CN115992238A - Gene modification detection method and application thereof - Google Patents

Gene modification detection method and application thereof Download PDF

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CN115992238A
CN115992238A CN202211277434.7A CN202211277434A CN115992238A CN 115992238 A CN115992238 A CN 115992238A CN 202211277434 A CN202211277434 A CN 202211277434A CN 115992238 A CN115992238 A CN 115992238A
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sample
disease
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林坚
陈航宇
许诺
陈龙
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Panshi Jincheng Biotechnology Beijing Co ltd
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Peking University
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Abstract

The application relates to a gene modification detection method and application thereof, which comprises the steps of obtaining a substance to be detected with a diameter of more than 200 nanometers in a sample to be detected, and detecting the quantity and/or the existence of hydroxymethyl cytosine in the substance to be detected. The application also provides application of the gene modification detection method.

Description

Gene modification detection method and application thereof
Technical Field
The application relates to the biomedical field, in particular to a gene modification detection method and application thereof.
Background
Extracellular vesicles (Extracellular Vesicle) are 50-1000nm membrane vesicles secreted by most cells and are present in biological fluids such as cell culture media, plasma, serum, saliva, urine, amniotic fluid and malignant ascites. Wherein the size of large extracellular vesicles is >200nm. There is growing evidence that these vesicles act as cellular messenger, conveying information to cells and tissues within the body. Extracellular vesicles contain cell-specific proteins, lipids, and RNAs, which are transported to other cells and alter the function or physiological effects of the other cells. These extracellular vesicles may play a role in mediating adaptive immune responses to infectious pathogens and tumors, tissue repair, neural communication, and metastasis of pathogenic proteins.
Clinically, tissue sampling is a major difficulty in limiting molecular diagnosis and accurate treatment of tumors. The liquid biopsy samples such as blood, urine and the like are easy to obtain, noninvasive and the like, so that the liquid biopsy samples are reliable sources of tumor DNA and are the hot spot field of the current tumor molecular diagnosis. Liquid biopsy samples mainly include plasma free DNA (cfDNA), circulating Tumor Cells (CTCs), and extracellular vesicles (Extracellular Vesicle). At present, cfDNA gene detection is mature and widely applied, however cfDNA is DNA fragments after apoptosis or death of tumor cells, and may not accurately reflect real-time gene information of tumor cells. In contrast, extracellular vesicle-derived nucleic acids (exorns) such as plasma and urine are actively released by living tumor cells, possibly reflecting the real-time dynamics of the tumor better.
DNA methylolation is an important form of epigenetic modification that has been demonstrated to be a stable epigenetic marker in the human genome, positively correlated with gene transcript levels, playing a vital role in cellular development, differentiation, maturation and self-renewal. 5-hydroxymethylcytosine (5 hmC) is a very promising biomarker, and is closely related to the occurrence and development of various diseases in human beings. Therefore, the development of 5hmC sequencing technology based on extracellular vesicle DNA is of great clinical significance.
Disclosure of Invention
The application provides a gene modification detection method and application thereof, and can comprise a 5hmC sequencing technical method based on extracellular vesicle DNA. The liquid biopsy samples such as blood, urine and the like are easy to obtain, noninvasive and the like, so that the liquid biopsy samples are reliable sources of tumor DNA, are also the hot spot field of the current tumor molecular diagnosis, however cfDNA is DNA fragments after tumor cell apoptosis or death, and the real-time gene information of tumor cells can not be accurately reflected. The extracellular vesicle-derived nucleic acid (exoRN) provided by the application is actively released by living tumor cells, wherein the preferred large extracellular vesicle has a size of >200nm, can serve as a cell messenger, can transfer information to cells and tissues in a body, and can better reflect the real-time dynamics of the tumor.
In one aspect, the present application provides an assay method comprising (S1) obtaining a test substance having a diameter of about 200nm or more in a test sample, and (S2) detecting the amount and/or presence of hydroxymethylcytosine in the test substance.
In another aspect, the present application provides a method for confirming the presence of a disease, assessing the risk of disease formation or formation, assessing the progression and/or prognosis of a disease and/or screening a population for a treatment, comprising (S1) obtaining a test substance having a diameter of about 200nm or more in a test sample, and (S2) detecting the number and/or presence of hydroxymethylcytosine in a test region in the test substance.
In another aspect, the present application provides an assay comprising detecting the amount and/or presence of hydroxymethylcytosine in a test substance having a diameter of about 200 nm or more in a test sample.
In another aspect, the present application provides a method for confirming the presence of a disease, assessing the risk of disease formation or formation, assessing the progression and/or prognosis of a disease and/or screening a population for a treatment, comprising detecting the amount and/or presence of hydroxymethylcytosine in a test substance having a diameter of about 200 nm or more in a test sample.
In another aspect, the present application provides a nucleic acid comprising a sequence capable of binding to a test region, or a region complementary thereto, or a fragment thereof, in a test substance having a diameter of about 200 nm or more in a test sample.
In another aspect, the present application provides a method for preparing a nucleic acid, comprising designing a nucleic acid capable of binding to a region to be measured, or a region complementary thereto, or a fragment thereof, in a substance to be measured having a diameter of about 200 nm or more in a sample to be measured.
In another aspect, the present application provides a kit comprising a nucleic acid of a pair of the present application.
In another aspect, the present application provides the use of a nucleic acid of the present application, and/or a kit of the present application, in the preparation of a disease detection product.
In another aspect, the present application provides the use of a nucleic acid of the present application, and/or a kit of the present application, for the preparation of a product for confirming the presence of a disease, assessing the risk of disease formation or formation, assessing the progression and/or prognosis of a disease, and/or screening a population for treatment.
In another aspect, the present application provides a database comprising sequences of regions to be measured, or complementary regions thereof, or fragments thereof, in a substance to be measured having a diameter of about 200 nm or more in a sample to be measured.
In another aspect, the present application provides a storage medium recording a program that can run the method of the present application.
In another aspect, the present application provides an apparatus comprising a storage medium as herein described.
Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.
Drawings
The specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention that are related to the present application will be better understood by reference to the exemplary embodiments and the drawings that are described in detail below. The drawings are briefly described as follows:
FIG. 1 shows the particle size distribution of isolated extracellular vesicles (> 200 nm) of the present application.
FIG. 2 shows the particle size distribution of the isolated exosomes (< 200 nm) of the present application.
FIG. 3 shows a graph of the quality control results of a library of 5-hydroxymethylcytosine (5 hmC) of DNA in extracellular vesicles (> 200 nm) isolated in the present application.
Figure 4 shows the particle size distribution of the dynamic light scattering assay of plasma isolated extracellular vesicles. Plasma isolated extracellular vesicle size (> 200 nm).
Figure 5 shows the particle size distribution of the dynamic light scattering assay of the plasma isolated exosomes. Plasma isolated exosome size (< 200 nm).
FIG. 6 shows the quality control of a 5-hydroxymethylcytosine (5 hmC) library of extracellular vesicle (> 200 nm) DNA: the results of the fully automated capillary electrophoresis system showed that the library size was about 306bp.
FIGS. 7A-7D show the results of tests for lung cancer patients and healthy persons. Fig. 7A: lung cancer and healthy person PCA plot: sequencing based on 30 lung cancer patients and 30 healthy human exosomes (< 200 nm) 5 hmC; fig. 7B: lung cancer and healthy person ROC graphs: distinguishing 30 lung cancer patients from 30 healthy people based on 5hmC markers in exosome DNA; fig. 7C: lung cancer and healthy person PCA plot: 5hmC sequencing based on 30 lung cancer patients and 30 healthy human extracellular vesicles (> 200 nm); fig. 7D: lung cancer and healthy person ROC graphs: 30 lung cancer patients and 30 healthy persons were distinguished based on 5hmC markers in extracellular vesicle DNA.
FIGS. 8A-8D show the results of tests for colorectal cancer patients and healthy persons. Fig. 8A: colorectal cancer and healthy person PCA profile: sequencing based on 25 colorectal cancer patients and 30 healthy human exosomes (< 200 nm) 5 hmC; fig. 8B: colorectal cancer and healthy person ROC graphs: distinguishing 25 colorectal cancer patients from 30 healthy people based on 5hmC markers in exosome DNA; fig. 8C: colorectal cancer and healthy person PCA profile: 5hmC sequencing based on 25 colorectal cancer patients and 30 healthy human exosomes (> 200 nm); fig. 8D: colorectal cancer and healthy person ROC graphs: 25 colorectal cancer patients and 30 healthy persons were distinguished based on 5hmC markers in extracellular vesicle DNA.
Detailed Description
Further advantages and effects of the invention of the present application will become apparent to those skilled in the art from the disclosure of the present application, from the following description of specific embodiments.
Definition of terms
In this application, the term "sample" generally refers to a material or mixture of materials, typically in liquid form or other form, which may contain one or more analytes of interest.
In this application, the term "extracellular vesicle" generally refers to an extracellular particle. For example, extracellular particles may refer to membranous particles released by a cell into the extracellular matrix. For example, the extracellular particles may have a diameter of about 30 nanometers to about 1000 nanometers. For example, extracellular vesicles may be a specific class of extracellular particles, e.g., extracellular vesicles may have diameters of about 200 nanometers or more.
In this application, the term "organoid" generally refers to a cell mass. For example, the organoids may have self-renewing and self-organizing capabilities, e.g., the organoids may have corresponding tissue organ functions. For example, the organoids may be cultured in matrigel with 3D stereo structure.
In this application, the terms "determine," "measure," "evaluate," "determine," and "analyze" are used interchangeably herein to generally refer to any form of measurement, including determining whether an element is present. These terms may both include quantitative and/or qualitative aspects. The evaluation may be relative or absolute. The "assessing … … for the presence" may include determining the amount of something present and determining the presence or absence thereof.
For example, a brief step of gene mutation detection may comprise taking a sample of a patient's tumor tissue, extracting DNA, detecting by a kit or other means, and determining whether the patient has a mutation; positive samples with mutations as clinical tests; as a clinical test negative sample, there was no mutation.
For example, a method of gene detection may comprise: polymerase chain reaction-restriction fragment length polymorphism analysis technique, pyrosequencing method, single-strand conformational isomerism polymorphism analysis technique, probe amplification blocking mutation system, high performance liquid chromatography, micro-digital polymerase chain reaction, high resolution melting curve analysis technique, and the like.
For example, the gene expression level detection may comprise detection according to a gene, a tumor condition selection kit, and other methods to determine whether a patient has a variation in gene expression level; positive samples for clinical examination with variation in gene expression level; the sample is used as a negative sample for clinical test without variation of gene expression.
For example, the genes for predicting occurrence of a disease or a symptom thereof may include genes related to a disease or a symptom thereof obtained by sequencing (including sequencing of DNA, RNA, etc.) of a clinical patient sample and analyzing the data, and genes capable of predicting occurrence of a disease or a symptom thereof by an expression level in the absence of occurrence of a disease or a symptom thereof. Samples predicted to have high correlation with diseases and symptoms related thereto may be used as positive samples for clinical tests, and samples predicted to have high correlation with diseases and symptoms related thereto may be used as negative samples for clinical tests.
For example, blood testing for tumor markers may involve blood collection to check the level of tumor markers in the blood. Or drainage of hydrothorax or ascites, detection of tumor markers, and comparison of the change of tumor marker content index in hydrothorax and ascites with the change of tumor marker index in blood of patients, thereby enhancing disease monitoring and diagnosis. Samples predicted to have high correlation with diseases and symptoms related thereto may be used as positive samples for clinical tests, and samples predicted to have high correlation with diseases and symptoms related thereto may be used as negative samples for clinical tests.
In the present application, the term "sequencing" generally refers to a method by which the identity of at least 10 consecutive nucleotides of a polynucleotide (e.g., the identity of at least 20, at least 50, at least 100, or at least 200 or more consecutive nucleotides) can be obtained.
In the present application, the term "UDP glucose modified with a chemoselective group" generally refers to UDP glucose that has been functionalized, which may be functionalized at the 6-hydroxy position, to include groups capable of participating in a 1, 3-cycloaddition (or "click") reaction. Such groups may include azido and alkynyl groups (e.g., cyclooctyne). For example, UDP-6-N3-Glu may be UDP glucose modified with a chemoselective group.
In the present application, the term "biotin moiety" is generally meant to include affinity tags of biotin or biotin analogues such as desthiobiotin, oxidized biotin, 2-iminobiotin, diaminobiotin, biotin oxysulfide, biotin, and the like. The biotin moiety may be bound to streptavidin.
In this application, the terms "cycloaddition" and "click reaction" are generally interchangeably described terms and generally refer to the 1, 3-cycloaddition between an azide and an alkyne group to form a five membered heterocyclic ring. In some embodiments, the alkynyl group may be strained (e.g., in a ring such as cyclooctyne), and the cycloaddition reaction may be performed in the absence of copper. Dibenzocyclooctyne (DBCO) and Difluorooctyne (DIFO) may be examples of alkynes capable of participating in a copper-free cycloaddition reaction.
In this application, the term "biotin-binding carrier" generally refers to a carrier (e.g., a bead, which may be magnetic) attached to streptavidin or avidin or a functional equivalent thereof.
In this application, the term "amplification" generally refers to the use of a target nucleic acid as a template to generate one or more copies of the target nucleic acid.
In this application, the term "copy of a fragment" generally refers to an amplified product, wherein the copy of the fragment may be the reverse complement of the fragment strand or may have the same sequence as the fragment strand.
In this application, the term "enriching" generally refers to partially purifying an analyte having a characteristic (e.g., a nucleic acid comprising hydroxymethylcytosine) from an analyte that does not have the characteristic (e.g., a nucleic acid comprising hydroxymethylcytosine). For example, enrichment will generally increase the concentration of an analyte having this characteristic (e.g., a nucleic acid comprising hydroxymethylcytosine) by at least a factor of 2, at least a factor of 5, or at least a factor of 10 relative to an analyte without the characteristic. After enrichment, at least 10%, at least 20%, at least 50%, at least 80%, or at least 90% of the analytes in the sample may have the characteristics used for enrichment. For example, in the enriched composition, at least 10%, at least 20%, at least 50%, at least 80%, or at least 90% of the nucleic acid molecules may comprise a strand having one or more hydroxymethylcytosine that has been modified to comprise a capture marker.
Detailed Description
In one aspect, the present application provides an assay method that may include obtaining a test substance having a diameter of about 200 nm or more in a test sample, and detecting the number and/or presence of hydroxymethylcytosine in the test substance.
In another aspect, the present application provides a method for confirming the presence of a disease, assessing the risk of disease formation or formation, assessing the progression and/or prognosis of a disease and/or screening a population for a treatment, which may comprise obtaining a test substance having a diameter of about 200 nm or more in a test sample, and detecting the number and/or presence of hydroxymethylcytosine in the test substance.
For example, a test sample of the present application may comprise plasma, urine, tissue culture fluid, cell culture fluid, and/or organoid culture fluid. For example, the test substances of the present application may comprise extracellular vesicles. For example, the extracellular vesicles of the present application may have diameters of about 200 nanometers or more. For example, the extracellular vesicles of the present application may have diameters of about 200 nm or more, 250 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 750 nm or more, 800 nm or more, 900 nm or more, or 1000 nm or more. For example, the extracellular vesicle diameter of the present application may be the average diameter of extracellular vesicles in a sample. For example, the test substances of the present application may contain nucleic acids. For example, the test substance of the present application may contain DNA.
For example, wherein the test sample may be of animal, plant and/or fungal origin. For example, wherein the test sample may comprise body fluids, tissues, cells and/or organoid culture supernatants. For example, wherein the body fluid in the sample to be tested may comprise plasma and/or urine. For example, wherein the tissue may comprise stomach tissue, liver tissue, brain tissue, kidney tissue, breast tissue, lung tissue, lymph node tissue, peritoneal tissue, and/or heart tissue.
For example, the methods of the present application may include the step of isolating extracellular vesicles. For example, the extracellular vesicle separation can include centrifugation, ultrafiltration, size exclusion chromatography, polymer precipitation, and/or affinity capture. For example, the centrifugation in the extracellular vesicle separation can include ultracentrifugation, density gradient centrifugation, sedimentation centrifugation, differential centrifugation, centrifugal panning, and/or zonal centrifugation.
For example, wherein said centrifugation in said extracellular vesicle separation can comprise a first centrifugation at a rotational speed of from about 300g to about 1000g. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a first centrifugation, said first centrifugation having a temperature of from about 2 ℃ to about 10 ℃. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a first centrifugation, said first centrifugation time being from about 5 minutes to about 20 minutes. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a first centrifugation at a rotational speed of from about 300g to about 1000g for from about 5 minutes to about 20 minutes at a temperature of from about 2 ℃ to about 10 ℃.
For example, the centrifugation in which the extracellular vesicles are separated may comprise a second centrifugation, which is performed using the supernatant obtained by the first centrifugation of the present application. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a second centrifugation at a rotational speed of from about 1500g to about 5000g. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a second centrifugation, said second centrifugation having a temperature of from about 2 ℃ to about 10 ℃. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a second centrifugation, said second centrifugation time being from about 10 minutes to about 30 minutes.
For example, the centrifugation in which the extracellular vesicles are separated may comprise a third centrifugation, which is performed using the supernatant obtained by the second centrifugation described herein. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a third centrifugation at a rotational speed of from about 10000g to about 15000g. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a third centrifugation, said third centrifugation having a temperature of from about 2 ℃ to about 10 ℃. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a third centrifugation, said third centrifugation time being from about 10 minutes to about 30 minutes. For example, wherein the third centrifugation may result in a tube bottom sediment, which may be a large vesicle. For example, wherein the diameter of the large vesicles in the third centrifugation may be greater than about 200nm.
For example, the centrifugation in the extracellular vesicle separation may include a fourth centrifugation, and the supernatant obtained by the above-described three centrifugation may be centrifuged. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a fourth centrifugation at a rotational speed of from about 50000g to about 150000g. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a fourth centrifugation, said fourth centrifugation having a temperature of from about 2 ℃ to about 10 ℃. For example, wherein said centrifugation in said extracellular vesicle separation can comprise a fourth centrifugation, said fourth centrifugation time being from about 60 minutes to about 100 minutes. For example, wherein the fourth centrifugation results in a tube bottom precipitate, the diameter of the precipitate may be less than about 200nm.
For example, the method of the present application may comprise the steps of: (S1-1) centrifuging the test sample at about 500g to obtain a supernatant; (S1-2) centrifuging the supernatant obtained in the step (S1-1) at about 3000g to obtain a supernatant; (S1-3) centrifuging the supernatant obtained in the step (S1-2) at about 12000g to obtain a precipitate containing the test substance. For example, the duration of step (S1-1) may be about 10 minutes. For example, the duration of step (S1-2) may be about 20 minutes. For example, the duration of step (S1-3) may be about 20 minutes.
For example, the method of the present application may comprise the steps of: (S1-1) centrifuging the test sample at about 1350g to obtain a supernatant; (S1-2) centrifuging the supernatant obtained in the step (S1-1) at about 3000g to obtain a supernatant; (S1-3) contacting the supernatant obtained in the step (S1-2) with a size-exclusion packing, and separating a component containing the substance to be tested. For example, the duration of step (S1-1) may be about 15 minutes. For example, the duration of step (S1-2) may be about 10 minutes. For example, the size-exclusion packing may comprise packing having a diameter of about 22 microns to 44 microns. For example, the step (S1-3) may collect the fraction eluted by size exclusion of two or more tubes, and determine the fraction containing the substance to be measured by dynamic light scattering.
For example, the hydroxymethylation sequencing method of the present application may comprise the steps of: (S2 a) extracting nucleic acid fragments in a sample, (S2 b) labeling nucleic acid fragments comprising hydroxymethylcytosine in the nucleic acid fragments, (S2 c) enriching the nucleic acid fragments comprising hydroxymethylcytosine, (S2 d) sequencing the enriched nucleic acid fragments comprising hydroxymethylcytosine.
For example, the labeling described herein comprises contacting the nucleic acid fragment with a DNA β -glucosyltransferase and UDP glucose modified with a chemoselective group. For example, the label of the present application may be such that the chemoselective group is attached to a nucleic acid fragment of the sample comprising hydroxymethylcytosine.
For example, the chemoselective groups described herein may comprise azido groups. For example, the chemoselective groups of the present application may comprise any chemical click group.
For example, the labeling described herein may further comprise contacting the nucleic acid fragment bearing a chemoselective group with a nucleic acid comprising biotin capable of reacting with the chemoselective group. For example, the biotin capable of reacting with the chemoselective group may comprise biotin comprising dibenzocyclooctyne modifications. For example, the label can be a first label attached to a nucleic acid fragment comprising hydroxymethylcytosine of the sample, and can comprise reattaching a second label to the nucleic acid fragment to which the first label is attached, the second label being selectively reactive with the first label.
For example, the enriching may comprise contacting the nucleic acid fragment with biotin with a magnetic bead comprising streptavidin. For example, the labeled nucleic acid fragment comprising hydroxymethylcytosine may be bound to a magnetic bead comprising streptavidin. For example, the enrichment may comprise magnetic bead separation of the nucleic acid fragment bearing the hydroxymethylcytosine by magnetic force.
For example, the methods of the present application may comprise detecting the number and/or presence of the nucleic acid fragment bearing the hydroxymethylcytosine of the test sample by a sequencing method. For example, the present application may sequence the test sample by a method selected from the group consisting of: digital PCR and high throughput sequencing. For example, the sequencing methods of the present application may be selected from any of the sequencing methods known in the art.
In another aspect, the present application provides an assay method that may include detecting the amount and/or presence of hydroxymethylcytosine in a test substance having a diameter of about 200 nm or more in a test sample.
In another aspect, the present application also provides a method for confirming the presence of a disease, assessing the risk of disease formation or formation, assessing the progression and/or prognosis of a disease and/or screening a population for a treatment, which may comprise detecting the number and/or presence of hydroxymethylcytosine in a test substance having a diameter of about 200 nm or more in a test sample.
In another aspect, the present application provides a nucleic acid that may comprise a sequence capable of binding to a test region, or a region complementary thereto, or a fragment thereof, in a test substance having a diameter of about 200 nm or more in a test sample.
In another aspect, the present application provides a method for preparing a nucleic acid, which may comprise a sequence of a region to be measured, or a region complementary thereto, or a fragment thereof, in a substance to be measured having a diameter of about 200 nm or more in a sample to be measured, and designing a nucleic acid capable of binding to the region to be measured, or the region complementary thereto, or the fragment thereof.
In another aspect, the present application provides a kit that may comprise a nucleic acid as described herein. For example, the kit may further comprise a blood collection tube. For example, the kit may be used in conjunction with a centrifuge. For example, the kit may further comprise reagents comprising a glycosylation group, e.g., the glycosylation group may comprise a UDP glucose group or derivative thereof, reagents comprising a labeling group, e.g., the labeling group may comprise a biotin group or derivative thereof, a linker, a medium, an amplification primer, and/or a buffer. For example, the kit may be used with a sequencing instrument.
In another aspect, the present application provides the use of a nucleic acid of the present application, and/or a kit of the present application, in the preparation of a disease detection product.
In another aspect, the present application provides the use of a nucleic acid of the present application, and/or a kit of the present application, in the preparation of a product that confirms the presence of a disease, evaluates the risk of disease formation or formation, evaluates the progression and/or prognosis of a disease, and/or screens a population for treatment.
In another aspect, the present application provides nucleic acids of the present application, and/or kits of the present application, which may be used for disease detection.
In another aspect, the present application provides nucleic acids of the present application, and/or kits of the present application, which may be used to confirm the presence of a disease, assess the risk of disease formation or formation, assess the progression and/or prognosis of a disease, and/or screen a population for treatment.
In another aspect, a method of disease detection of the present application may comprise providing a nucleic acid of the present application, and/or a kit of the present application.
In another aspect, the present methods of confirming the presence of a disease, assessing disease formation or risk of formation, assessing the progression and/or prognosis of a disease and/or screening a population having a corresponding treatment may comprise providing a nucleic acid as described herein, and/or a kit as described herein.
For example, a disease of the present application may comprise a tumor.
In another aspect, the present application provides a database that may include sequences of regions to be measured, or complementary regions thereof, or fragments thereof, in a substance to be measured having a diameter of about 200 nm or more in a sample to be measured.
In another aspect, the present application provides a storage medium that may record a program that may run the methods of the present application.
In another aspect, the present application provides an apparatus that may comprise a storage medium as herein described. For example, the non-volatile computer-readable storage medium may include a floppy disk, a flexible disk, a hard disk, a Solid State Storage (SSS) (e.g., solid State Drive (SSD)), a Solid State Card (SSC), a Solid State Module (SSM)), an enterprise-level flash drive, a tape, or any other non-transitory magnetic medium, etc. The non-volatile computer-readable storage medium may also include punch cards, paper tape, optical discs (or any other physical medium having a hole pattern or other optically recognizable indicia), compact disc read-only memory (CD-ROM), rewritable optical discs (CD-RW), digital Versatile Discs (DVD), blu-ray discs (BD), and/or any other non-transitory optical medium.
For example, an apparatus as described herein further comprises a processor coupled to the storage medium, the processor configured to execute based on a program stored in the storage medium to implement the methods described herein. For example, the database system may implement various mechanisms to ensure that the methods described herein executing on the database system produce the correct results. In this application, the database system may use a disk as the persistent data store. In this application, the database system may provide database storage and processing services for a plurality of database clients. The database client may store database data across multiple shared storage devices and/or may utilize one or more execution platforms having multiple execution nodes. The database system may be organized such that storage and computing resources may be effectively infinitely extended.
Without intending to be limited by any theory, the following examples are meant to illustrate the methods and uses of the present application, and the like, and are not intended to limit the scope of the invention of the present application.
Examples
Example 1 extracellular vesicle DNA 5-hydroxymethylcytosine detection method
Library construction of (one) 5-hydroxymethylcytosine (5 hmC)
Extracellular vesicle isolation
Extracellular vesicles can be isolated from plasma samples:
1. whole blood (8 mL) was taken from 10mL Kangzhi (CW 2815M) cfDTM free nucleic acid tube (please operate at room temperature, store at room temperature after blood was taken;
2. centrifuging for the first time, centrifuging at 500g and 4 ℃ for 10min, carefully taking out pale yellow supernatant (removing blood cells and platelets), and transferring to a 2mLDNase free sterile centrifuge tube;
3. a second centrifugation, 3000g, centrifugation at 4℃for 20min, careful removal of the supernatant (removal of leukocytes and cell debris), transfer to a 2-3 tube 2mL DNase free sterile centrifuge tube;
4. centrifuging for the third time, centrifuging for 20min at 12000g and 4 ℃, carefully taking out the supernatant, transferring the supernatant to a 2-3-tube 2mL DNase free sterile centrifuge tube, collecting tube bottom sediment (large vesicle with diameter of more than 200 nm), adding 1mLPBS (1X), mixing uniformly, and freezing in a-80 DEG refrigerator;
5. centrifuging for the fourth time, centrifuging for 70min at 100000g and low temperature of 4deg.C, removing supernatant, collecting tube bottom precipitate (exosomes with diameter less than 200 nm), adding 1mLPBS (1X), mixing, and freezing in-80 ° refrigerator;
Extracellular vesicles can be isolated from urine samples:
1. taking 10mL of urine;
2. centrifuging for 10min at low temperature of 500g and 4 ℃, taking out pale yellow supernatant (removing cells) carefully, and transferring to a 2mLDNase free sterile centrifuge tube;
3. a second centrifugation, 3000g, at 4℃for 20min, carefully removing the supernatant (removing cell debris), transferring to a 15mL sterile centrifuge tube;
4. centrifuging for the third time, centrifuging for 20min at 12000g and 4 ℃, carefully taking out the supernatant, transferring the supernatant into a 15mL sterile centrifuge tube, collecting tube bottom sediment (large vesicle with the diameter of more than 200 nm), adding 1mLPBS (1X), mixing uniformly, and freezing in a-80 DEG refrigerator;
5. centrifuging for the fourth time, centrifuging for 70min at 100000g and low temperature of 4deg.C, removing supernatant, collecting tube bottom precipitate (exosomes with diameter less than 200 nm), adding 1mLPBS (1X), mixing, and freezing in-80 ° refrigerator;
extracellular vesicles can be isolated from tissue, cells, primary cells, organoid culture supernatant samples:
1. taking 10mL of tissue, cells, primary cells and organoid culture supernatant in a 15mL centrifuge tube;
2. centrifuging for the first time, centrifuging for 10min at 500g and 4 ℃ and carefully taking out the supernatant (removing cells), and transferring into a 15mL sterile centrifuge tube;
3. a second centrifugation, 3000g, at 4℃for 20min, carefully removing the supernatant (removing cell debris), transferring to a 15mL sterile centrifuge tube;
4. Centrifuging for the third time, centrifuging for 20min at 12000g and 4 ℃, carefully taking out the supernatant, transferring the supernatant into a 15mL sterile centrifuge tube, collecting tube bottom sediment (large vesicle with the diameter of more than 200 nm), adding 1mLPBS (1X), mixing uniformly, and freezing in a-80 DEG refrigerator;
5. centrifuging for the fourth time, centrifuging for 70min at 100000g and 4 deg.C, removing supernatant, collecting tube bottom precipitate (exosomes with diameter less than 200 nm), adding 1mLPBS (1X), mixing, and freezing in-80 deg.C refrigerator.
Extracellular vesicles can be isolated by exclusion chromatography:
1. the collected non-anticoagulated whole blood is placed in a 4-degree refrigerator for 4 hours;
centrifuging 2.1350 g for 15 minutes, and separating to obtain a plasma sample;
3. centrifugation of 3000g of plasma sample for 10 min, removal of cell debris;
4. packing the size exclusion column with a superdex packing from Sigma;
5. the pre-packed columns were equilibrated with PBS buffer;
6. adding the cell debris-removed plasma sample into a size exclusion column, eluting with PBS, collecting the eluted components in 0.5mL per tube, eluting large extracellular vesicles first, eluting small extracellular vesicles later;
7. dynamic light scattering was performed to characterize the particle size of each tube, i.e. to distinguish between large extracellular vesicles (> 200 nm) and exosomes (< 200 nm).
Extracellular vesicle (> 200 nm) gDNA extraction
1. Extracting about 20ng of genomic DNA from the extracellular vesicle sample prepared by any one of the above methods using a Quick-DNA miniprep plus kit (ZYMO, D4069) kit;
2. extracellular vesicles and particle gDNA concentrations were determined using qubit 3.0.
Breaking gDNA, end-to-end alignment and ligating to sequencing adaptors
According to the KAPA HyperPlus Library Preparation Kit (KK 8514) specification, the brief operation is as follows:
1. breaking large fragment gDNA, wherein the input amount of gDNA is 10ng, the sample volume is 12 mu L, and the reactants are 2 mu L of 0.6% FCS, 2 mu L of fragment Buffer and 4 mu L of fragment Enzyme; water bath at 37 ℃ for 20 minutes;
2. mu.L of a reaction mixture (total volume: 24. Mu.L) containing 10ng of genomic DNA, 2.8. Mu.L of End Repair & A-stirring Buffer and 1.2. Mu.L of End Repair & A-Tailing Enzyme mix was prepared; incubate at 20℃for 30 minutes, then at 65℃for 30 minutes;
3. the following ligation reaction mixture was configured in a.5 mL low adsorption EP tube: 2. Mu. L Nuclease free water (nuclease free water), 12. Mu. L Ligation Buffer and 4. Mu.L DNA Ligase; to 18. Mu.L of ligation reaction mixture was added 2. Mu.L of sequencing KAPA index (PKR 2015, PKR2016 and PKR 2017), mixed, added to 24. Mu.L of reaction samples, and heated at 20℃for 4 hours;
4. The reaction product was purified using a DNA Clean & Concentrator 5 (ZYMO, D4014) purification kit and eluted with 20. Mu.L of elution buffer to obtain the final DNA ligation samples.
5hmC tag
1. A total volume of 4 μl of the labeling reaction mixture was prepared: mu.L of 50uM UDP-N3-Glu (hmC labeled substrate), 2.5. Mu.L of beta. GT enzyme (NEB) and 2.5. Mu.L of HEPES buffer (pH 8.0, final concentration of 50 mM) were added to 20. Mu.L of the DNA ligation sample. The mixture is put in a water bath at 37 ℃ for 1 hour;
2. taking out the mixed solution, and purifying the reaction product by using a DNA Clean & Concentrator 5 (ZYMO, D4014) purification kit to obtain purified 30 mu L of DNA;
3. then, 1. Mu.L of 45. Mu.L of DBCO-PEG4-Biotin (Click Chemistry Tools) was added to the above purified 30. Mu.L of DNA, and the mixture was subjected to a water bath at 37℃for 1 hour;
4. the reaction product was purified using a DNA Clean & Concentrator 5 (ZYMO, D4014) purification kit to obtain 30. Mu.L of purified labeled product.
Enrichment of 5hmC
1. The bound beads were equilibrated as follows: 2.5. Mu.L Dynabeads (Invitrogen, 65306) was removed and 100. Mu.L of wash buffer (5 mM Tris (pH 7.5), lM NaCl and 0.02% Tween 20) was added, the mixture was blown down and mixed well, placed on a 1.5mL magnetic rack, the bound beads were washed 3 times with 100. Mu.L of wash buffer repeatedly, finally 100. Mu.L of wash buffer was added, the beads were mixed well, and vortexed for 30min;
After 2.30 min, the beads were washed 3 times with 100uL of wash buffer, and finally 32 uL of binding buffer (10 mM Tris (pH 7.5), 2M NaCl and 0.04% tween 20) was added and mixed well;
3. adding the purified marked product obtained in the above steps into the magnetic bead mixed solution, and mixing for 30min in a rotary mixer to fully combine the marked product;
4. finally, the beads were washed 5 times with 100. Mu.L of wash buffer, and 23.8. Mu.L of RNase-Free water was added.
PCR amplification
1. To the final system of the above procedure, 25. Mu.L of 2 XPCR master mix and 1.25. Mu.L of PCR primers (total volume 50. Mu.L) were added, and amplification was performed at the temperature and under the following PCR reaction cycle conditions:
Figure BDA0003897022600000131
2. the amplified product was purified with AmpereXP beads (KAPA, KK 8001) and finally eluted with 20uL of eluent and a final 5hmC library was obtained. Library concentration assays can be performed with Qubit 3.0.
High throughput sequencing of 5hmC library after quality control
1. Performing Fragment AnalyzerTM full-automatic capillary electrophoresis system quality control (kit: DNF-900; using software: fragment Analyzer instrument control software, PROSize data analysis software) on the obtained 5hmC library, and determining whether the DNA fragment size in the library contains impurities (about 300bp in library size);
2. qPCR concentration determination (kit: KAPA SYBR FAST Universal qPCR Kit (KK 4601)) is performed to determine whether the sample library meets the on-machine sequencing standard;
the method comprises the following specific steps:
a. preparing a sample: preparing 5 1.5mL EP tubes, mixing every 4 samples to be sequenced into one EP tube, and adding 20 samples (index cannot be repeated) to the 5 tubes; 5ng of each 5hmC library sample was pipetted, the total volume of each EP tube was 20uL, and the final concentration was 1ng/uL;
b. the sample reaction system (20 uL) was as follows: 4uL were pipetted into each EP tube Chinese pool for qPCR quantification.
Figure BDA0003897022600000132
Figure BDA0003897022600000141
Qpcr program settings:
Figure BDA0003897022600000142
d. analysis of results: analyzing according to qPCR operation software, judging whether the 5hmC library is degraded or not, and judging whether the sequencing requirement is met or not;
3. the library (16 uL) passing the quality test was sequenced with I1 luminea NextSeq500 using a sequencing kit of High Output Kit v (75 cycles) with a sequencing throughput of 1.5Gb for each sample and a sequencing band size of 75bp.
Raw sequencing data alignment
1. Each original sequencing FASTQ data is trimmed by using Trimmomatic software, and then is aligned to human genome hg19 by using Bowtie2 software;
2. MACS software was used to identify the reads peak containing 5hmC, with the parameters: effective genome size =2.72e+09; tag size=38; band width=100; model fold = 10; p value cutoff=1.00 e-05, and call peaks are performed according to the P value cutoff=1.00 e-05, so that a count file is generated;
3. The count files from the positive samples and the negative samples are compared by using the DEseq2 software, and the found 5hmC peak areas with the reads number larger than 50 are respectively obtained according to the absolute log2FoldChange absolute > =0.5 and the pvalue absolute <0.05, so that the differential biomarkers of up-and-down regulation of 5hmC are respectively obtained.
Clinical sample detection
The sample of the clinical test positive patient and the sample of the clinical test negative patient are collected, and the copy number of the methylolation site of the differential biomarker in extracellular vesicles (> 200 nm) of different sample sources is detected by a 5hmC sequencing technical method based on extracellular vesicle DNA. The results show that the 5hmC sequencing technical method based on extracellular vesicle DNA can more accurately distinguish clinical detection positive patients from clinical detection negative patients.
Example 2
1. Sample collection and extracellular vesicle extraction:
1 sample of healthy human blood (8-10 mL) was collected and plasma (4-5 mL) was isolated; extracting extracellular vesicles (> 200 nm) from plasma by gradient centrifugation; then extracting DNA in extracellular vesicles to construct a 5hmC library; finally, the 5hmC library was sequenced using I1lumina NextSeq500, the sequencing throughput for each sample was 1.5Gb, and the sequencing band size was 75bp;
2. extracellular vesicles (> 200 nm) and exosomes (< 200 nm) characterization:
The extracted extracellular vesicles and exosomes were diluted into PBS at a concentration of 0.2mg/mL, and then the sizes of the extracellular large vesicles and exosomes were determined by dynamic light scattering using Zeta sizer Nano ZS of Malven company.
FIG. 1 shows the particle size distribution of extracellular vesicles (> 200 nm) isolated in the present application, and FIG. 2 shows the particle size distribution of exosomes (< 200 nm) isolated in the present application. The results show that the method of the present application can isolate extracellular vesicles (> 200 nm).
3.5hmC library quality control:
the obtained 5hmC library was subjected to quality control by a Fragment AnalyzerTM full-automatic capillary electrophoresis system (kit: DNF-900; using software: fragment Analyzer instrument control software, PROSize data analysis software), and the size of the DNA fragments in the library was determined as to whether impurities were contained (library size: about 300 bp). FIG. 3 shows a graph of the quality control results of a 5-hydroxymethylcytosine (5 hmC) library of DNA in extracellular vesicles (> 200 nm) isolated in the present application, and the results of a fully automatic capillary electrophoresis system show that the library size is about 306 bp. The results show that the extracellular vesicle (> 200 nm) based detection method of the present application can yield a qualified 5hmC library.
Example 3
Detection of the sample may be by hydroxymethylation site screening for the corresponding test site.
1. Determining a clinical sample group: detecting the results of drug treatment targets (such as MET) according to the methods of clinical Immunohistochemistry (IHC), in situ hybridization Fluorescence (FISH) and the like, and dividing clinical samples into two groups of MET positive and MET negative;
2. screening 5hmC differential markers according to the screening conditions of |log2FoldChange| > =0.5 and pvalue <0.05 through the comparative analysis of the positive group and the negative group, and searching for a methylolation site (MET and the like) corresponding to the MET;
3. synthesis of MET methylolation site primer (Optimus Trimereship, prime Biotech Co., ltd.)
Digital PCR detection based on cfDNA methylolation sites
(1) Sample system preparation (20 uL)
1. Sample 5hmC library addition was 10ng, primer standard concentration was 10uM, dye premix (Yongno, S0200020301);
reactants Volume (mu L)
Sample of X
Nuclease-free water 9.2 subtracting X
Primer F1 0.4
Primer F2 0.4
Dye premix 10
(1) Droplet generation
1. Chip preparation using a Yonno sample preparation universal consumable (S0100010101), 50uL of microdroplet generating oil was added to the first row of 8 wells of the chip, 20uL of sample system was added to the second row of 8 wells of the chip, and then 5uL of sealant was added to the second row of sample wells;
reactants Volume (mu L) Chip position
Droplet generation oil 50 First row
Sample system 20 Second row
Sealing agent
5 Second row
2. Droplet generation was performed using a Yongno Microdrop-100 digital PCR system;
(2) Sealing film
1. Transferring the prepared microdroplet (50 uL) to a 96-well PCR plate (S0100030101), and attaching a membrane;
2. sealing the membrane at 190 ℃ by using a Yongno Microdrop-100 digital PCR system;
(3) PCR amplification
PCR amplification procedure:
Figure BDA0003897022600000161
2. carrying out PCR amplification on the sample;
(4) Digital PCR detection
1. And detecting and analyzing the PCR product by using a Yongno Microdrop-100 digital PCR system.
The methylolation detection of the sample to be detected can be not limited to a specific methylolation site, and the methylolation site at any position on the chromosome can be subjected to single-gene digital PCR detection.
Example 4
Detection of extracellular vesicles and exosomes samples
1. Sample collection and extracellular vesicle extraction:
1 sample of healthy human blood (8-10 mL) was collected and plasma (4-5 mL) was isolated; extracting extracellular vesicles (> 200 nm) from plasma by gradient centrifugation; then extracting DNA in extracellular vesicles to construct a 5hmC library; finally, the 5hmC library was sequenced using I1lumina NextSeq500, the sequencing throughput for each sample was 1.5Gb, and the sequencing band size was 75bp;
2. extracellular vesicles (> 200 nm) and exosomes (< 200 nm) characterization:
The extracted extracellular vesicles and exosomes were diluted into PBS at a concentration of 0.2mg/mL, and then the sizes of the extracellular large vesicles and exosomes were determined by dynamic light scattering using Zeta sizer Nano ZS of Malven company.
3.5hmC library quality control:
performing Fragment AnalyzerTM full-automatic capillary electrophoresis system quality control (kit: DNF-900; using software: fragment Analyzer instrument control software, PROSize data analysis software) on the obtained 5hmC library, and determining whether the DNA fragment size in the library contains impurities (about 300bp in library size);
4. results
As the results of fig. 4 and 5 show, the present application isolates plasma isolated extracellular vesicle size (> 200 nm); plasma isolated exosome size (< 200 nm). As the results in fig. 6 show, the extracellular vesicles of the present application construct libraries.
Example 5
Detection of lung cancer samples
Sample collection and 5hmC-Seal sequencing:
blood samples (8-10 mL) were collected from 60 lung cancer patients and 60 healthy persons, and plasma (4-5 mL) was isolated. Extracellular vesicles (> 200 nm) and exosomes (< 200 nm) were isolated from plasma using gradient centrifugation methods and DNA was extracted for 5hmC-Seal sequencing. Wherein 30 lung cancer and 30 healthy human samples isolate extracellular vesicles (> 200 nm); extracellular vesicles (< 200 nm) were isolated from 30 lung cancer and 30 healthy human samples. The sequencing flux of each sample was 1.5Gb, and the sequencing band size was 75bp; FIGS. 7A-7D show the results of tests for lung cancer patients and healthy persons. The results show that the application has significantly improved accuracy, detection sensitivity and specificity for 5hmC detection of extracellular vesicles, which are higher than those of exosomes.
Example 6
Detection of colorectal cancer samples
Sample collection and 5hmC-Seal sequencing:
50 colorectal cancer patients and 60 healthy human blood samples (8-10 mL) were collected and plasma (4-5 mL) was isolated. Extracellular vesicles (> 200 nm) and exosomes (< 200 nm) were isolated from plasma using gradient centrifugation methods and DNA was extracted for 5hmC-Seal sequencing. Wherein 25 colorectal cancers and 30 healthy human samples isolate extracellular vesicles (> 200 nm); extracellular vesicles (< 200 nm) were isolated from 25 colorectal cancers and 30 healthy human samples. The sequencing flux of each sample was 1.5Gb, and the sequencing band size was 75bp; FIGS. 8A-8D show the results of tests for colorectal cancer patients and healthy persons. The results show that the application has significantly improved accuracy, detection sensitivity and specificity for 5hmC detection of extracellular vesicles, which are higher than those of exosomes.
The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Numerous variations of the presently exemplified embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and equivalents thereof.

Claims (35)

1. An analytical method comprising (S1) obtaining a substance to be measured having a diameter of about 200nm or more in a sample to be measured, and (S2) detecting the number and/or presence of hydroxymethylcytosine in the substance to be measured.
2. A method for confirming the presence of a disease, assessing the risk of disease formation or formation, assessing the progression and/or prognosis of a disease and/or screening a population for a treatment comprising (S1) obtaining a test substance having a diameter of about 200 nm or more in a test sample, (S2) detecting the number and/or presence of hydroxymethylcytosine in a test region in said test substance.
3. The method of any one of claims 1-2, wherein the sample to be tested comprises plasma, urine, tissue culture fluid, cell culture fluid, and/or organoid culture fluid.
4. A method according to any one of claims 1 to 3, wherein the test substance comprises extracellular vesicles.
5. The method of any one of claims 1-4, wherein the test substance comprises a nucleic acid.
6. The method of any one of claims 1-5, wherein the test substance comprises DNA.
7. The method according to any one of claims 1-6, said step (S1) comprising the steps of: (S1-1) centrifuging the test sample at about 500g to obtain a supernatant; (S1-2) centrifuging the supernatant obtained in the step (S1-1) at about 3000g to obtain a supernatant; (S1-3) centrifuging the supernatant obtained in the step (S1-2) at about 12000g to obtain a precipitate containing the test substance.
8. The method of claim 7, wherein the duration of step (S1-1) is about 10 minutes.
9. The method of any one of claims 7-8, wherein the duration of step (S1-2) is about 20 minutes.
10. The method of any one of claims 7-9, wherein the duration of step (S1-3) is about 20 minutes.
11. The method according to any one of claims 1-6, said step (S1) comprising the steps of: (S1-1) centrifuging the test sample at about 1350g to obtain a supernatant; (S1-2) centrifuging the supernatant obtained in the step (S1-1) at about 3000g to obtain a supernatant; (S1-3) contacting the supernatant obtained in the step (S1-2) with a size-exclusion packing, and separating a component containing the substance to be tested.
12. The method of claim 11, wherein the duration of step (S1-1) is about 15 minutes.
13. The method of any one of claims 11-12, wherein the duration of step (S1-2) is about 10 minutes.
14. The method of any of claims 11-13, the size-exclusion packing comprising a diameter of about 22 microns to 44
And a filler of microns.
15. The method according to any one of claims 11 to 14, wherein the step (S1-3) collects the fractions eluted by size exclusion of two or more tubes, and the fraction containing the substance to be measured is determined by dynamic light scattering.
16. The method of any one of claims 1-15, wherein step (S2) comprises determining the number and/or presence of nucleic acid fragments comprising hydroxymethylcytosine in the test substance by a hydroxymethylation sequencing method.
17. The method of claim 16, the hydroxymethylation sequencing method comprising the steps of: (S2 a) extracting nucleic acid fragments in the substance to be detected, (S2 b) marking nucleic acid fragments containing hydroxymethyl cytosine in the nucleic acid fragments, (S2 c) enriching the nucleic acid fragments containing hydroxymethyl cytosine, and (S2 d) sequencing the enriched nucleic acid fragments containing hydroxymethyl cytosine.
18. The method of claim 17, wherein the labeling comprises contacting the nucleic acid fragment with a DNA β -glycosyltransferase and UDP glucose modified with a chemoselective group.
19. The method of claim 18, wherein the chemoselective group comprises an azide group.
20. The method of any one of claims 18-19, the labeling further comprising contacting the nucleic acid fragment bearing a chemoselective group with a nucleic acid comprising biotin capable of reacting with the chemoselective group.
21. The method of claim 20, wherein the biotin capable of reacting with the chemoselective group comprises dibenzocyclooctyne modified biotin.
22. The method of any one of claims 17-21, wherein the enriching comprises contacting the nucleic acid fragment with biotin with a magnetic bead comprising streptavidin.
23. The method of any one of claims 17-22, wherein the enriching comprises magnetically separating magnetic beads of the nucleic acid fragment bearing the hydroxymethylcytosine.
24. The method of any one of claims 1-23, wherein the test substance is sequenced by a method selected from the group consisting of: digital PCR and high throughput sequencing.
25. An analytical method comprising detecting the amount and/or presence of hydroxymethylcytosine in a test substance having a diameter of about 200 nm or more in a test sample.
26. A method for confirming the presence of a disease, assessing the risk of disease formation or formation, assessing the progression and/or prognosis of a disease and/or screening a population for a treatment, comprising detecting the number and/or presence of hydroxymethylcytosine in a test substance having a diameter of about 200 nm or more in a test sample.
27. A nucleic acid comprising a sequence capable of binding to a test region in a test substance having a diameter of about 200 nm or more in a test sample, or a region complementary thereto, or a fragment thereof.
28. A method for preparing a nucleic acid, comprising the step of designing a nucleic acid capable of binding to a region to be measured, a region complementary thereto, or a fragment thereof in a substance to be measured having a diameter of about 200 nm or more in a sample to be measured.
29. A kit comprising the nucleic acid of claim 27.
30. Use of a nucleic acid according to claim 27, and/or a kit according to claim 29, in the preparation of a disease detection product.
31. Use of a nucleic acid according to claim 27, and/or a kit according to claim 29, for the preparation of a product for confirming the presence of a disease, assessing the risk of disease formation or formation, assessing the progression and/or prognosis of a disease and/or screening a population for treatment.
32. A database comprising sequences of regions to be measured, or complementary regions thereof, or fragments thereof, in a substance to be measured having a diameter of about 200 nm or more in a sample to be measured.
33. A storage medium carrying a program operable to perform the method of any one of claims 1 to 26.
34. An apparatus comprising the storage medium of claim 33.
35. The apparatus of claim 34, further comprising a processor coupled to the storage medium, the processor configured to execute to implement the method of any one of claims 1-26 based on a program stored in the storage medium.
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