CN117625745A - DNA methylation conversion method, methylase conversion kit and use method thereof - Google Patents
DNA methylation conversion method, methylase conversion kit and use method thereof Download PDFInfo
- Publication number
- CN117625745A CN117625745A CN202311637502.0A CN202311637502A CN117625745A CN 117625745 A CN117625745 A CN 117625745A CN 202311637502 A CN202311637502 A CN 202311637502A CN 117625745 A CN117625745 A CN 117625745A
- Authority
- CN
- China
- Prior art keywords
- dna
- conversion
- cytosine
- reaction solution
- methylation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 100
- 238000000034 method Methods 0.000 title claims abstract description 46
- 230000007067 DNA methylation Effects 0.000 title claims abstract description 22
- 230000011987 methylation Effects 0.000 claims abstract description 22
- 238000007069 methylation reaction Methods 0.000 claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 13
- 108020004414 DNA Proteins 0.000 claims description 58
- 239000000243 solution Substances 0.000 claims description 52
- 238000007254 oxidation reaction Methods 0.000 claims description 43
- 239000012295 chemical reaction liquid Substances 0.000 claims description 41
- 238000006481 deamination reaction Methods 0.000 claims description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 27
- 102000053642 Catalytic RNA Human genes 0.000 claims description 25
- 108090000994 Catalytic RNA Proteins 0.000 claims description 25
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 25
- 108091092562 ribozyme Proteins 0.000 claims description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 claims description 20
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 16
- 239000011324 bead Substances 0.000 claims description 15
- 238000000746 purification Methods 0.000 claims description 15
- 239000000523 sample Substances 0.000 claims description 15
- 102000053602 DNA Human genes 0.000 claims description 13
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 12
- 229940104302 cytosine Drugs 0.000 claims description 12
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 claims description 11
- 238000004925 denaturation Methods 0.000 claims description 10
- 230000036425 denaturation Effects 0.000 claims description 10
- 238000003752 polymerase chain reaction Methods 0.000 claims description 8
- 229940035893 uracil Drugs 0.000 claims description 8
- SBHSUMUTJOPRIK-HPFNVAMJSA-N 5-(beta-D-glucosylmethyl)cytosine Chemical compound NC1=NC(=O)NC=C1CO[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 SBHSUMUTJOPRIK-HPFNVAMJSA-N 0.000 claims description 7
- BLQMCTXZEMGOJM-UHFFFAOYSA-N 5-carboxycytosine Chemical compound NC=1NC(=O)N=CC=1C(O)=O BLQMCTXZEMGOJM-UHFFFAOYSA-N 0.000 claims description 7
- 102000000311 Cytosine Deaminase Human genes 0.000 claims description 7
- 108010080611 Cytosine Deaminase Proteins 0.000 claims description 7
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 claims description 7
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 claims description 7
- 239000012488 sample solution Substances 0.000 claims description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims description 6
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 6
- 239000012148 binding buffer Substances 0.000 claims description 6
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 6
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- 108030004080 Methylcytosine dioxygenases Proteins 0.000 claims description 5
- 241001515965 unidentified phage Species 0.000 claims description 5
- FHSISDGOVSHJRW-UHFFFAOYSA-N 5-formylcytosine Chemical compound NC1=NC(=O)NC=C1C=O FHSISDGOVSHJRW-UHFFFAOYSA-N 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 claims description 3
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims description 3
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 108010067770 Endopeptidase K Proteins 0.000 claims description 3
- 239000002211 L-ascorbic acid Substances 0.000 claims description 3
- 235000000069 L-ascorbic acid Nutrition 0.000 claims description 3
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 3
- 229960005070 ascorbic acid Drugs 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- -1 ferrous salt compound Chemical class 0.000 claims description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 3
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 3
- 239000011654 magnesium acetate Substances 0.000 claims description 3
- 235000011285 magnesium acetate Nutrition 0.000 claims description 3
- 229940069446 magnesium acetate Drugs 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 235000011056 potassium acetate Nutrition 0.000 claims description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 3
- 235000011009 potassium phosphates Nutrition 0.000 claims description 3
- 229940113082 thymine Drugs 0.000 claims description 3
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 2
- 230000003197 catalytic effect Effects 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 238000006911 enzymatic reaction Methods 0.000 claims description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 abstract description 11
- 230000002255 enzymatic effect Effects 0.000 abstract description 10
- 238000012163 sequencing technique Methods 0.000 abstract description 9
- 238000012164 methylation sequencing Methods 0.000 abstract description 4
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 abstract description 3
- 238000007405 data analysis Methods 0.000 abstract description 3
- 102000039446 nucleic acids Human genes 0.000 abstract description 3
- 108020004707 nucleic acids Proteins 0.000 abstract description 3
- 150000007523 nucleic acids Chemical class 0.000 abstract description 3
- 238000010276 construction Methods 0.000 abstract description 2
- 230000005778 DNA damage Effects 0.000 abstract 1
- 231100000277 DNA damage Toxicity 0.000 abstract 1
- 150000003839 salts Chemical class 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000011084 recovery Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000012165 high-throughput sequencing Methods 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 230000009615 deamination Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108020000992 Ancient DNA Proteins 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 108091029430 CpG site Proteins 0.000 description 1
- 101000653374 Homo sapiens Methylcytosine dioxygenase TET2 Proteins 0.000 description 1
- 102100030803 Methylcytosine dioxygenase TET2 Human genes 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000001190 Q-PCR Methods 0.000 description 1
- 108091061939 Selfish DNA Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000001369 bisulfite sequencing Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000000749 co-immunoprecipitation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000019975 dosage compensation by inactivation of X chromosome Effects 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000011365 genetic imprinting Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000007855 methylation-specific PCR Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 229940005657 pyrophosphoric acid Drugs 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention discloses a DNA methylation conversion method, a methylation enzyme conversion kit and a use method thereof, and the enzymatic conversion and bisulphite conversion results of the scheme are similar, so that the downstream application cannot be influenced, and the original data analysis tool can be adopted. However, compared to WGBS, the invention can achieve higher library yields in fewer PCR cycles, contain fewer sequencing repeats, produce more available sequences, increase effective coverage of the genome, have more standard GC bias, and have higher data quality. The methylation enzyme conversion kit can specifically identify and convert 5-mC and 5hmC, and the methylation library-building kit can be connected in the later stage, so that the construction of a methylation sequencing library can be completed, and DNA methylation can be effectively identified with single base resolution. Compared with the phase-specific sulfite conversion method, the method has the advantages of higher conversion efficiency, milder reaction conditions and effective reduction of DNA damage, thereby reducing the requirement of the input amount of DNA in a nucleic acid sample.
Description
Technical Field
The invention relates to the field of DNA methylation, in particular to a DNA methylation conversion method, a methylase conversion kit and a use method thereof.
Background
DNA methylation is a form of chemical modification of DNA, and is one of the most important epigenetic modes of regulation, in which genetic expression can be altered without altering the DNA sequence.
Briefly, DNA methylation refers to the covalent bonding of a methyl group at the cytosine-5 carbon of a genomic CpG dinucleotide under the action of DNA methyltransferase (DNMT). Numerous studies have shown that DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression. The DNA methylation modification has a crucial role in maintaining the functions of normal cells, the X chromosome inactivation of female individuals, the inhibition of parasitic DNA sequences, the structural stability of genomes, genetic imprinting, embryo development, and the tight correlation of the occurrence and development of tumors and diseases, and is one of the research hot spots for a long time at present and in the future.
Currently, the analysis methods for DNA methylation mainly include: methylation sensitive restriction enzyme-PCR/Southern method, bisulfite sequencing, methylation specific PCR, methylation fluorescent method, pyrophosphoric acid sequencing, methylated DNA co-immunoprecipitation, bisulfite binding restriction enzyme method, etc.
Wherein whole genome bisulfite methylation sequencing (WGBS) can accurately detect the methylation level of all individual cytosine bases (C bases) over the whole genome, has long been the gold standard for methylation profiling. The principle is that treatment of DNA with bisulfite converts cytosine residues (C) to uracil (U), but that 5-methylcytosine residues (abbreviated as 5-mC) are resistant thereto and do not undergo conversion. Wherein, 5-hydroxymethyl cytosine is abbreviated as: 5-hmC.
Thus, bisulfite treatment introduces specific changes in the DNA sequence depending on the methylation state of individual C residues, thereby generating single nucleotide resolution information about the methylation state of DNA fragments.
However, the heavy sulfite buffer solution can cause larger damage to sample DNA, the sites with high GC regions which are not subjected to methylation modification are easy to break, the sites with methylation modification are easy to form hairpin structures, and the DNA fragments are not converted, so that poor sequence diversity, target enrichment deviation and higher sequencing error are caused;
meanwhile, the column purification bisulphite conversion method is complex in flow and operation, and has higher requirements on experimental staff.
The above disadvantages limit the application of whole genome bisulfite methylation sequencing in low initial sample single cell, FFPRDNA, cfDNA, paleo DNA and other analyses.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provide a DNA methylation conversion method, a methylase conversion kit and a use method thereof, wherein the DNA methylation conversion method has high conversion efficiency and can effectively reduce the damage of DNA, thereby reducing the requirement on the input amount of DNA in a nucleic acid sample.
In order to solve the technical problems, the invention adopts the technical method that: the invention discloses a DNA methylation conversion method, which comprises the following steps:
s1, performing an enzyme reaction on sample DNA by using human methyl cytosine dioxygenase 2 to realize a catalytic process from 5-methyl cytosine to 5-hydroxymethyl cytosine to 5-formyl cytosine and finally to 5-carboxyl cytosine;
s2, completely transferring glucose groups of uridine diphosphate glucose to 5-hydroxymethylcytosine groups in double-stranded DNA by using T4 bacteriophage beta-glucosyltransferase to form beta-glucosyl-5-hydroxymethylcytosine;
s3, performing deamination reaction, namely using cytosine deaminase to deaminate cytosine in single-stranded DNA into uracil under the condition of not affecting 5-carboxyl cytosine and beta-glucosyl-5-hydroxymethyl cytosine, so as to realize the distinction of cytosine in the original sequence from 5-methyl cytosine and 5-hydroxymethyl cytosine, and further realize the identification of DNA methylation sites by single base resolution.
Further, deaminated single-stranded DNA is amplified by polymerase chain reaction, modified 5-carboxycytosine and beta-glucosyl-5-hydroxymethylcytosine remain cytosine, and uracil is converted to thymine, thereby recognizing methylation sites C.fwdarw.C and unmethylation sites C.fwdarw.T.
The invention also discloses a methylase conversion kit which comprises a first oxidation reaction liquid, a second oxidation reaction liquid providing ionic conditions for the first oxidation reaction liquid, a termination reaction liquid, a denaturation reaction liquid for changing a DNA double chain into a single chain, a first deamination reaction liquid, a second deamination reaction liquid providing ionic conditions for the first deamination reaction liquid, a purification reaction liquid and non-ribozyme water;
the first oxidation reaction solution comprises non-ribozyme water, human methyl cytosine dioxygenase 2, T4 bacteriophage beta-glucosyltransferase, 10mM tris (hydroxymethyl) aminomethane, 20mM potassium phosphate, 200mM sodium chloride, 0.25mM dithiothreitol, 0.1mM ethylenediamine tetraacetic acid and 50% glycerol;
the first deamination reaction solution comprises cytosine deaminase, 20mM of tris hydrochloride, 50mM of sodium chloride, 1mM of dithiothreitol, 0.1mM of ethylenediamine tetraacetic acid, 50% of glycerol and water without ribozyme;
further, the second oxidation reaction solution comprises non-ribozyme water, 100mM tris hydrochloride, 500mM sodium chloride and 10mM alpha-ketoglutarate; 10mM L-ascorbic acid, 10mM adenosine triphosphate, 5mM dithiothreitol, 0.1mM uridine diphosphate glucose, and 20mM ferrous salt compound;
the second deamination reaction solution comprises 50mM potassium acetate, 20mM tris acetate, 10mM magnesium acetate, 1mM dithiothreitol, 1% BSA and ribozyme-free water.
Further, the termination reaction solution comprises 200mg/mL proteinase K, 20mM tris hydrochloride, 1mM calcium chloride, 50% glycerol and water without ribozyme;
the denaturation reaction liquid comprises non-ribozyme water and formamide;
the binding buffer comprises 30% PEG 8000, 1M NaCl and 0.2M Tris-HCl, pH 8.0, and water without ribozyme.
Further, the purification reaction solution comprises magnetic beads and a magnetic bead binding buffer solution, wherein the magnetic beads are selected from 1 mu m MagBeads50mg/mL.
The invention also discloses a using method of the methylase conversion kit, which comprises the following steps based on the methylase conversion kit,
sa1, uniformly mixing a sample solution containing DNA molecules with a first oxidation reaction solution and a second oxidation reaction solution, and incubating at 37 ℃ for 1 hour to perform oxidation reaction;
after the oxidation reaction is finished, adding a stopping reaction solution into the reaction system, and incubating for 15min at 50 ℃ to digest the residual enzyme in the first oxidation reaction solution to stop the reaction;
sa3, after Sa2 is completed in the step, adding a denaturation reaction solution, and denaturing the sample at 85 ℃ for 10 minutes to change the DNA molecule from a double-stranded structure to a single-stranded structure, and immediately transferring the DNA molecule to ice; then adding the first deamination reaction liquid and the second deamination reaction liquid which are prepared by precooling, and incubating for 3 hours at 37 ℃ to carry out deamination reaction;
sa4 purifying and recovering DNA.
Further, in the step Sa2, the method further comprises purifying and recovering the DNA after terminating the reaction.
Further, in the step Sa1, the ratio of the sample solution containing the DNA molecules to the first oxidation reaction solution and the second oxidation reaction solution is: 15:2:8.
Further, in the step Sa3, the ratio of the sample solution containing DNA molecules, the first deamination reaction solution, the second deamination reaction solution, and the water without ribozyme is 20:1:11:68.
The beneficial effects are that:
1. the enzymatic conversion of the present invention is similar to the result of bisulfite conversion and therefore does not affect downstream applications, and may also employ the original data analysis tools. However, compared to WGBS, the invention can achieve higher library yields in fewer PCR cycles, contain fewer sequencing repeats, produce more available sequences, increase effective coverage of the genome, have more standard GC bias, and have higher data quality.
2. The methylation enzyme conversion kit can specifically identify and convert 5-mC and 5hmC, and the methylation library-building kit can be connected in the later stage, so that the construction of a methylation sequencing library can be completed, and DNA methylation can be effectively identified with single base resolution. Compared with the phase-specific sulfite conversion method, the method has higher conversion efficiency and milder reaction conditions, effectively reduces the damage of DNA, thereby reducing the requirement of the input amount of DNA in a nucleic acid sample, and being more suitable for methylation research of trace samples including single cells, cfDNA, FFPE DNA, ancient DNA and the like, wherein the input amount of DNA can be as low as 1ng or even lower.
3. Compared with the prior column purification scheme, the magnetic bead purification scheme is adopted in the kit provided by the invention, so that the purification efficiency is higher, the requirements on laboratory equipment are lower, and an automatic application scheme is expected to be realized.
Drawings
FIG. 1 is a schematic diagram of a DNA methylation conversion method according to the present invention;
FIG. 2 is a graph showing comparison of recovery efficiency of methylase conversion and salt conversion in example 2 of the present invention;
FIG. 3 is a graph showing comparison of conversion efficiencies of methylase and salt conversions in example 2 of the present invention;
FIG. 4 is a graph showing the comparison of the number of library amplification cycles with library yields for the methylation enzymatic conversion and salt conversion in example 3 of the present invention;
FIG. 5 is a chart showing GC coverage analysis of high throughput sequencing after methylation enzymatic conversion and salt conversion in example 3 of the present invention.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings and detailed description.
As shown in FIG. 1, the invention discloses a DNA methylation conversion method, in FIG. 1A, in the first oxidation reaction, human methylcytosine dioxygenase 2 (TET 2) is used for catalyzing and completing the process of 5-methylcytosine (5-mC) →5-hydroxymethylcytosine (5-hmC) →5-formylcytosine (5-fC) →5-carboxycytosine (5-caC) with high efficiency;
in FIG. 1B, the T4 bacteriophage beta-glucosyltransferase (T4-BGT) can rapidly and completely transfer the glucose moiety of uridine diphosphate glucose (UDP-glucose) to the 5-hydroxymethylcytosine (5-hmC) moiety in double-stranded DNA, forming beta-glucosyl-5-hydroxymethylcytosine (5-ghmC);
these reactions protect 5-mC and 5hmC from downstream deamination.
As shown in FIG. 1C, cytosine (C), 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5 hmC) in single stranded DNA can be deaminated to uracil (U) using cytosine deaminase.
Through denaturation, in the second deamination reaction, cytosine deaminase (APOBEC 3A) deaminates cytosine, 5-mC and to a small extent 5hmC (without affecting 5-caC and 5-ghmC), and changes cytosine deamination in single-stranded DNA into uracil, so that cytosine in the original sequence is distinguished from 5-methylcytosine and 5-hydroxymethylcytosine, and DNA methylation sites are identified with single base resolution.
Subsequent amplification by downstream Polymerase Chain Reaction (PCR), modified 5-caC and 5-ghmC remain cytosine, while uracil (U) is converted to thymine (T), thereby identifying the methylation (C.fwdarw.C) and unmethylation (C.fwdarw.T) sites.
Enzymatic conversion is similar to the result of bisulfite conversion and therefore does not affect downstream applications, and may also employ prior data analysis tools.
However, the enzyme transfer process is milder, has little damage to DNA, can retain more complete DNA fragments compared with the traditional bisulfite conversion, is superior to a bisulfite library in specific indexes such as coverage, repeatability, sensitivity and the like, can be matched with a magnetic bead purification mode, and is expected to realize automatic operation.
When the initial amount of DNA is as low as 1ng or even lower, the DNA can be normally built into libraries and keep advantages, and a new way is opened for methylation research and clinical application.
The invention also discloses a methylase conversion kit which comprises the following components: the device comprises a first oxidation reaction liquid, a second oxidation reaction liquid, a termination reaction liquid, a denaturation reaction liquid, a first deamination reaction liquid, a second deamination reaction liquid, a purification reaction liquid and non-ribozyme water.
The first oxidation reaction solution comprises non-ribozyme water, TET2, T4-BGT, 10mM tris, 20mM potassium phosphate, 200mM sodium chloride, 0.25mM dithiothreitol, 0.1mM ethylenediamine tetraacetic acid, and 50% glycerol.
The second oxidation reaction solution comprises water without ribozyme, 100mM tris hydrochloride, 500mM sodium chloride, 10mM alpha-ketoglutarate; 10mM L-ascorbic acid, 10mM adenosine triphosphate, 5mM dithiothreitol, 0.1mM uridine diphosphate glucose and 20mM ferrous salt compound.
Wherein the first oxidation reaction liquid is used for providing enzyme needed by oxidation reaction, and the second oxidation reaction liquid is used for providing ion conditions needed by oxidation reaction; the stop reaction solution comprises 200mg/mL proteinase K, 20mM tris hydrochloride, 1mM calcium chloride, 50% glycerol and water without ribozyme.
The denaturing reaction liquid includes water without ribozyme and formamide.
The first deamination reaction solution comprises cytosine deaminase, 20mM of tris hydrochloride, 50mM of sodium chloride, 1mM of dithiothreitol, 0.1mM of ethylenediamine tetraacetic acid, 50% of glycerol and water without ribozyme.
The second deamination reaction solution comprises 50mM potassium acetate, 20mM tris acetate, 10mM magnesium acetate, 1mM dithiothreitol, 1% BSA, and water without ribozyme.
Wherein the first deamination reaction solution is used for providing enzyme needed by deamination reaction, and the second deamination reaction solution is used for providing ionic conditions needed by deamination reaction.
The purification reaction solution comprises magnetic beads and a magnetic bead binding buffer solution, wherein the magnetic beads are preferably 1 mu m MagBeads50 mg/mL; the binding buffer included 30% PEG 8000, 1M NaCl and 0.2M Tris-HCl, pH 8.0, no ribozyme water.
The invention relates to a use method of a mild methylase conversion kit, which comprises the following steps:
oxidation reaction: uniformly mixing a sample solution containing DNA molecules with a first oxidation reaction solution and a second oxidation reaction solution, and incubating at 37 ℃ for 1 hour to perform oxidation reaction;
after the oxidation reaction is finished, adding a stopping reaction liquid into the reaction system, and incubating for 15min at 50 ℃ to digest the rest enzyme to stop the reaction;
then purifying and recovering DNA;
the recovery mode refers to magnetic bead recovery, column recovery or extraction precipitation recovery, wherein the magnetic bead method is adopted for purification, the operation is simple, and the purification effect is optimal.
Deamination reaction: adding a denaturation reaction liquid into the reaction system of the previous step, denaturing a sample to be tested at 85 ℃ for 10 minutes, and immediately transferring the sample to ice; and then adding the first deamination reaction liquid and the second deamination reaction liquid which are prepared by precooling, incubating for 3 hours at 37 ℃ for deamination reaction, and purifying and recovering DNA after the reaction is finished, thus carrying out downstream experiments.
Example-method of Using methylation enzyme conversion kit
1. Oxidation reaction
(1) On ice, the following oxidation reaction system was formulated:
| reagent name | Volume of |
| cfDNA sample | 30μl |
| First oxidation reaction liquid | 4μl |
| Second oxidation reaction liquid | 16μl |
| Together, a total of | 50μl |
(2) After 1 hour of reaction at 36℃1. Mu.L of stop reaction solution was added thereto, and the mixture was incubated at 37℃for 15 minutes to terminate the oxidation reaction.
2. Purification reaction
(1) The reaction solution was vortexed to resuspend the beads therein.
(2) 90 μl of the resuspended purified reaction solution was added to the sample, and the mixture was then blown up and down with a pipette for 10 times or more.
(3) After incubating the mixed samples for 5 minutes at room temperature, the centrifuge tube was placed on a suitable magnet rack.
(4) After waiting 5 minutes or the solution became clear, the supernatant was carefully aspirated and removed.
(5) The centrifuge tube was kept on a magnetic rack, 200. Mu.l of freshly prepared 80% alcohol was added to the centrifuge tube, incubated at room temperature for 30 seconds, and the supernatant carefully aspirated and removed.
(6) Repeating the operation step (5) once and rinsing twice.
(7) The tube was microcentrifuged, replaced on a magnetic rack, and the residual supernatant carefully aspirated with a 10 μl gun head to remove residual ethanol.
(8) The centrifuge tube is kept on a magnetic frame, a centrifuge tube cover is opened, and the centrifuge tube is dried for 1-2 minutes at room temperature.
(9) The centrifuge tube was removed from the magnet rack and 17. Mu.l of nuclease-free water was added.
(10) Blowing and sucking the mixture for more than 10 times by a pipette, and incubating for 1-2 minutes at room temperature.
(11) The tube was placed on a magnet stand and either left to stand for 3 minutes or the solution became clear, 16 μl of supernatant was transferred to a new PCR tube.
3. Deamination reaction
(1) Mu.l of the denaturation reaction solution was added to the purified sample, and incubated at 85℃for 10 minutes.
(2) Immediately after the end, the sample was placed on ice and allowed to cool completely for use.
(3) On ice, the following deamination reaction system was formulated:
(4) The reaction was carried out at 37℃for 3 hours.
Wherein the centrifuge tube is placed on ice.
4. Purification reaction
(1) The reaction solution was vortexed to resuspend the beads therein.
(2) 180 μl of the resuspended purified reaction solution was added to the sample, and the mixture was then blown up and down with a pipette for 10 times or more.
(3) After incubating the mixed samples for 5 minutes at room temperature, the centrifuge tube was placed on a suitable magnet rack.
(4) After waiting 5 minutes or the solution became clear, the supernatant was carefully aspirated and removed.
(5) The centrifuge tube was kept on a magnetic rack, 200. Mu.l of freshly prepared 80% alcohol was added to the centrifuge tube, incubated at room temperature for 30 seconds, and the supernatant carefully aspirated and removed.
(6) Repeating the operation step (5) once and rinsing twice.
(7) The tube was microcentrifuged, replaced on a magnetic rack, and the residual supernatant carefully aspirated with a 10 μl gun head to remove residual ethanol.
(8) The centrifuge tube is kept on a magnetic frame, a centrifuge tube cover is opened, and the centrifuge tube is dried for 1-2 minutes at room temperature.
(9) The centrifuge tube was removed from the magnet rack and 17. Mu.l of nuclease-free water was added.
(10) Blowing and sucking the mixture for more than 10 times by a pipette, and incubating for 1-2 minutes at room temperature.
(11) The tube was placed on a magnet stand and either left to stand for 3 minutes or the solution became clear, 16 μl of supernatant was transferred to a new PCR tube.
The obtained DNA sample after enzymatic conversion can be directly used for downstream second-generation sequencing library establishment or Q-PCR experiments to verify methylation conversion effects.
Example 2
After enzymatic conversion and salt conversion respectively using 200ng of unmethylated Lambda DNA and CpG methylated pUC19 DNA, the kit of the invention was used for enzymatic conversion, and the Zymo Research EZ DNA Methylation-Gold kit was used for salt conversion, and a second generation sequencing library was constructed for high throughput sequencing after conversion.
Referring to FIGS. 2 and 3, the enzyme conversion method has higher recovery efficiency and conversion efficiency for unmethylated Lambda DNA (enzyme conversion method 1) and CpG-methylated pUC19 DNA (enzyme conversion method 2) than the salt conversion method.
Example 3
After enzymatic and salt conversions using 10, 50 and 200ng of NA12878 genomic DNA, respectively, a second generation sequencing library was constructed for high throughput sequencing.
As shown in fig. 4 and 5, the enzymatic transfer method can obtain higher library yields in fewer PCR cycles and can significantly improve uniformity of GC coverage without AT over-sequencing and GC under-sequencing compared to the salt transfer method.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A DNA methylation conversion method, characterized in that: the method comprises the following steps:
s1, performing an enzyme reaction on sample DNA by using human methyl cytosine dioxygenase 2 to realize a catalytic process from 5-methyl cytosine to 5-hydroxymethyl cytosine to 5-formyl cytosine and finally to 5-carboxyl cytosine;
s2, completely transferring the glucose group of uridine diphosphate glucose to a 5-hydroxymethylcytosine group in double-stranded DNA by using T4 bacteriophage beta-glucosyltransferase to form beta-glucosyl-5-hydroxymethylcytosine;
s3, performing deamination reaction, namely using cytosine deaminase to deaminate cytosine in single-stranded DNA into uracil under the condition of not affecting 5-carboxyl cytosine and beta-glucosyl-5-hydroxymethyl cytosine, so as to realize the distinction of cytosine in the original sequence from 5-methyl cytosine and 5-hydroxymethyl cytosine, and further realize the identification of DNA methylation sites by single base resolution.
2. The DNA methylation conversion method according to claim 1, wherein: the deaminated single-stranded DNA is amplified by polymerase chain reaction, the modified 5-carboxycytosine and beta-glucosyl-5-hydroxymethylcytosine remain cytosine, and uracil is converted to thymine, thereby recognizing methylation sites C.fwdarw.C and unmethylation sites C.fwdarw.T.
3. A methylase conversion kit, characterized in that: comprises a first oxidation reaction liquid, a second oxidation reaction liquid providing ionic conditions for the first oxidation reaction liquid, a termination reaction liquid, a denaturation reaction liquid for changing DNA double chains into single chains, a first deamination reaction liquid, a second deamination reaction liquid providing ionic conditions for the first deamination reaction liquid, a purification reaction liquid and non-ribozyme water;
the first oxidation reaction solution comprises non-ribozyme water, human methyl cytosine dioxygenase 2, T4 bacteriophage beta-glucosyltransferase, 10mM tris, 20mM potassium phosphate, 200mM sodium chloride, 0.25mM dithiothreitol, 0.1mM ethylenediamine tetraacetic acid and 50% glycerol;
the first deamination reaction solution comprises cytosine deaminase, 20mM tris hydrochloride, 50mM sodium chloride, 1mM dithiothreitol, 0.1mM ethylenediamine tetraacetic acid, 50% glycerol and water without ribozyme.
4. A methylase conversion kit according to claim 3, wherein: the second oxidation reaction solution comprises non-ribozyme water, 100mM of tris hydrochloride, 500mM of sodium chloride and 10mM of alpha-ketoglutarate; 10mM L-ascorbic acid, 10mM adenosine triphosphate, 5mM dithiothreitol, 0.1mM uridine diphosphate glucose, and 20mM ferrous salt compound;
the second deamination reaction solution comprises 50mM potassium acetate, 20mM tris acetate, 10mM magnesium acetate, 1mM dithiothreitol, 1% BSA and water without ribozyme.
5. A methylase conversion kit according to claim 3, wherein: the termination reaction solution comprises 200mg/mL proteinase K, 20mM tris hydrochloride, 1mM calcium chloride, 50% glycerol and water without ribozyme;
the denaturation reaction liquid comprises non-ribozyme water and formamide;
the binding buffer comprises 30% PEG 8000, 1M NaCl and 0.2M Tris-HCl, pH 8.0, and water without ribozyme.
6. The methylase conversion kit according to claim 5, wherein: the purification reaction solution comprises magnetic beads and a magnetic bead binding buffer solution, wherein the magnetic beads are 1 mu m MagBeads50mg/mL.
7. The method for using the methylase conversion kit is characterized by comprising the following steps of: based on the methylase conversion kit according to any one of claims 3 to 6, further comprising the step of,
sa1, uniformly mixing a sample solution containing DNA molecules with a first oxidation reaction solution and a second oxidation reaction solution, and incubating at 37 ℃ for 1 hour to perform oxidation reaction;
after the oxidation reaction is finished, adding a termination reaction solution into the reaction system, and incubating for 15min at 50 ℃ to digest the residual enzyme in the first oxidation reaction solution to terminate the reaction;
after Sa2 is completed in the step Sa3, adding a denaturation reaction liquid, and denaturing the sample at 85 ℃ for 10 minutes to change the DNA molecule from a double-chain structure to a single-chain structure, and immediately transferring the DNA molecule to ice; then adding the first deamination reaction liquid and the second deamination reaction liquid which are prepared by precooling, and incubating for 3 hours at 37 ℃ to carry out deamination reaction;
sa4 purifying and recovering DNA.
8. The method of using the methylase conversion kit according to claim 7, wherein: in step Sa2, the method further comprises purifying and recovering the DNA after terminating the reaction.
9. The method of using the methylase conversion kit according to claim 7, wherein: in the step Sa1, the ratio of the sample solution containing the DNA molecules to the first oxidation reaction solution and the second oxidation reaction solution is: 15:2:8.
10. The method of using a methylase conversion kit according to claim 9, wherein: in step Sa3, the ratio of the sample solution containing DNA molecules, the first deamination reaction solution, the second deamination reaction solution and the water without ribozyme is 20:1:11:68.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311637502.0A CN117625745A (en) | 2023-12-01 | 2023-12-01 | DNA methylation conversion method, methylase conversion kit and use method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311637502.0A CN117625745A (en) | 2023-12-01 | 2023-12-01 | DNA methylation conversion method, methylase conversion kit and use method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117625745A true CN117625745A (en) | 2024-03-01 |
Family
ID=90019655
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311637502.0A Pending CN117625745A (en) | 2023-12-01 | 2023-12-01 | DNA methylation conversion method, methylase conversion kit and use method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117625745A (en) |
-
2023
- 2023-12-01 CN CN202311637502.0A patent/CN117625745A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Vaisvila et al. | Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA | |
| JP7136899B2 (en) | Cytosine-modified, bisulfite-free, base resolution specific | |
| AU2016202081B2 (en) | Methods for detection of nucleotide modification | |
| US8728733B1 (en) | Methyl-CpG island-associated genome signature tags | |
| US11274335B2 (en) | Methods for the epigenetic analysis of DNA, particularly cell-free DNA | |
| CN107760762B (en) | Fluorescent chemical sensor for detecting DNA adenine methyltransferase and detection method thereof | |
| Lv et al. | Advances in mapping the epigenetic modifications of 5‐methylcytosine (5mC), N6‐methyladenine (6mA), and N4‐methylcytosine (4mC) | |
| CN108866174B (en) | Method for detecting low-frequency mutation of circulating tumor DNA | |
| WO2023193357A1 (en) | Method for constructing free dna methylation sequencing library and use thereof | |
| CN113943763B (en) | Method for reducing nucleic acid and detection method thereof | |
| CN113881739B (en) | Method for oxidizing nucleic acid molecules containing jagged ends, reduction method and library construction method | |
| CN113862333B (en) | Composition and method for oxidizing 5-methylcytosine by using same | |
| WO2020135347A1 (en) | Method for detecting dna methylation, test kit, device and application | |
| CN117625745A (en) | DNA methylation conversion method, methylase conversion kit and use method thereof | |
| CN113846160A (en) | Forward-screening cytosine methylation rapid detection method and application of peroxytungstate in oxidation of 5hmC to thT | |
| WO2019233450A1 (en) | Detection method for dna methylation | |
| WO2019233451A1 (en) | Dna methylation detection probe | |
| CN112301118B (en) | Method and kit for simultaneously obtaining RNA abundance and active RNA polymerase sites in full transcriptome range | |
| CN116926167A (en) | Target nucleic acid capture probe, kit, method and application in targeted capture urine sample | |
| Bai et al. | Chemical-Assisted Epigenome Sequencing | |
| WO2024064915A2 (en) | Methods for enriching nucleic acid target sequences | |
| CN115418391A (en) | Resolution positioning analysis method for 5-methylcytosine modification single base in DNA assisted by artificially reconstructed deaminase | |
| CN113249446A (en) | Quantitative method of 5hmC level of whole genome based on nucleic acid isothermal amplification and application thereof | |
| CN115044651A (en) | Method for co-detecting DNA methylation and DNA mutation | |
| CN115976160A (en) | Click chemistry-based enrichment and localization analysis method for 5-carboxyl cytosine (5 caC) in DNA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |