in altered gene expression and impaired cell differentiation. Ivosidenib and Enasidenib, two small-molecule oral targeted therapies, have been approved by the U.S. Food and Drug Administration for relapsed or refractory AML with IDH1 or IDH2 mutations.

[0007] Currently, clinically used methods for detecting gene mutations mainly include restriction enzyme digestion, Sanger sequencing, and next-generation sequencing (NGS). Restriction enzyme digestion is a qualitative method; although easy to perform, it cannot be used to determine mutation rates and lacks sensitivity. While Sanger sequencing can easily determine mutation rates, its sensitivity is poor, making it difficult to detect mutations below 5%. The widespread application of NGS in mutation detection has greatly improved the efficiency of gene mutation detection in cancer patients. Its advantage is the ability to detect multiple gene mutations simultaneously, but it also suffers from high cost, long processing times, insufficient sensitivity, and the need for expensive NGS sequencers. In general, existing methods all suffer from insufficient sensitivity, with interference from normal wild-type sequences in the patient's DNA being a major problem. Establishing a simple and rapid method for specifically enriching mutant sequence fragments is of great significance for detecting gene mutations such as IDH1 / 2. Summary of the Invention

[0008] This application designs and develops a method for the specific enrichment of mutant gene fragments by combining streptavidin magnetic beads with restriction endonucleases, called MICAPS ( magnetic isolated cleaved amplified polymorphic sequencesTo enrich mutant genes using restriction endonucleases, restriction sites were created near the IDH1-R132H site using derived cleaved amplified polymorphic sequences (dCAPS) technology. By combining streptavidin-dependent magnetic beads with restriction endonucleases, the enrichment of mutant genes in samples was achieved, significantly improving the final detection sensitivity, thus completing this application.

[0009] Therefore, in a first aspect, this application relates to a method for enriching a target nucleic acid in a sample, wherein the target nucleic acid contains a nucleic acid region to be detected, and the nucleic acid region to be detected is the region where the gene mutation to be detected is located. The method includes: (1) providing a nucleic acid sample containing the target nucleic acid; (2) using the nucleic acid sample containing the target nucleic acid in step (1) as a template, amplifying a fragment containing the target nucleic acid by PCR and introducing a restriction endonuclease recognition site into the product, wherein one of the PCR primers is tagged with biotin, thereby obtaining a biotin-tagged amplified fragment product; (3) digesting the amplified fragment product obtained in step (2) with a restriction endonuclease, thereby obtaining a digested product; (4) purifying the digested product obtained in step (3) using a solid support such as magnetic beads with streptavidin, to obtain an enriched sample containing the target nucleic acid. In a specific embodiment, the gene mutation is an IDH1 gene mutation.

[0010] Secondly, this application relates to a method for detecting gene mutations, comprising enriching a sample using the method of the first aspect, and then detecting the enriched sample. In a specific embodiment, the gene mutation is an IDH1 gene mutation, particularly the IDH1-R132H mutation.

[0011] Thirdly, this application provides a kit for enrichment comprising (a) PCR primers for amplifying a fragment containing a target nucleic acid, wherein one of the PCR primers is biotin-tagged and capable of introducing a previously non-existent restriction enzyme recognition site into the product; (b) one or more restriction endonucleases; and (c) a solid support such as magnetic beads with streptavidin, wherein the target nucleic acid contains the nucleic acid region to be detected.

[0012] The applicant discovered that effective enrichment of the target gene can be achieved through restriction enzyme differential digestion combined with affinity purification. This method requires the presence of restriction enzyme cleavage sites in the wild-type sequence corresponding to the mutant. For many genes whose natural sequences do not contain restriction enzyme recognition sites at the mutation sites, cleavage sites can be created using derived cleaved amplified polymorphic sequences (dCAPS) technology, making them suitable for the restriction enzyme-based affinity purification enrichment scheme.

[0013] The method described in this application requires readily available instruments and reagents, and the experimental cost is low. Regarding instruments, only a standard PCR instrument and a magnetic rack are needed. As for reagents, the cost of DNA extraction is approximately 12 yuan per sample (whole genome DNA extraction kit), the cost of enzyme reactions is approximately 12-20 yuan per sample (including Taq enzyme, EcoRV / XhoI endonuclease, buffer, primers, etc.), and the cost of streptavidin magnetic bead enrichment is approximately 10 yuan per sample (including streptavidin magnetic beads and buffer, etc.). In total, the enrichment cost is approximately 34-42 yuan per sample.

[0014] The method described in this application can be used in various downstream analytical methods, especially conventional and lower-cost ones. For example, the enrichment method described in this application can be combined with downstream detection methods such as Sanger sequencing, AS-PCR, or AS-qPCR, at a cost far lower than that of next-generation sequencing (approximately 2000 RMB per sample). The method described in this application can achieve a detection rate that is better than that of next-generation sequencing at a much lower cost.

[0015] The method described in this application can shorten the detection cycle. In terms of time consumption, the detection method described in this application can be completed within one day, which is much shorter than the time required for second-generation sequencing (approximately 2 weeks).

[0016] The method described in this application exhibits high sensitivity. It can detect as low as 1% of FLT3 D835Y / H mutations in human genomic DNA, significantly outperforming the 10% sensitivity of Sanger sequencing. Specifically, 1. This application is the first to use this method to detect the mutant IDH1-R132H. This application first uses primer mismatches to create cleavage sites for specific restriction endonucleases (e.g., PvuI) with the wild-type gene fragment. This allows the restriction endonuclease to cleave the wild-type gene fragment but not the mutant gene fragment. By adding a cleavage step between two amplification steps, the wild-type IDH1-R132 and mutant IDH1-R132 are significantly separated. Further purification steps can enrich the mutant gene fragment in large quantities for subsequent mutation detection. 2. Regarding the sensitivity of the method in this application, compared with methods without enrichment, enriching mutant gene fragments using the method of this application, combined with Sanger detection and / or AS-PCR, can significantly improve the sensitivity of the method used in this application for detecting gene mutations. For example, for the IDH1-R132H mutation, it is generally difficult to detect gene mutations with a mutation rate of less than 10% (as shown in the data of Example 2); however, when enriching and detecting IDH1-R132H using the method of this application, the sensitivity of the detection method is as low as 1% (as shown in the data of Example 2), which is at least 10 times or even higher than that of the detection method without enrichment. 3. Regarding the purification method in this application, this application uniquely utilizes the affinity principle of streptavidin and biotin to simply and efficiently separate wild-type IDH1-R132 and mutant IDH1-R132, further ensuring the sensitivity of the detection method. In some of the applicant's studies, it was found that compared with traditional purification steps, such as using gel electrophoresis to separate and purify wild-type IDH1-R132 and mutant IDH1-R132, the method of this application can further improve the sensitivity of the detection method by 10-1000 times, or even higher. Therefore, this application, by introducing mismatched bases + streptavidin magnetic beads-biotin separation and purification + restriction endonuclease digestion, can achieve rapid and efficient enrichment of mutant IDH1-R132 (e.g., IDH1-R132H) and high-sensitivity detection of IDH1-R132 mutations. Attached Figure Description

[0017] Figure 1 A flowchart illustrating the MICAPS method of this application is provided.

[0018] Figure 2 The results of Sanger sequencing of the standard samples enriched by the MICAPS method in Example 2 are shown (construction of PvuI restriction site method).

[0019] Figure 3The results of Sanger sequencing of the standard samples enriched by the MICAPS method in Example 3 are shown (construction of BceAI restriction site method). Invention Details

[0020] definition

[0021] In the context of this application, the term "gene" has the same meaning as it is known in the art, referring to a segment of nucleotides that has protein-coding function. In a specific embodiment of this application, the gene refers to the IDH1 gene, namely the gene of citrate dehydrogenase (IDH) isotype 1.

[0022] "Alleles" refer to one of two or more different forms of a gene that occupies the same location on a chromosome, and these differences are caused by mutations.

[0023] The "wild type" of a gene refers to the predominant allele genotype found in nature. In contrast, the "mutant" of a gene, in the context of this paper, refers to a gene that has a nucleotide sequence different from the wild type due to mutation. Mutants can exhibit changes relative to the wild type through base deletions, additions, or substitutions. Due to codon degeneracy, some mutant genes do not produce polypeptide sequences different from the wild type and are therefore called "silent mutations." In the context of this paper, the specific nucleotide that results in a mutant gene is called a "mutant nucleotide" or "mutant base," that is, a specific nucleotide that differs from the sequence of the wild-type gene.

[0024] "IDH" refers to isocitrate dehydrogenase (IDH) (EC 1.1.1.42 and EC 1.1.1.41), which participates in citrate metabolism. IDH catalyzes the oxidative decarboxylation of isocitrate, converting it to α-ketoglutarate (αKG). αKG participates in a variety of biological reactions.

[0025] "IDH1 / 2 mutation" refers to a mutation in either of the two isocitrate dehydrogenases (IDH), namely IDH1 and IDH2. The mutated IDH converts αKG to 2-hydroxyglutarate, which is a competitive inhibitor of the αKG-dependent enzyme and is known to be a carcinogenic metabolite.

[0026] "Mutation frequency" refers to the proportion of mutant DNA in a population of DNA fragments. In a specific implementation, a sample with a predetermined mutation frequency is obtained by mixing wild-type DNA with mutant DNA, wherein the mutation frequency is the weight percentage of mutant DNA in the total DNA in the sample.

[0027] In this article, "genomic DNA" refers to chromosomal DNA, which is distinct from extrachromosomal DNA such as cell-free DNA. This term emphasizes the state and origin of the DNA, without limiting the DNA to include the entire genome. For example, genomic DNA can be a part of a complete genome.

[0028] In the context of this application, "target nucleic acid" refers to a nucleic acid sequence from a subject sample that contains the region where the gene mutation to be detected is located.

[0029] “MICAPS” refers to magnetically isolated cleave-amplified polymorphic sequences, a technique for the specific enrichment and detection of mutated genes.

[0030] "Streptavidin" is a protein derived from Streptomyces avidinii. Its binding to biotin is one of the strongest known covalent interactions that occurs naturally. Furthermore, the streptavidin-biotin complex is stable, making this combination useful in various biological applications, particularly for the purification and detection of molecules.

[0031] In this article, "solid support" refers to a solid material capable of supporting reagents, cells, etc., and it is usually chemically stable. Optionally, the solid support can be magnetic beads.

[0032] "Restriction endonuclease," "restriction nuclease," or "restriction enzyme" refers to an endonuclease that recognizes and cuts specific sequences. The site recognized by a restriction endonuclease is called a "restriction site."

[0033] "Derived cleaved amplified polymorphic sequences (dCAPS)" or "dCAPS technology" refers to a labeling technique that uses primers to introduce mismatched bases, thereby constructing or removing restriction endonuclease recognition sites.

[0034] "AS-PCR" refers to allele-specific PCR.

[0035] Sample source

[0036] The samples used for testing in this application may be biological samples, including but not limited to body fluids, blood, cells, tissues, and nucleotides, as long as they contain the gene fragment to be detected. The nucleotide samples used in this application may be whole-genome DNA or nucleic acid fragments including IDH1 / 2 that have undergone amplification. Methods for obtaining DNA samples from biological samples are well known to those skilled in the art.

[0037] Enrichment methods

[0038] The enrichment method described in this application can be used to enrich nucleic acid samples to improve the sensitivity of subsequent gene mutation detection.

[0039] The main steps include: amplifying the nucleic acid fragment containing the target site and introducing a restriction enzyme recognition site by adding a biotin tag through amplification; differentially digesting the nucleic acid containing the target mutation and the nucleic acid without the target mutation, such as wild type, using a restriction enzyme digestion step; affinity purification, for example, affinity purification using biotin-streptavidin and magnetic beads; and further amplifying the nucleic acid fragment containing the target site by PCR.

[0040] The first step of the enrichment method in this application, the amplification process, is crucial. The fundamental purpose of this PCR amplification step is to amplify the nucleic acid containing the target site from the sample. More importantly, it introduces the restriction enzyme recognition site into the amplified sequence, which originally did not contain such a site. Furthermore, leveraging the sequence differences between wild-type and mutant genes, the recognition site is introduced only in products containing wild-type genes, while it is not introduced in products containing mutant genes. On the other hand, this step also simultaneously adds a biotin tag to the PCR product for subsequent affinity purification.

[0041] To amplify nucleic acid fragments containing the mutation sites to be detected, PCR primers can be designed using standard methods. The DNA sequence of human IDH is known; see GeneBank accession number NG_023319.2. For example, nucleic acid fragments with a total length of approximately 100-3000 bp can be designed for amplification, such as 150-2000 bp, preferably 200-1000 bp.

[0042] In a specific implementation, the mismatched bases are introduced using derivatized enzyme digestion amplification polymorphic sequence (dCAPS) technology, and restriction enzyme recognition sites are created in the amplification products where the target site is wild-type.

[0043] In the amplified nucleic acid fragment, since a restriction enzyme site needs to be introduced through a mismatched base contained in the primer, and the mismatched base together with the wild-type base of the site to be detected can form a restriction enzyme digestion site, the site to be detected needs to be located at one end of the amplified fragment, such as within 30 bases at the 5' or 3' end, preferably within 20 bases.

[0044] For example, in a specific implementation, the mutation to be detected is the IDH1-R132H mutation. When amplifying using dCAPS, primers as shown in SEQ ID NO:1 and SEQ ID NO:2 are used. Specifically, the sequence near the wild-type IDH1-R132 site is as follows:

[0045]

[0046] After amplification using SEQ ID NO:1 and SEQ ID NO:2, the sequence of the amplified product near this site became the specific recognition site of PvuI (the arrow indicates the restriction enzyme cleavage site), as follows:

[0047]

[0048] In contrast, the amplification product of the mutated IDH1-R132H, due to the presence of a point mutation in G395A, has the following sequence at the corresponding position, and therefore cannot form a specific recognition site for PvuI:

[0049]

[0050] For subsequent affinity purification, this amplification step uses a biotin-containing primer to introduce a biotin tag into the amplification product. In a preferred embodiment, the biotin-containing primer is one that is closer to the mutation site than another primer.

[0051] In a specific implementation, the primers used in the amplification step are shown in SEQ ID NO:1 and SEQ ID NO:2, and SEQ ID NO:1 is a 5' primer with biotin.

[0052] This application specifically utilizes the restriction enzyme recognition site formed by the wild-type IDH1-R132 codon and the introduced mismatched bases. Specifically, in the differential restriction enzyme digestion step of this application, PvuI enzyme can be used for the differential restriction enzyme digestion step.

[0053] PvuI is a restriction endonuclease derived from Escherichia coli that recognizes and cleaves the following sites in double-stranded nucleotides, producing sticky ends.

[0054] 5'…CGAT^CG…3'

[0055] 3'…GC^TAGC…5'

[0056] As mentioned above, in wild-type IDH1, the middle position of the codon encoding arginine at position 132 is G, which can form a PvuI recognition sequence with the primer. When the IDH1-R132H mutation occurs, the middle position of the nucleotide codon corresponding to position 132 becomes A, thus failing to form this recognition sequence. In other words, even if the mutated sequence undergoes dCAPS amplification, it cannot be recognized and cleaved by PvuI. The method of this application cleverly utilizes this sequence characteristic, distinguishing between wild-type and mutant types by recognizing the restriction enzyme-cleaved wild-type fragment, and enabling subsequent simple detection of their presence and relative proportions. In subsequent steps, after the wild-type sequence is specifically cleaved with the restriction endonuclease PvuI, the cleaved fragment without biotin labeling is eluted, while the biotin-labeled fragment is captured by magnetic beads. The biotin-labeled mutant sequence remains intact and is also captured by magnetic beads after elution. The two primers for the second round of PCR are designed to be identical to those for the first round of PCR. Therefore, the wild-type sequence, which has already been cut, cannot be amplified; while the biotin-tagged mutant sequence remains intact and can be amplified. Through this process, the mutant sequence is enriched and becomes easier to detect.

[0057] Another important step in the enrichment method of this application is to further enrich the nucleic acid fragment by utilizing the affinity of biotin-streptavidin. Biotin has been added to the end of the nucleic acid fragment to be enriched through a prior amplification reaction. Streptavidin can be attached to a solid support to react with biotin. The solid support can be magnetic beads or a solid matrix known in the art for affinity purification, such as agarose gel.

[0058] Following affinity chromatography, PCR amplification further increases the concentration of the target nucleic acid in the sample. The purpose of this PCR step is to ensure the target nucleic acid concentration reaches the level required for subsequent sequencing. This PCR step can be performed according to conventional procedures in the art. The primers used should only amplify sequences that were not cleaved during the restriction enzyme digestion step, and not sequences that have been cleaved due to the absence of the IDH gene mutation R132H. In other words, the two primers should be positioned upstream and downstream of the restriction enzyme site, respectively. In a preferred embodiment, the two primers in the second PCR step have the same sequence as the two primers in the aforementioned PCR step that introduced the mismatched bases, thus simplifying the entire method.

[0059] Result Evaluation

[0060] The nucleic acid fragments containing the target site obtained by the enrichment method of this application can be used in conventional sequencing methods.

[0061] Sanger sequencing estimates mutation rates by observing base peaks at mutation sites in the sequencing map. Sanger sequencing can be performed using conventional reagents, equipment, and procedures known in the art. Exemplary experimental conditions are shown in the examples.

[0062] In the context of this application, sensitivity is expressed as a percentage, referring to the lowest detection limit achievable by a certain detection method. Specifically, the detection method can detect the presence of a mutation when the percentage of mutant copies in the total copy number is not less than this detection limit. Therefore, the lower the percentage value, the higher the sensitivity of the detection method. Specifically, when the detection method can detect 10 mutant copies per 100 DNA copies, the sensitivity of the method is 10%; when the detection method can detect 1 mutant copy per 100 DNA copies, the sensitivity of the method is 1%; and so on. The standards of this application are prepared by mixing plasmid DNA or patient genomic DNA at known concentrations. Accordingly, the detection method of this application, combined with Sanger sequencing, can detect 1% of the IDH1-R132H mutation in plasmid DNA or human genomic DNA. In contrast, the sensitivity of conventional Sanger sequencing is approximately 10%, while next-generation sequencing requires a longer processing time and higher costs. Therefore, for example, the method of this application can improve the detection sensitivity of the target mutation by at least 10 times compared with detection methods that directly perform Sanger sequencing or allele-specific PCR amplification, for example, from 10% to 1%.

[0063] In the context of this application, sensitivity is expressed as a percentage, referring to the lowest detection limit achievable by a detection method. Specifically, the detection method can detect the presence of a mutant when the percentage of mutant copies in the total copy number is not less than this detection limit. Therefore, a lower percentage indicates higher sensitivity. Specifically, when a detection method can detect one mutant copy per 100 DNA copies, the sensitivity of the method is 1%; and so on. The standards of this application are prepared by mixing plasmid DNA at a known concentration. Accordingly, the detection method of this application, combined with Sanger sequencing, can detect 1% of the IDH1-R132H mutation in plasmid DNA. In contrast, the sensitivity of conventional Sanger sequencing is approximately 10%. Therefore, for example, the method of this application can improve the detection sensitivity of the target mutation by at least 10 times compared to direct Sanger sequencing, for example, from 10% to 1%.

[0064] use

[0065] Therefore, the detection method described in this application is suitable for efficiently, accurately, and inexpensively detecting the presence of mutations in genes associated with diseases such as IDH. Identifying IDH mutations, such as the positive IDH1-R132H mutation, is of great significance for AML patients before, after, and during treatment.

[0066] IDH1-R132H can be used to assess the prognosis of AML patients. Isocitrate dehydrogenases (IDHs) are homodimeric enzymes. IDH1 is located in the cytoplasm and peroxisomes and participates in various cellular regulatory processes, including hypoxia adaptation, histone demethylation, and DNA modification. IDH1 protein catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), generating NADPH from NADP+. Many dioxygenases depend on sufficient levels of α-KG to complete various cellular regulatory processes and epigenetic regulation. Cancer-associated IDH mutations are heterozygous missense mutations, characterized by alloenzymatic activity and the production of 2-hydroxyglutarate (2HG). The most common mutation type in IDH1 / 2 in myeloid malignancies is heterozygous missense mutation, including R132 in IDH1 and R140 or R172 in IDH2. These mutant products have decreased affinity for isocitrate and increased affinity for α-KG and NADPH, resulting in D-2-hydroxyglutarate (D-2HG). It is speculated that D-2HG provides biological advantages that promote malignant transformation of cells: inhibiting 2-OG-dependent dioxygenases, promoting DNA methylation, promoting histone methylation, affecting collagen synthesis, and regulating hypoxia-inducible factor signaling.

[0067] In recent years, numerous targeted drug studies have been conducted on IDH1-R132 mutations. Ivosidenib (AG-120) is a first-in-class selective small-molecule allosteric inhibitor of IDH1-R132 mutations, approved by the U.S. Food and Drug Administration as a single-drug therapy for adults with relapsed or refractory acute myeloid leukemia (AML) and those susceptible to IDH1 mutations. In in vitro experiments, AG-120 can induce differentiation of primary blast cells with IDH1 mutations in AML patients, manifested as enhanced colony differentiation capacity in methylcellulose assays, increased levels of cell surface differentiation markers, and an increased proportion of mature myeloid cells. In in vivo experiments, in a human IDH1-mutant tumor cell xenograft model, AG-120 effectively reduced tumor D-2HG levels, demonstrating favorable pharmacokinetic properties and tolerability. Therefore, developing a highly sensitive detection technology for IDH1-R132H could effectively guide targeted therapy for AML.

[0068] This application relates to the following:

[0069] 1. A method for enriching a target nucleic acid, wherein the target nucleic acid comprises a nucleic acid region to be detected, comprising:

[0070] (1) Provide a nucleic acid sample containing the target nucleic acid;

[0071] (2) Amplify using a nucleic acid sample containing the target nucleic acid in step (1) as a template, including the step of making the amplified fragment contain mismatched bases, and one of the amplified primers in the amplification primer pair is biotin-labeled, thereby obtaining a biotin-labeled amplified fragment product.

[0072] (3) The amplified fragment product obtained in step (2) is digested using a restriction endonuclease to obtain the digested product;

[0073] (4) The digestion product obtained in step (3) was purified using a solid support containing streptavidin to obtain an enriched sample containing the target nucleic acid; and

[0074] (5) Amplify the sample obtained in step (4) to further enrich the target nucleic acid in the sample;

[0075] Preferably, the biotin-tagged amplification primers have the biotin tag located at the 5' end of the PCR primers;

[0076] Preferably, the step of incorporating mismatched bases into the amplified fragment includes amplifying a fragment containing the target nucleic acid using derivatized enzyme digestion amplification polymorphic sequence (dCAPS) technology.

[0077] 2. The method according to item 1, wherein the nucleic acid sample containing the target nucleic acid is genomic DNA or a fragment thereof containing the site of the mutation to be detected.

[0078] 3. The method according to item 2, wherein the genomic DNA is mammalian genomic DNA, preferably human or non-human mammalian genomic DNA.

[0079] 4. The method according to item 2 or 3, wherein the nucleic acid sample is derived from cells, tissues, body fluids or blood.

[0080] 5. The method according to item 4, wherein the nucleic acid sample is derived from peripheral blood.

[0081] 6. The method according to any one of items 1-5, wherein the length of the amplified fragment product containing the target nucleic acid in step (2) is 200 bp to 1000 bp.

[0082] 7. The method according to any one of items 1-6, wherein in step (2), in the amplified fragment product containing the target nucleic acid amplified by PCR, the nucleic acid region to be detected is located within the last 30 bases of the 5' or 3' end of the fragment containing the target nucleic acid.

[0083] 8. The method according to any one of items 1-7, wherein the nucleic acid region to be detected includes the region where the gene mutation to be detected is located.

[0084] 9. The method according to item 8, wherein the gene mutation is a disease-related gene mutation; preferably, the disease-related gene mutation is an AML-related gene mutation; more preferably, the AML-related gene mutation is a mutation in the IDH1 / 2 gene, preferably including the R132H mutation of IDH1.

[0085] 10. The method according to any one of items 1-9, wherein in step (3), the restriction endonuclease is capable of cleaving only when the amplified fragment product is wild-type at the position corresponding to the gene mutation, and is not capable of cleaving when the amplified fragment product contains the gene mutation.

[0086] 11. The method according to item 10, wherein the mismatched bases in step (2) together with the wild-type sequence corresponding to the gene mutation constitute the recognition site of the restriction endonuclease in step (3).

[0087] 12. The method according to any one of items 1-11, wherein the restriction endonuclease is a PvuI enzyme.

[0088] 13. The method according to any one of items 1-12, wherein the two amplification primers used in step (5) are located upstream and downstream of the restriction endonuclease cleavage site, respectively.

[0089] 14. The method according to any one of items 1-13, wherein the enriched sample containing the target nucleic acid obtained in step (5) is used for sequencing.

[0090] 15. The sequencing method according to item 14 is Sanger sequencing.

[0091] 16. The method according to any one of items 1-15, wherein the biotin-labeled amplification primer in step (2) is closer to the mutation site than the other amplification primer.

[0092] 17. The method according to any one of items 1-26, wherein the nucleotide sequences of the amplification primers used in step (2) are as shown in SEQ ID NO:1 and / or SEQ ID NO:2; and SEQ ID NO:3.

[0093] 18. The method according to item 17, wherein primers with sequences such as SEQ ID NO:1 and SEQ ID NO:2 are tagged with biotin and mismatched bases.

[0094] 19. The method according to any one of items 1-18, wherein the nucleotide sequence of the primer used in step (5) is as shown in SEQ ID NO:1 and / or SEQ ID NO:2; and SEQ ID NO:3.

[0095] 20. A method for detecting gene mutations in a sample, comprising enriching the gene mutation to be detected in the sample using any one of the methods in items 1 to 19, and performing Sanger sequencing.

[0096] 21. A reagent kit comprising the following:

[0097] (a) PCR primers for amplifying a fragment containing a target nucleic acid, wherein one of the PCR primers is biotinylated;

[0098] (b) one or more restriction endonucleases; and

[0099] (c) Magnetic beads containing streptavidin,

[0100] (d) Amplification primer pair for amplifying a fragment containing the target nucleic acid, wherein the amplification primer pair may or may not contain a biotin tag.

[0101] The target nucleic acid contains the nucleic acid region to be detected.

[0102] 22. The kit described in item 21, wherein the nucleic acid region to be detected is the region where the gene mutation to be detected is located;

[0103] Preferably, the gene mutation is a disease-related gene mutation;

[0104] More preferably, the disease-related gene mutation is an AML-related gene mutation; even more preferably, the AML-related gene mutation is a mutation in the IDH1 / 2 gene, preferably a gene mutation encoding the R132H mutation of IDH1.

[0105] 23. The kit according to item 21 or 22, wherein the restriction endonuclease is capable of cleaving the amplified fragment product only when the position corresponding to the gene mutation is wild-type, and cannot cleave the amplified fragment product when it contains the gene mutation.

[0106] 24. The kit according to item 23, wherein the wild-type bases at the corresponding positions of the gene mutation constitute part of the restriction site.

[0107] 25. The kit according to item 24, wherein the amplification primers are capable of introducing mismatched bases into the amplified fragment product, the mismatched bases forming part of the restriction site of the restriction endonuclease.

[0108] 26. The kit according to item 25, wherein the amplification primers are primers for performing derivatized enzyme digestion amplification polymorphic sequence (dCAPS) technology.

[0109] 27. The kit according to any one of items 21-26, wherein the restriction endonuclease is PvuI enzyme.

[0110] 28. The method according to any one of items 21-27, wherein the nucleotide sequence of the amplification primer in (a) is as shown in SEQ ID NO:1 and / or SEQ ID NO:2; and SEQ ID NO:3.

[0111] 29. The method according to any one of items 21-28, wherein the nucleotide sequence of the amplification primer in (d) is as shown in SEQ ID NO:1 and / or SEQ ID NO:2; and SEQ ID NO:3. Example

[0112] To provide a more comprehensive understanding and application of this application, the following description will refer to embodiments and accompanying drawings. These embodiments are merely illustrative and not intended to limit the scope of the application. The scope of this application is specifically defined by the appended claims.

[0113] Example 1. Procedure for enriching IDH1-R132H mutations using the MICAPS method

[0114] This embodiment details the experimental procedure for enriching IDH1-R132H mutations using the MICAPS method described in this application.

[0115] (1) Extraction of whole genome DNA

[0116] Routine whole-genome DNA extraction was performed from peripheral blood samples using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0. Other methods and kits for whole-genome DNA extraction are well known to those skilled in the art.

[0117] (2) dCAPS PCR

[0118] Genomic DNA was amplified by dCAPS PCR, and the gene fragment containing the target site IDH1 (R132H) was amplified. Simultaneously, a biotin tag was introduced for subsequent streptobiotin enrichment. The following primers were used:

[0119] Forward primer, 5'-GTGAGTGGATGGGTAAAACCTATCATCAT C GA T-3' (SEQ ID NO:1) (5' end carries a biotin tag for constructing the PvuI restriction site); or

[0120] Forward primer, 5'-GTGAGTGGATGGGTAAAACCTATCATCAT A G GC C-3' (SEQ ID NO:2) (5' end carries a biotin tag for constructing the BceAI restriction site); and

[0121] Reverse primer, 5'-GTGTTGAGATGGACGCCTATTTG-3' (SEQ ID NO:3).

[0122] In the forward primer SEQ ID NO:1, the bases highlighted in bold underline at the 3' end are the bases used to introduce mismatches. The last four bases at the 3' end, including these two bases, together with the following C and G bases in the wild-type sequence, constitute the recognition site for PvuI. These following C and G bases are precisely the C and G in the arginine codon CGT at position 132. In the IDH1-R132H mutation, the codon at position 132 is mutated to CAT, where G changes to A, thus the amplified product cannot be recognized by PvuI.

[0123] In the forward primer SEQ ID NO:2, the bases highlighted in bold underline at the 3' end are used to introduce mismatches. The last three bases at the 3' end, including this base, along with the following G and T bases in the wild-type sequence, constitute the BceAI recognition site. The last three CGT bases encode the arginine codon at position 132. In the IDH1-R132H mutation, the codon at position 132 is mutated to CAT, where G changes to A, thus the amplified product cannot be recognized by BceAI.

[0124] The reaction system is as follows, where the final concentration of each primer is 0.25 μM.

[0125] 2x Taq Mix (NEB) 12.5μl <![CDATA[ddH2O]]> 11μl DNA template (approximately 500 ng) 1μl Primers 0.5μl Total 25μl

[0126] The PCR reaction conditions were: 95℃ for 2 minutes; 25 cycles of 95℃ for 30 seconds, 56℃ for 30 seconds, and 68℃ for 20 seconds; and 68℃ for 5 minutes.

[0127] (3) Restriction enzyme digestion

[0128] The dCAPS PCR product was digested with PvuI or BceAI at 37℃ for 15 min. The specific reaction system is as follows:

[0129] PCR products 1μl <![CDATA[H2O]]> 7μl 10X buffer 1μl PvuI or BceAI 1μl Total 10μl

[0130] (4) Purification of streptavidin

[0131] The enzyme digestion products were purified using streptavidin magnetic beads. The reaction system is shown in the table below.

[0132]

[0133] The reaction was incubated at room temperature for 10 minutes, followed by elution three times with 50 μl of 1x elution buffer. The product purified by streptavidin magnetic beads was diluted to 50 μl with H2O.

[0134] Optionally, using 5 μl of streptavidin purified product as a template, repeat the aforementioned steps (2), (3), and (4).

[0135] (5) Sequencing

[0136] The products obtained through the aforementioned enrichment steps are used for downstream sequencing. Sanger sequencing will be used as an example to illustrate the subsequent steps.

[0137] The streptavidin magnetic bead purified product was subjected to PCR to amplify the product to a level suitable for sequencing. The primers used were the same as those used in the previous PCR, but without the biotin tag:

[0138] Forward primer, 5'-GTGAGTGGATGGGTAAAACCTATCATCATCGAT-3'

[0139] (SEQ ID NO:1); or

[0140] Forward primer, 5'-GTGAGTGGATGGGTAAAACCTATCATCAT A G G CC-3'(SEQ ID NO:2); and

[0141] Reverse primer, 5'-GTGTTGAGATGGACGCCTATTTG-3' (SEQ ID NO:3).

[0142] The final concentrations of both primers were 2 μM. The PCR reaction system is as follows:

[0143] 2x Master Mix(Thermo Fisher F632L) 20μl <![CDATA[H2O]]> 11μl Purified product 5μl Primers 4μl Total 40μl

[0144] The reaction conditions were: 95℃ for 2 minutes; 40 cycles of 95℃ for 30 seconds, 56℃ for 30 seconds, and 68℃ for 20 seconds; followed by 5 minutes at 68℃. The PCR products were purified using a SteadyPure PCR reaction solution purification kit (AG, China) and then subjected to Sanger sequencing. The mutation rate was estimated based on the base peaks at the mutation sites of the genes to be detected in the sequencing data.

[0145] Example 2. Detection of IDH1-R132H mutation in plasmid standards using MICAPS combined with Sanger sequencing (PvuI restriction site construction method)

[0146] This embodiment demonstrates the performance of samples enriched by the method of this application in Sanger sequencing.

[0147] The gene fragment encoding IDH1 R132 (SEQ ID NO:4) was inserted into plasmid pBluescriptKS (Guangzhou Bioyard Biotechnology Development Co., Ltd.) to obtain plasmid PKS-IDH1 WT. The amino acid sequence of the polypeptide encoded by SEQ ID NO:4 is shown in SEQ ID NO:7. Point mutations of PKS-IDH1 R132H were induced by PCR using primers D1_Hm5 (5'-ATCATAGGTCATCATGCTTATG; SEQ ID NO:5) and D1_Hm3 (5'-GATAGGTTTTACCCATCC; SEQ ID NO:6). Finally, plasmid standards with mutation rates of 50%, 1%, and 0% were obtained by mixing PKS-IDH1 WT and PKS-IDH1 R132H in different proportions.

[0148] Various plasmid standards were enriched using the MICAPS method as described in Example 1. The enriched samples were amplified by PCR using primer pairs SEQ ID NO:1 and SEQ ID NO:2, and the products were subjected to Sanger sequencing. The unenriched samples, serving as plasmid standards, were directly amplified by PCR using primer pairs SEQ ID NO:1 and SEQ ID NO:2, and the products were subjected to Sanger sequencing. The results are shown below. Figure 2 .

[0149] like Figure 2 As shown, the MICAPS method combined with Sanger sequencing detected the IDH1-R132H mutation in plasmid standards. The IDH1-R132H mutation is c.395G>A (marked in the dashed box, complementary strand sequencing result). Without MICAPS enrichment, Sanger sequencing detected a negative result for the IDH1-R132H plasmid standard with a 1% mutation rate. However, after one enrichment with MICAPS, Sanger sequencing could clearly detect the standard with a 1% mutation rate.

[0150] Example 3. Detection of IDH1-R132H mutation in plasmid standards using MICAPS combined with Sanger sequencing (construction of BceAI restriction site method)

[0151] This embodiment demonstrates the performance of samples enriched by the method of this application in Sanger sequencing.

[0152] The gene fragment encoding IDH1 R132 (SEQ ID NO:4) was inserted into plasmid pBluescriptKS (Guangzhou Bioyard Biotechnology Development Co., Ltd.) to obtain plasmid PKS-IDH1 WT. The amino acid sequence of the polypeptide encoded by SEQ ID NO:4 is shown in SEQ ID NO:7. Point mutations of PKS-IDH1 R132H were induced by PCR using primers D1_Hm5 (5'-ATCATAGGTCATCATGCTTATG; SEQ ID NO:5) and D1_Hm3 (5'-GATAGGTTTTACCCATCC; SEQ ID NO:6). Finally, plasmid standards with mutation rates of 50%, 1%, and 0% were obtained by mixing PKS-IDH1 WT and PKS-IDH1 R132H in different proportions.

[0153] Various plasmid standards were enriched using the MICAPS method as described in Example 1. The enriched samples were amplified by PCR using primer pairs SEQ ID NO:2 and SEQ ID NO:3, and the products were sequenced by Sanger sequencing. The unenriched samples, serving as plasmid standards, were directly amplified by PCR using primer pairs SEQ ID NO:2 and SEQ ID NO:3, and the products were sequenced by Sanger sequencing. The results are shown below. Figure 3 .

[0154] like Figure 3 As shown, the MICAPS method combined with Sanger sequencing detected the IDH1-R132H mutation in plasmid standards. The IDH1-R132H mutation is c.395G>A (marked in the dashed box). Without MICAPS enrichment, Sanger sequencing detected a negative result for the IDH1-R132H plasmid standard with a 1% mutation rate. However, after one enrichment with MICAPS, Sanger sequencing could clearly detect the standard with a 1% mutation rate.

[0155] IDH Sequence Description

[0156]

[0157]

[0158] The description in this disclosure is provided for illustrative and descriptive purposes only and is not intended to be exhaustive or to limit the disclosure to its forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of this disclosure and to enable those skilled in the art to understand this disclosure and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for enriching target nucleic acids containing the IDH gene mutation R132H in a nucleic acid sample, comprising: (1) Provide a nucleic acid sample containing the target nucleic acid; (2) Amplify using a nucleic acid sample containing the target nucleic acid in step (1) as a template, including the step of making the amplified fragment contain mismatched bases, and one of the amplified primers in the amplification primer pair is biotin-labeled, thereby obtaining a biotin-labeled amplified fragment product. (3) The amplified fragment product obtained in step (2) is digested using a restriction endonuclease to obtain the digested product; (4) The digestion product obtained in step (3) was purified using a solid support containing streptavidin to obtain an enriched sample containing the target nucleic acid; and (5) Amplify the sample obtained in step (4) to further enrich the target nucleic acid in the sample. The mismatched bases in step (2) and the wild-type sequence corresponding to the gene mutation together constitute the recognition site of the restriction endonuclease in step (3); the restriction endonuclease can only cleave the amplified fragment product when the position corresponding to the gene mutation is wild-type, and cannot cleave the amplified fragment product when it contains the gene mutation. Preferably, the biotin-tagged amplification primers have the biotin tag located at the 5' end of the PCR primers; Preferably, the step of incorporating mismatched bases into the amplified fragment includes amplifying a fragment containing the target nucleic acid using derivatized enzyme digestion amplification polymorphic sequence (dCAPS) technology.

2. The method according to claim 1, wherein the nucleic acid sample containing the target nucleic acid is genomic DNA or a fragment thereof containing the site of the mutation to be detected.

3. The method according to any one of claims 1 or 2, wherein the nucleic acid sample is derived from cells, tissues, body fluids or blood, preferably peripheral blood.

4. The method according to any one of claims 1 to 3, wherein the length of the amplified fragment product containing the target nucleic acid in step (2) is 200 bp to 1000 bp.

5. The method according to any one of claims 1 to 4, wherein the restriction endonuclease is PvuI enzyme and / or BceAI enzyme.

6. The method according to any one of claims 1 to 5, wherein the nucleotide sequences of the amplification primers used in step (2) are as shown in SEQ ID NO:1 and / or SEQ ID NO:2; and SEQ ID NO:

3.

7. The method according to any one of claims 1 to 6, wherein the primers used in step (5) have the same sequence as the primers in step (2), and wherein either of the amplification primers is free of or one of the primers is biotinylated.

8. A method for detecting the presence and / or frequency of the IDH gene mutation R132H in a sample, comprising enriching the target nucleic acid containing the gene mutation in the nucleic acid sample using the method of any one of claims 1 to 7, and detecting the IDH gene mutation in the enriched sample by sequencing or PCR amplification.

9. A kit for enriching the IDH gene mutation R132H, comprising the following items: (a) A PCR primer pair for amplifying a fragment containing a target nucleic acid, wherein one of the primers in the PCR primer pair is tagged with biotin; (b) PvuI enzyme and / or BceAI enzyme; and (c) Solid support containing streptavidin; (d) an amplification primer pair for amplifying a fragment containing a target nucleic acid, wherein either amplification primer in the amplification primer pair is free of or one of the primers is biotinylated. The target nucleic acid contains the nucleic acid region where the IDH gene is located.

10. The kit according to claim 9, wherein the sequences of the amplification primer pair in step (a) and the amplification primer pair in step (d) are as shown in SEQ ID NO:1 and / or SEQ ID NO:2; and SEQ ID NO:3.

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