39 results about "5-Hydroxymethylcytosine" patented technology

The present invention relates generally to the field of molecular biology. More particularly, it concerns methods and compositions for detecting, evaluating, and / or mapping 5-hydroxymethyl-modified cytosine bases within a nucleic acid molecule.

The present invention relates to methods and kits for the detection of 5-hydroxymethylcytosine (5hmC) and / or 5-methylcytosine (5meC). In some embodiments, the present invention relates to methods and kits for detection of 5hmC and / or 5meC in nucleic acid (e.g., DNA, RNA). In some embodiments, the present invention relates to detection of 5hmC in genomic DNA, e.g., mammalian genomic DNA

The present invention provides a method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine, comprising the following steps: (1) DNA purification and fragmentation pretreatment: the target DNA is extracted and then broken to an average of 50 nucleotides to 10,000 nucleotides in length; (2) the repair of trace amount of DNA and the ligation thereof to the adaptor: the pre-treated DNA fragments are repaired and ligated with the sequencing adaptor required for the second-generation sequencing, (3) covalently labeling 5-methylcytosine and 5-hydroxymethylcytosine, (4) solid-phase enrichment of the labeled DNA fragments having cytosine with 5-position modification; (5) the PCR amplification of the solid-phase enriched DNA fragments, the PCR product is obtained and purified to obtain a library for the second-generation sequencing, after mapping the sequencing reads to the genome, the distribution map of the cytosine with 5-position modification in the DNA sample could be generated. The present invention greatly enhances the selectivity and efficiency of binding of the solid-phase surface with the DNA modified base.

The present invention relates to the field of cancer. More specifically, the present invention provides methods and compositions useful for diagnosing or predicting cancer in a patient. In one embodiment, a method for identifying a patient as having cancer comprises the steps of (a) providing a formalin-fixed, paraffin-embedded or fresh frozen sample of patient tissue; (b) steaming the sample in antigen retrieval buffer; (c) incubating the sample in hydrochloric acid (HCl); (d) incubating the sample with an affinity reagent specific for 5hmC under conditions to form a complex between the affinity reagent and 5-hydroxymethylcytosine (5hmC) present in the sample; (e) detecting the complexes formed between 5hmC and the affinity reagent with secondary detection reagents; (f) quantifying 5hmC levels; and (g) identifying the patient as having cancer if the 5hmC levels in the sample are reduced as compared to a control.

Provided herein are methods for detecting and quantifying 5-hydroxymethylated cytosine bases in a DNA molecule.

The invention discloses a photoelectrochemical biosensor for detecting 5-hydroxymethylcytosine (5hmC) deoxyribonucleotide and a preparation method thereof. The photoelectrochemical biosensor comprisesan electrode and tungsten sulfide, polydopamine, mercaptobenzoic acid, 5hmC, phos-tag-biotin and streptavidin which are sequentially modified on the surface of the electrode. High sensitivity and high specificity detection of 5 hmC can be realized by use of the good photoelectric activity and biocompatibility of the tungsten sulfide, the excellent electronic donor properties and biocompatibilityof the polydopamine, the specific recognition and binding properties of phos-tag-biotin to phosphoric acid groups, and the specific reaction of hydroxymethyl and sulfydryl groups on the 5hmC catalyzedby M.HhaI methyltransferase. The method can well eliminate the interference of 5mC to the detection of the 5hmC. The detection method is simple, instrument miniaturization is realized, the photoelectrochemical biosensor is easy to operate, and the detection of the 5hmC can be realized by simple treating of the surface of the electrode.

A method of detecting the level of 5-hydroxymethylcytosines (5-hmc) in a DNA molecule of a cell having a 5-hmc prevalence lower than 0.002% of total DNA bases is provided. The method comprising:(a) attaching a 5-hmc labeling agent to the DNA molecule; and(b) subjecting the DNA molecule to an imaging method suitable for detecting the labeling agent, thereby detecting the level of 5-hmc in the DNA molecule. Also provided is a method of diagnosing cancer in a subject in need thereof, the method comprising:(a) providing a DNA sample of a cell of the subject;(b) detecting the level of 5-hmc in the DNA sample as described herein;wherein a significant decrease in the level of 5-hmc in the DNA sample, as compared to a control DNA sample from a healthy subject is indicative that the subject has cancer.

The present invention relates to methods and kits for the detection of 5-hydroxymethylcytosme (5hmC). In some embodiments, the present invention relates to methods and kits for detection of 5hmC in nucleic acid (e.g., DNA, RNA). In some embodiments, the present invention relates to detection of 5hmC in genomic DNA, e.g., mammalian genomic DNA.

The present invention relates to methods and kits for the detection of 5-hydroxymethylcytosme (5hmC). In some embodiments, the present invention relates to methods and kits for detection of 5hmC in nucleic acid (e.g., DNA, RNA). In some embodiments, the present invention relates to detection of 5hmC in genomic DNA, e.g., mammalian genomic DNA.

The present invention is related to the use of 5-hydroxymethylcytosine or a biomolecule having general structure of 5-hydroxymethycytosine as a biomarker for the detection, treatment monitoring, and prognostic prediction of cancer.

The invention relates to the technical field of clinical molecular diagnosis, in particular to a superficial modification based 5hmC (5-hydroxymethylcytosine) polymolecular marker and a CRC (colorectal cancer) early diagnosis model. With the adoption of a 5-hmC high throughput sequencing technology, 5-hmC expression quantities of peripheral blood plasma DNA whole genomes of a CRC patient and a healthy person are checked, 5-hmC differential expression of genetic loci of the whole genomes of the two groups of samples are compared, the genetic locus of the part with the most obvious differentialexpression is screened as the gene marker of the diagnosis model, and then the CRC early diagnosis model is constructed. Verification proves that the diagnosis sensitivity and specificity are both higher, and compared with a stool occult blood detection kit and a spetin9 kit in the market currently, sensitivity and specificity are improved by about 20% and 8% respectively. The diagnosis method hasthe advantages of being noninvasive in detection and convenient, can be applied to the first-line clinic and is used for early screening of CRC.

The invention provides a fluorescent detection method of 5-hydroxymethylcytosine based on FRET (Fluorescence Resonance Energy Transfer) principle with non-diagnostic purposes. The fluorescent detection method comprises the following steps of: firstly, oxidizing the 5-hydroxymethylcytosine in a DNA into 5-aldehyde cytosine by using potassium perruthenate; secondly, reacting a BODIPY (Boron Trifluoride Complex Dipyrromethene) compound with fluorescence with the 5-aldehyde cytosine in the DNA; and finally, adding a cationic fluorescent polymer, and measuring the content of the 5-hydroxymethylcytosine in the DNA through fluorescent detection. The detection method has the advantages of simpleness and rapidity, and low cost.

The invention discloses a method for detecting 5-aldehyde cytosine and 5-hydroxymethylcytosine in DNA (deoxyribonucleic acid) by utilizing a piperidine aqueous solution. Piperidine specifically reacts with 5-aldehyde cytosine in DNA with a fluorescent mark; the 5-aldehyde cytosine can be detected while the 5-aldehyde cytosine in the DNA is oxidized into 5-aldehyde cytosine through potassium perruthenate, and subjected to piperidine treatment, and the position and the quantity of the 5-hydroxymethylcytosine can be obtained by comparing two reacting DNA bands in a DNA polyacrylamide modified adhesive. By adopting the method, detection of the 5-aldehyde cytosine and the 5-hydroxymethylcytosine can be rapidly, effectively and sensitively achieved.

The invention relates to a gene marker, a kit and a detection method for detecting pancreas cancer. The gene marker comprises one or two or more of the following genes: SI, CLEC4C, SNX7, MEOX2, FAT1, FMO3, CFTR, PLPPR3, AFM and COL5A2. The content of 5-hydroxymethylcytosine in the pancreas cancer gene marker is detected through high-throughput sequencing, so as to determine whether the pancreas caner exists. The gene marker for detecting pancreas cancer has the advantages of being safe and noninvasive, having a wide DNA source, being highly accurate, being convenient to operate, and providing good user experience. The gene marker can be combined with other clinical indexes, and provides more accurate determining results for screening, diagnosis, treatment and prognosis of pancreas cancer.

The present invention relates to a diagnostic reagent and a kit for cancer. The present invention discloses that the existence condition of 5-hydroxymethylcytosine (hmc) in organizations is closely related to the incidence and development of cancer, the significant decrease of the hmc level in the organization indicates incidence or development of cancer, and also the hmc level indicates the severity of cancer. Therefore, the hmc detection reagent can be well applied to cancer diagnosis, prognosis or parting, and has a high clinical application value.

The present invention relates to the field of cancer. More specifically, the present invention provides methods and compositions useful for diagnosing or predicting cancer in a patient. In one embodiment, a method for identifying a patient as having cancer comprises the steps of (a) providing a formalin-fixed, paraffin-embedded or fresh frozen sample of patient tissue; (b) steaming the sample in antigen retrieval buffer; (c) incubating the sample in hydrochloric acid (HCl); (d) incubating the sample with an affinity reagent specific for 5hmC under conditions to form a complex between the affinity reagent and 5-hydroxymethylcytosine (5hmC) present in the sample; (e) detecting the complexes formed between 5hmC and the affinity reagent with secondary detection reagents; (f) quantifying 5hmC levels; and (g) identifying the patient as having cancer if the 5hmC levels in the sample are reduced as compared to a control.

The invention discloses a method and kit for detecting 5-hydroxymethyl cytosine in DNA through boric acid mediated polymerase chain reactions. On the basis of glucosylation reactions, boric acid and derivative reagents can carry out covalent effect with vicinal diol in glycosylated 5-hydroxymethyl cytosine; thus the volume of the duplicated base unit is prominently increased, the polymerase amplification reactions are effectively inhibited; boric acid mediated polymerase chain amplification reactions are provided, and the 5-hydromethyl cytosine in specific gene / sections can be specifically recognized. The invention further provides a kit on the basis of the method, and the kit can rapidly and sensitively detect the 5-hydroxymethyl cytosine and can be applied to various cell analysis. Compared with the conventional detection kits, the provided kit is not limited by the cleavage sites, and can be used to analyze and identify 5-hydroxymethyl in various sequences. The kit has a wide application prospect, and has an important meaning for the further research on the biological functions of 5-hydroxymethyl cytosine.

The invention relates to a method and a reagent for adjusting the methylation / demethylation state of nucleic acid. An inventor proves that 5-methylcystein (mC) and 5-hydroxymethylcytosine (hmC) in genome deoxyribonucleic acid (DNA) can be oxidized into 5-carboxyl cytosine (caC) by Tet dioxygenase for the first time. Thymine DNA glycosylase (TDG) can specifically identify the 5caC and remove the 5caC from the genome to ensure that DNA is demethylated actively. Therefore, the methylation / demethylation effect of DNA in the genome can be adjusted, and the reagent for adjusting methylation / demethylation effect of DNA in the genome can be prepared. Through verification, the genome DNA in a cell has 5caC modification, a 5caC modification spectrum can be used for monitoring the state of the cell, and adenosine triphosphate (ATP) and analogues thereof can be used as regulators of Tet enzyme. Moreover, by Tet mutation in leukemia, the 5caC activity is reduced or the 5caC is inactivated.

Provided are a method and a kit for detecting 5-hydroxymethylcytosine in a nucleic acid. The method is a method for detecting 5-hydroxymethylcytosine in a nucleic acid, comprising the steps of: (1) oxidizing 5-hydroxymethylcytosine in a nucleic acid sample by treating the nucleic acid sample with a tungstic acid-based oxidizing agent comprising peroxotungstic acid, tungstic acid, a salt thereof, or a combination thereof with a reoxidizing agent; and (2) determining the position of the oxidized 5-hydroxymethylcytosine in the nucleic acid sample.

The invention discloses application of 5-hydroxymethylcytosine and a method for detecting distribution of 5-hydroxymethylcytosine in mouse embryo. The detection method comprises the following steps: (a) allowing a mouse to perform natural insemination or in vitro fertilization, and extracting the mouse embryo; (b) dyeing the mouse embryo; and (c) observing staining by virtue of a microscope or performing quantitative analysis. By adopting the detection method, the distribution of 5hmc in a mouse pre-implanted embryo is analyzed, the dynamical changing situation of 5mc and 5hmc in paternal and maternal chromosomes in the mouse pre-implanted embryo developing phase can be proved, and the important role of 5hmc to epigenetic reprogramming process in the mouse pre-implanted embryo developing phase can be disclosed, so that a new prospect is opened for studying a DNA (deoxyribonucleic acid) demethylation mechanism, and the 5-hydroxymethylcytosine can be used as a male nucleus marker or a female nucleus marker.

Provided is a sequencing method for the genetic mapping of DNA 5-methylcytosine and 5-hydroxymethylcytosine. The method employs next generation sequencing and library construction technology for trace free DNA and a high-efficiency chemical bioorthogonal reaction, which greatly enhances the selectivity and efficiency of a solid-phase surface binding to a DNA modified base.

Provided are a method and a kit for detecting 5-hydroxymethylcytosine in a nucleic acid. The method is a method for detecting 5-hydroxymethylcytosine in a nucleic acid, comprising the steps of: (1) oxidizing 5-hydroxymethylcytosine in a nucleic acid sample by treating the nucleic acid sample with a tungstic acid-based oxidizing agent comprising peroxotungstic acid, tungstic acid, a salt thereof, or a combination thereof with a reoxidizing agent; and (2) determining the position of the oxidized 5-hydroxymethylcytosine in the nucleic acid sample.

The invention discloses a single-base resolution localization analysis method for 5-carboxycytosine modification in DNA assisted by deaminase. The cytosine deaminase APOBEC3A protein is used to efficiently deaminate normal cytosine, 5-methylcytosine, 5-hydroxymethylcytosine and 5-aldehyde cytosine in DNA, except 5-carboxycytosine. Then PCR amplification is carried out, followed by sequencing to obtain the site information of 5-carboxycytosine. This method has the advantages of high sensitivity, high selectivity and simple operation, and can be adopted to directly obtain the single base resolution location of 5-carboxycytosine in DNA without the treatment of a DNA sample with bisulfite.

The present invention includes mutant AID, APOBEC, and Tet enzymes with improved functions. In one aspect the invention provides APOBEC fusion proteins comprising hyperactive deamination activity. In another aspect, the invention provides AID mutant proteins comprising hyperactive deamination activity. In yet another aspect, the invention provides mutant Tet proteins capable of stalling oxidation at a 5-hydroxymethylcytosine (hmC).

The invention provides a method for simultaneously detecting genomic DNA polymorphism and methylation. The method comprises the following steps: performing DNA fragmentation, and digesting the 3' endof the DNA fragment with DNA exonuclease; adding a dNTPs mixture containing 5-hydroxymethylcytosine deoxyribonucleotide, and performing completion the 3' end of the DNA fragment; performing bisulfiteconversion treatment on the modified DNA fragment and connecting an adaptor adapted to a high-throughput sequencing platform, and finally constructing a sequencing library by PCR amplification. By combining a double-end sequencing mode of the high-throughput sequencing platform, genomic DNA polymorphism and methylation detection can be performed simultaneously, and the goal of simultaneous detection of genome and epigenome is achieved.

The invention discloses a method for identifying and detecting 5-hydroxymethyl cytosine (5hmC) and 5-formyl cytosine (5fC) in DNA and belongs to the fields of molecular biology, nucleic acid chemistryand epigenetics, The method includes: adding to-be-detected DNA into a mixed solution containing potassium perruthenate and alkali, performing reaction under an ice water bath condition to allow the5hmC in the DNA to be oxidized into the 5fC; purifying the DNA after the reaction, adding into a hot alkali solution to perform heat-preservation reaction to achieve DNA cleavage, and performing gel analysis to measure the total number and site of the 5hmC and 5fC in the DNA; directly adding the to-be-detected DNA into the hot alkali solution without potassium perruthenate oxidation to perform heat-preservation reaction to achieve DNA cleavage, and performing gel analysis to measure the total number and site of the 5fC in the DNA; comparing the two results to obtain the number and site of the5hmC and 5fC in the DNA. By the method high in sensitivity and wide in application range, the defects that existing detection methods are high in equipment requirements, complex in operation and the like.

The invention relates to the technical field of biochemistry, in particular to a method for detecting 5-methylcystein in RNA and a kit thereof. The method for detecting 5-methylcystein in RNA comprises the steps that total RNA and a modified reagent are mixed and modified, and modified RNA is obtained; the modified RNA is combined on a nitrocellulose membrane, drying and sealing are carried out, and an antigen membrane is obtained; the antigen membrane and a 5-hydroxymethylcytosine antibody are subjected to first incubation, then the antigen membrane and a second specific antibody are subjected to second incubation, development is carried out, and a detecting result is obtained. According to the detecting method, 5-methylcystein in RNA can be detected and analyzed fast, high sensitivity is achieved, operation is easy, and sample preparation is easy.