阅读说明：本技术 一种dna甲基化靶向富集的方法及其应用 (Method for DNA methylation targeted enrichment and application thereof ) 是由 李小刚 贾鑫淼 肖盟 徐英春 于 2021-01-04 设计创作，主要内容包括：本发明公开了一种DNA甲基化靶向富集的方法,特异性富集甲基化修饰DNA片段。通过本发明所得到的靶向富集后的DNA可广泛应用于下游诸多分子诊断,如基于检测母体血浆中胎儿游离DNA的无创性产前分子诊断(NITP),病原微生物检测,肿瘤伴随诊断等。(The invention discloses a method for the methylation targeted enrichment of DNA, which specifically enriches methylation modified DNA fragments. The DNA obtained by the invention after targeted enrichment can be widely applied to downstream molecular diagnosis, such as noninvasive prenatal molecular diagnosis (NITP) based on detection of fetal free DNA in maternal plasma, pathogenic microorganism detection, tumor concomitant diagnosis and the like.)

1. A method for the targeted enrichment of DNA methylation, which is an in vitro method, comprising the following steps:

(1) fragmenting sample DNA;

(2) treating the fragmented DNA with thymine DNA glycosylase, excising 5-carboxyl cytosine in the DNA, and adding unmethylated cytosine at the excision site of the 5-carboxyl cytosine through a DNA shearing repair way;

(3) covalently linking the saccharide having a glucosyl group to the hydroxymethyl group of the 5-hydroxymethylcytosine of the DNA obtained in step (2) by using transglycosylase to form β -glucosyl-5-hydroxymethylcytosine;

(4) converting 5-methylcytosine in the DNA to 5-carboxycytosine or 5-formylcytosine by treating the DNA with a dioxygenase or by other chemical means;

(5) and (4) continuously enriching the product obtained in the step (4) by using a chemical labeling method, and then performing library construction and sequencing.

2. The method of claim 1, wherein the thymine DNA glycosylase comprises: a full-length thymine DNA glycosylase or a fragment or derivative thereof that retains enzymatic activity.

3. The method of claim 1, wherein the transglycosylase includes, but is not limited to, β GT, α GT, and recombinases thereof; the saccharide of glucosyl group includes but is not limited to uracil diphosphate glucose or other modified saccharides.

4. The method of claim 1, wherein the dioxygenase comprises Tet or other dioxygenase ALKB family or the like, said Tet dioxygenase comprising: tet1, Tet2, or Tet3, or fragments or derivatives thereof that retain the catalytic domain and have catalytic activity.

5. The method according to claim 1, wherein the chemical labeling method is continued with enrichment, in particular, the formation of an amide through the carboxyl group of 5caC and the modification of small molecules with biotin having a primary amine group.

6. Use of the method of any one of claims 1 to 5 for typing identification of a microorganism.

7. Use of the method of any one of claims 1 to 5 in molecular diagnostics.

8. Use according to claim 7, wherein said molecular diagnostics include non-invasive prenatal molecular diagnostics, pathogenic microorganism detection, tumor-associated diagnostics.

9. A DNA targeting enrichment and detection kit is characterized by comprising: transglycosylase, oxidase, a purification reagent and a primer joint.

Technical Field

The invention relates to the technical field of molecular detection, in particular to a method for DNA methylation targeted enrichment and application thereof.

Background

Since Mander and Metais discovered that extracellular free DNA and RNA exist in human peripheral blood in 1948, the detection of the free nucleic acid is widely applied to various clinical aspects as an effective minimally invasive diagnosis and treatment means, such as occurrence and development of tumors, pregnancy-related diseases, autoimmune diseases, transplant rejection, wound emergency medicine and the like, and has important significance in the aspects of early diagnosis, staging, treatment detection, prognosis judgment, prenatal molecular diagnosis and the like of diseases.

Researchers from the university of pennsylvania noted enzymatic deamination of AID/APOBEC family DNA deaminases. This deamination can achieve the bisulfite effect without damaging the DNA. Based on the above, researchers developed a brand-new epigenetic sequencing method ACE-seq (APOBEC-coupled epigenetic sequencing), and the detection and localization of 5-hydroxymethylcytosine (5hmC) can be carried out under the condition of single base resolution without bisulfite, so as to obtain a 5hmC map with high confidence. More importantly, the method requires 1000 times less DNA sample size than current gold standard techniques. The ACE-seq method uses a deaminase called "APOBEC". The deaminase can effectively distinguish different cytosine modification states and simultaneously avoid damaging DNA. The method is verified to have similar performance to the recognition of cytosine modification state by bisulfite. An important property of ACE-seq is the ability to stably distinguish 5hmC from cytosine and 5-methylcytosine (5mC), which makes a highly reliable analysis of 5hmC possible.

CN 105648537B reports a method for enriching by oxidizing methylcytosine to 5-hydroxymethylcytosine using murine Tet oxidase or a recombinase thereof and biotin-labeling 5-hydroxymethylcytosine. However, the Tet oxidase cannot completely convert 5-methylcytosine into 5-hydroxymethylcytosine at present, and can further oxidize the 5-formylcytosine and the 5-carboxycytosine, so that the method has certain loss.

Methylation enrichment of other free DNA is not reported at present, so that better methylated DNA fragments are developed in a targeted enrichment manner, and further methylation specific fragment DNA library sequencing is constructed, so that the methylation specific fragment DNA library sequencing has important significance in the aspects of early diagnosis, staging, treatment detection, prognosis judgment, prenatal molecular diagnosis and the like of diseases.

Disclosure of Invention

The invention aims to provide a method for the targeted enrichment of DNA methylation, which is used for specifically enriching methylation modified DNA fragments.

The pathogenic microorganism contains 5-methylcytosine (5mC), but does not contain 5-hydroxymethylcytosine (5hmC), 5-aldehyde cytosine (5fC), 5-carboxyl cytosine (5caC), so that the DNA content of the pathogenic microorganism in the DNA sample can be improved by capturing 5mC in a targeted manner.

Based on this, the invention provides a method for targeted enrichment of DNA methylation, which is an in vitro method comprising the steps of:

(1) fragmenting sample DNA;

(2) treating the fragmented DNA with thymine DNA glycosylase, excising 5-carboxyl cytosine in the DNA, and adding unmethylated cytosine at the excision site of the 5-carboxyl cytosine through a DNA shearing repair way; the purpose of this step is to remove the 5-carboxycytosine contained in the DNA itself.

(3) Covalently linking the saccharide having a glucosyl group to the hydroxymethyl group of 5-hydroxymethylcytosine of the DNA obtained in step (2) by using transglycosylase to form β -glucosyl-5-hydroxymethylcytosine (5 gmC);

the step can avoid the interference of 5hmC, which is very important for the diagnosis of pathogenic microorganisms.

(4) Converting 5-methylcytosine in the DNA to 5-carboxycytosine or 5-formylcytosine by treating the DNA with a dioxygenase or by other chemical means;

(5) and (4) continuing to enrich the product obtained in the step (4) by using a chemical labeling method, and then sequencing.

Preferably, the use of the thymine DNA glycosylase of step (1) for excising 5-carboxycytosine in DNA; or for preparing a composition for cleaving 5-carboxycytosine in DNA. (preferably, the use is non-therapeutic and non-diagnostic).

In another preferred embodiment, the thymine DNA glycosylase comprises: full-length thymine DNA glycosylase or a fragment or derivative thereof that retains the activity of an enzyme, such as Flag-TDG.

Preferably, the transglycosylases include, but are not limited to, β GT, α GT, and recombinases thereof; the saccharide of glucosyl group includes but is not limited to uracil diphosphate glucose (UDP-Glc) or other modified saccharides.

Preferably, the present invention provides the use of a dioxygenase enzyme for converting 5-methylcytosine (5mC) in DNA to 5-carboxycytosine (5 caC); or for the preparation of a composition for converting 5-methylcytosine (5mC) to 5-formylcytosine (5fC) in DNA, as shown in figure 1. Preferably, the use is non-therapeutic and non-diagnostic.

The specific steps for oxidizing 5mC to 5Cac are as follows:

for example, 50ng of DNA was incubated in 50. mu.l of a solution containing 50mM MOPS buffer (pH6.9), 100. mu.M ammonium iron (II) sulfate, 1mM alpha-ketoglutarate, 2mM ascorbic acid, 1mM dithiothreitol, 50mM NaCland 5. mu.M NgTET1 for 1 hour at 37 ℃. Thereafter, 4U proteinase K (New England Biolabs) were added to the reaction mixture and incubated at 37 ℃ for 30 minutes. The product was then purified.

50ng DNA 50. mu.l of a reaction containing 50mM HEPES buffer (pH 8.0), 100. mu.M ammonium iron (II) sulfate, 1mM α -ketoglutarate, 2mM ascorbic acid, 1mM dithiothreitol, 100mM NaCl, 1.2mM ATP and 4. mu.M was incubated with mTet1CD for 80 minutes at 37 ℃. Thereafter, 0.8U of ProteinaseK (New England Biolabs) was added to the reaction mixture and incubated at 50 ℃ for 1 hour. And then purifying.

In another preferred embodiment, the dioxygenase comprises Tet or other dioxygenase ALKB family or the like, said Tet dioxygenase comprising: tet1, Tet2 or Tet3 or fragments or derivatives thereof that retain the catalytic domain and have catalytic activity, such as Tet1CD, Tet2CD or Tet3 CD.

Preferably, the chemical labeling method continues enrichment, and specifically, the amide formation is formed by carboxyl of 5caC and biotin modified small molecules with primary amine groups;

for example, 5caC DNA was incubated with 4-methylaminophenyl azide (10mM), EDC (2mM), Mes (pH 6.0,75mM) at 37 ℃ for 1 hour. mu.M biotin-modified dibenzocyclooctyne was added and incubated at 37 ℃ for 2 hours.

Preferably, the catalytic reactant includes, but is not limited to, 1-ethyl-3- [ 3-dimethylaminopropyl ] -carbodiimide hydrochloride (EDC) catalyst.

Then, the solid phase material comprises magnetic beads with the diameter of 1nm to 100um, agarose beads with the diameter of 1nm to 100um, and artificial polymer beads with the diameter of 1nm to 100um, and the beads are provided with surface modification (streptavidin or other ligand receptors which are adjusted according to the matched small molecule modification).

Preferably, the method for constructing a library in step (4) comprises a novel single base resolution technology.

The novel single base resolution technology carries out the library construction process as follows: a commercially available methylation pooling kit or DNA, bisulfite sequencing Qiagen (180502QIAsaq Mehtyl library kit) or a commercially available kit for NEB (E7120S NEBNext enzymatic conversion methylation pooling kit) was used.

Further, the invention also provides application of the method in microbial typing identification and molecular diagnosis.

The molecular diagnosis comprises noninvasive prenatal molecular diagnosis, pathogenic microorganism detection and tumor concomitant diagnosis.

Furthermore, the invention also provides a DNA targeting enrichment and detection kit, which comprises: transglycosylase, oxidase, a purification reagent and a primer joint.

Advantageous effects

The kit and the method remove 5-hydroxymethylcytosine (5hmC), 5-aldehyde cytosine (5fC) and 5-carboxyl cytosine (5caC) in the sample DNA in a targeted manner, thereby achieving the purpose of specifically and targeted enrichment of methylated DNA fragments and improving the sensitivity and accuracy of enrichment. The DNA after targeted enrichment by the kit and the method can be widely applied to downstream molecular diagnosis, such as noninvasive prenatal molecular diagnosis (NITP) based on detection of fetal free DNA in maternal plasma, pathogenic microorganism detection, tumor-associated diagnosis and the like.

Drawings

FIG. 15 illustrates the selective oxidation of mC to 5 caC;

FIG. 2 agarose electrophoresis detection results after fragmentation;

FIG. 3 results of methylation analysis of Cryptococcus neoformans and Cryptococcus Gothii.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1:

a method of targeted enrichment of DNA methylation, the method being an in vitro method comprising the steps of:

(1) fragmenting sample DNA;

(2) treating DNA extracted from a sample by thymine DNA glycosylase, cutting 5-carboxyl cytosine in the DNA, and adding unmethylated cytosine at a 5-carboxyl cytosine cutting site through a DNA cutting and repairing way; the purpose of this step is to remove the 5-carboxycytosine contained in the DNA itself.

(3) Covalently linking the saccharide having a glucosyl group to the hydroxymethyl group of 5-hydroxymethylcytosine of the DNA obtained in step (2) by using transglycosylase to form β -glucosyl-5-hydroxymethylcytosine (5 gmC);

this step avoids human 5hmC interference, which is important for the diagnosis of pathogenic microorganisms.

(4) Treating the DNA with Tet dioxygenase or converting 5-methylcytosine in the DNA into 5-carboxycytosine or 5-formylcytosine by other chemical methods;

(5) and (4) continuously enriching the product obtained in the step (4) by using a chemical labeling method, and then performing library construction and sequencing.

Example 2:

1. genomic DNA from Cryptococcus cati (NIH76, ATCC) and Cryptococcus neoformans H99(BNCC290220, Benna organisms) was used as a model.

200ng of genomic DNA (fungal genomic DNA extraction kit, Beijing Solebao technology Co., Ltd., product number: D2300) was mixed with quality control DNA (CpG methylated pUC19 and unmethylated lambda DNA in 50. mu.L of 0.1 XTE buffer solution pH 8.0.

2. The mixed DNA was fragmented by Covaris ultrasonication 240-290 bp.

Oxidative capture moiety

3.50ng DNA mTet1CD was incubated in 50. mu.l reaction with 50mM HEPES buffer (pH 8.0), 100. mu.M ammonium iron (II) sulfate, 1mM α -ketoglutarate, 2mM ascorbic acid, 1mM dithiothreitol, 100mM NaCl, 1.2mM ATP and 4. mu.M for 80 minutes at 37 ℃. Thereafter, 0.8U of ProteinaseK (New England Biolabs) was added to the reaction mixture and incubated at 50 ℃ for 1 hour. And then purifying.

4. The product of step 3, 5caC DNA, was incubated with 4-methylaminophenyl azide (10mM), EDC (2mM), Mes (pH 6.0,75mM) at 37 ℃ for 1 hour.

5. mu.M biotin-modified dibenzocyclooctyne was added, incubated at 37 ℃ for 2 hours, and purified using the QIAquick Nucleotide Removal Kit (QIAGEN). 100mM DTT was added and incubated at 37 ℃ for 2 hours.

Direct sequencing

Ligation to the sequencing adapter primer (NEB) required for the second-generation sequencing was carried out as described in the NEBNext enzymic Methyl-seq kit (NEB, E7120S), PCR cycles were performed according to the following procedure, and the beads were purified and then subjected to sequencing on the machine.

TABLE 1 PCR cycling program

CYCLE STEP	TEMP	TIME	CYCLES
				Initial Denaturation	98℃	30seconds	1
Denaturation	98℃	10seconds	4-8*
				Annealing	62℃	30seconds
Extension	65℃	60seconds
				Final Extension	65℃	5minutes	1
Hold	4℃

Bioinformatics analysis

And performing bioinformatics analysis by using R package software, performing sample quality control, filtering data, comparing with a reference genome of NCBI (national center for Biotechnology information) to perform differential analysis and detecting and annotating differential methylation regions. FIG. 3 shows that the methylation levels and sites of the two species of bacteria are significantly different, which can be used as species typing identification.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.