CN110669824A - Kit and method for methylation library construction of low-initial-amount plasma free DNA - Google Patents
Kit and method for methylation library construction of low-initial-amount plasma free DNA Download PDFInfo
- Publication number
- CN110669824A CN110669824A CN201910963720.0A CN201910963720A CN110669824A CN 110669824 A CN110669824 A CN 110669824A CN 201910963720 A CN201910963720 A CN 201910963720A CN 110669824 A CN110669824 A CN 110669824A
- Authority
- CN
- China
- Prior art keywords
- reaction
- dna
- optionally
- methylation
- reagent
- 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
- 230000011987 methylation Effects 0.000 title claims abstract description 46
- 238000007069 methylation reaction Methods 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000010276 construction Methods 0.000 title abstract description 18
- 238000006243 chemical reaction Methods 0.000 claims abstract description 48
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 44
- 102000012410 DNA Ligases Human genes 0.000 claims abstract description 25
- 108010061982 DNA Ligases Proteins 0.000 claims abstract description 25
- 102000004190 Enzymes Human genes 0.000 claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 claims abstract description 21
- 101000889901 Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) Tetrahedral aminopeptidase Proteins 0.000 claims abstract description 19
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 15
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 14
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 14
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 14
- 238000000746 purification Methods 0.000 claims abstract description 11
- 230000008439 repair process Effects 0.000 claims abstract description 8
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 13
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 13
- 229960003237 betaine Drugs 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
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 12
- 238000012408 PCR amplification Methods 0.000 claims description 11
- 102000000311 Cytosine Deaminase Human genes 0.000 claims description 10
- 108010080611 Cytosine Deaminase Proteins 0.000 claims description 10
- 238000005580 one pot reaction Methods 0.000 claims description 10
- 239000011535 reaction buffer Substances 0.000 claims description 10
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 8
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 8
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 claims description 8
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 claims description 8
- 108010006785 Taq Polymerase Proteins 0.000 claims description 8
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 8
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 8
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 7
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- 238000004925 denaturation Methods 0.000 claims description 6
- 230000036425 denaturation Effects 0.000 claims description 6
- 238000006911 enzymatic reaction Methods 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- 238000007259 addition reaction Methods 0.000 claims description 4
- 229940035893 uracil Drugs 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- -1 dNTP Chemical compound 0.000 claims description 3
- 230000002708 enhancing effect Effects 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims 1
- 238000012163 sequencing technique Methods 0.000 abstract description 12
- 230000008569 process Effects 0.000 abstract description 5
- 230000006378 damage Effects 0.000 abstract description 3
- 239000000047 product Substances 0.000 description 19
- 206010028980 Neoplasm Diseases 0.000 description 8
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 6
- 101000653374 Homo sapiens Methylcytosine dioxygenase TET2 Proteins 0.000 description 6
- 102100030803 Methylcytosine dioxygenase TET2 Human genes 0.000 description 6
- 239000011324 bead Substances 0.000 description 5
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 229910017052 cobalt Inorganic materials 0.000 description 3
- 239000010941 cobalt Substances 0.000 description 3
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 230000007067 DNA methylation Effects 0.000 description 2
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 2
- 101000653360 Homo sapiens Methylcytosine dioxygenase TET1 Proteins 0.000 description 2
- 102100030819 Methylcytosine dioxygenase TET1 Human genes 0.000 description 2
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 2
- 229960001570 ademetionine Drugs 0.000 description 2
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 2
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 229940104302 cytosine Drugs 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 108010028143 Dioxygenases Proteins 0.000 description 1
- 102000016680 Dioxygenases Human genes 0.000 description 1
- 101000653369 Homo sapiens Methylcytosine dioxygenase TET3 Proteins 0.000 description 1
- 102100030812 Methylcytosine dioxygenase TET3 Human genes 0.000 description 1
- 102000055027 Protein Methyltransferases Human genes 0.000 description 1
- 108700040121 Protein Methyltransferases Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 231100000118 genetic alteration Toxicity 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000012164 methylation sequencing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000007481 next generation sequencing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 238000007039 two-step reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an enzymatic methylation library construction method, which reduces the damage of DNA in the methylation library construction process to a certain extent through TET enzyme methylation treatment, thereby reducing the input requirement of the DNA in a nucleic acid sample; in addition, by further matching with the terminal repair and A reaction one-step reagent, one-tube library construction can be realized, and purification steps are reduced; by further matching with the DNA ligase enhancement solution, the ligation efficiency can be improved, and the library construction quality and the sequencing quality can be improved; the method has low initial database building amount and DNA input amount as low as 1ng, and is suitable for micro database building including cfDNA.
Description
Technical Field
The invention belongs to the field of methylation sequencing, and particularly relates to a methylation library construction method and a library construction kit.
Background
Blood free DNA (cfDNA) is a DNA fragment discharged into blood from human tissues, a small amount of free DNA can be found in plasma of healthy people, and the fragment is mainly 160-175 bp or an integral multiple thereof. Free DNA in healthy human plasma is mainly derived from apoptotic cells. Tumor patients have elevated cfDNA content compared to healthy people, which can be from tumor tissue, circulating tumor cells and normal tissue. ctdna (circulating tumor dna) is one of the total free circulating dna (cfdna) in body fluids, a free genomic fragment that carries tumor-specific genetic alterations released only by tumor cells. ctDNA can reveal comprehensive genetic information of tumor, accurately reflect heterogeneity of tumor tissue and tumor load, and has longitudinal monitoring capability of repeated detection on the same patient.
DNA methylation refers to the process of transferring a methyl group to a specific base by the action of DNA methyltransferase, using S-adenosylmethionine (SAM) as a methyl donor. The research shows that the methylation abnormality can be used as a tumor marker for detecting free DNA (cfDNA) in blood, and the DNA methylation detection plays an important role in early tumor screening, dynamic detection and prognosis guidance.
The current major technique for detecting methylation by next generation sequencing is to treat genomic DNA with Bisulfite (bisufite method), whereas treatment of DNA with Bisulfite results in a large loss of DNA, which leads to the problem that methylation detection requires a high DNA input and a high number of amplification cycles. Because the content of cfDNA is very trace, it is often difficult to effectively meet the DNA input requirement of bisulfite-based methylation banking method, and too high number of amplification cycles introduces more sequencing errors, the conventional cfDNA methylation banking quality is often very low.
The present invention has been made based on this.
Disclosure of Invention
The invention aims to provide a methylation database building method and a database building kit.
The technical scheme adopted by the invention is as follows:
a methylation library building method, comprising the steps of:
(1) simultaneously carrying out end repair and A addition reaction on a nucleic acid sample;
(2) carrying out methylation joint connection on the product obtained in the step (1);
(3) carrying out enzymatic methylation treatment on the product obtained in the step (2), wherein the enzymatic methylation treatment comprises TET enzyme treatment, TET enzyme reaction termination, DNA denaturation treatment and cytosine deaminase treatment in sequence;
(4) and (4) carrying out PCR amplification on the product obtained in the step (3).
Optionally, in step (1), the amount of DNA in the nucleic acid sample is as low as 1 ng.
Optionally, the methylated linker ligation reagent comprises T4 DNA ligase, T4 DNA ligase buffer, and methylated linker.
Optionally, the T4 DNA ligase buffer comprises 40-80mM Tris-HCl, 8-15mM MgCl2、3-7mM DTT，0.8-1.5mM ATP。
Optionally, the methylated linker ligation reagent further comprises a DNA ligase enhancement solution comprising the following composition of a) or b):
a) hexaammine cobalt chloride
b) Hexaammine cobalt chloride and betaine;
alternatively, the initial concentration of cobalt hexammine chloride in the reaction system is 0.1 to 1 mM.
Optionally, the initial concentration of betaine in the reaction system is 0.5-3M.
Optionally, the end-repair and the addition of a reaction is a one-step reaction;
the reagents of the one-step reaction comprise: ER/dA enzyme mixture, and ER/dA reaction buffer.
Optionally, the ER/dA enzyme mixture comprises T4 polynucleotide kinase, Taq DNA polymerase, T4 DNA polymerase.
Alternatively, the initial concentration in the reaction system: the T4 polynucleotide kinase is 0.2-1U/. mu.l, the Taq DNA polymerase is 0.01-0.8U/. mu.l, and the T4 DNA polymerase is 0.1-1U/. mu.l.
Alternatively, the ER/dA reaction buffer comprises: dATP, dNTP, Tris-HCl, MgCl2、DTT。
Optionally, the initial concentration in the reaction system is 0.1-1nmol/μ l dATP, 0.1-1nmol/μ l dNTP, 50-100mM Tris-HCl and MgCl25-20mM and DTT 2-10 mM.
Optionally, the Enzymatic methylation reagent of step (3) comprises an enzymic Methyl-seq Kit.
Optionally, the PCR amplification reagent of step (4) comprises i5 Index Primer, Universal Primer and KAPAHiFi HotStart Uracil + ReadyMix.
Optionally, a purification step is further included after at least one of the steps of linker ligation, termination of the TET enzyme reaction, and cytosine deaminase treatment.
A methylation library building kit comprising:
end repairing and adding A reaction reagent, methylated joint connecting reagent, enzymatic methylation treatment reagent and PCR amplification reagent; the agent is as defined in any one of the above.
Optionally, the kit further comprises a purification reagent.
A DNA ligase enhancement solution comprising the following composition of a) or b):
a) hexaammine cobalt chloride
b) Hexaammine cobalt chloride and betaine;
optionally, the initial concentration of the hexaammine cobalt chloride in the reaction system is 0.1-1 mM;
optionally, the initial concentration of betaine in the reaction system is 0.5-3M.
The invention has the beneficial effects that:
the invention provides an enzymatic methylation library construction method, which reduces the damage of DNA in the methylation library construction process to a certain extent through TET enzyme methylation treatment, thereby reducing the input requirement of the DNA in a nucleic acid sample; in addition, by further matching with the terminal repair and A reaction one-step reagent, one-tube library construction can be realized, and purification steps are reduced; by further matching with the DNA ligase enhancement solution, the ligation efficiency can be improved, and the library construction quality and the sequencing quality can be improved; the method has low initial database building amount and DNA input amount as low as 1ng, and is suitable for micro database building including cfDNA.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
In view of the defects of large damage to DNA, low recovery rate, large DNA input amount, high cycle number of amplification for establishing a database of a trace nucleic acid sample and poor database establishing quality in the prior art based on bisulfite treatment.
The invention provides a methylation database building method, which comprises the following steps:
(1) simultaneously carrying out end repair and A addition reaction on a nucleic acid sample;
(2) carrying out methylation joint connection on the product obtained in the step (1);
(3) carrying out enzymatic methylation treatment on the product obtained in the step (2), wherein the enzymatic methylation treatment comprises TET enzyme treatment, TET enzyme reaction termination, DNA denaturation treatment and cytosine deaminase treatment in sequence;
(4) and (4) carrying out PCR amplification on the product obtained in the step (3).
The invention reduces the DNA damage in the methylation library building process to a certain extent through TET enzyme treatment, thereby reducing the input requirement of DNA in a nucleic acid sample.
TET enzyme is an alpha-ketoglutaric acid (alpha-KG) and Fe existing in organisms2+Dependent upon the dioxygenase, there are three members of the TET enzyme family, TET1, TET2 and TET3, all of which have the ability to convert 5-mC to 5-hmC. The TET enzyme is optionally TET1, TET2 or TET 3.
In the step (1), the terminal repairing and adding a reaction may be a one-step reaction or a two-step reaction, and as an embodiment, the terminal repairing and adding a reaction is a one-step reaction, and the one-step reaction reagent includes: ER/dA enzyme mixture, and ER/dA reaction buffer.
In one embodiment, the ER/dA enzyme mixture comprises T4 polynucleotide kinase, Taq DNA polymerase, T4 DNA polymerase; alternatively, the initial concentration in the reaction system: the T4 polynucleotide kinase is 0.2-1U/. mu.l, the Taq DNA polymerase is 0.01-0.8U/. mu.l, and the T4 DNA polymerase is 0.1-1U/. mu.l.
As one embodiment, the ER/dA reaction buffer comprises: dATP, dNTP, Tris-HCl, MgCl2DTT; optionally, the initial concentration in the reaction system is 0.1-1nmol/μ l dATP, 0.1-1nmol/μ l dNTP, 50-100mM Tris-HCl and MgCl25-20mM and DTT 2-10 mM.
The one-step reaction condition is that the incubation is carried out for 25-40min at 25-35 ℃, and then the incubation is carried out for 25-40min at 70-75 ℃.
The one-step reaction developed by the invention does not need Klenow Fragment, can save the purification step by the one-step reaction, reduces the loss of DNA samples, and realizes one-tube library construction.
In the step (2), the methylated linker connecting reagent comprises: t4 DNA ligase, T4 DNA ligase buffer solution and methylated linker.
In one embodiment, the T4 DNA ligase buffer comprises 40-80mM Tris-HCl, 8-15mM MgCl2、3-7mM DTT，0.8-1.5mM ATP。
The concentration of T4 DNA ligase in the reaction system is 20-50U/. mu.l.
A methylated linker refers to a linker wherein the C base has been methylated.
As an embodiment, the linker is formed by annealing linker 1 and linker 2, and the linker is 10-30 pmol/. mu.l in the reaction system
The sequence of the joint 1 is as follows: 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3'
The sequence of the linker 2 is: 5 '-TACACTCTTTCCCTACACGACGCTCTTCCGATC T-3'
As an embodiment, the methylated linker attachment reagent further comprises: a DNA ligase enhancement solution comprising the following composition of a) or b):
a) hexaammine cobalt chloride
b) Hexaammine cobalt chloride and betaine;
optionally, the initial concentration of the hexaammine cobalt chloride in the reaction system is 0.1-1 mM; specifically, it is used at a concentration typically but not limited to 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0 mM; the invention discovers that the hexaammine cobalt chloride can improve the library quality and the sequencing quality.
Optionally, the initial concentration of betaine in the reaction system is 0.5-3M; specifically, the concentration of the compound is typically, but not limited to, 0.5M, 0.8M, 1M, 1.5M, 1.8M, 2M, 2.5M, 3M; the invention finds that the betaine and the hexaammine cobalt chloride can synergistically improve the library quality and the sequencing quality.
The methylated linker ligation conditions are typically incubation for 20-40min at 25-30 ℃.
In one embodiment, the Enzymatic methylation reagent of step (3) comprises enzymic Methyl-seqKit.
As one embodiment, the PCR amplification reagents of step (4) include i5 Index Primer, Universal Primer and KAPA HiFi HotStart Uracil + ReadyMix.
Wherein i5 Index Primer and Universal Primer are illumina adapter primers, and Index can be designed by self and is a conventional technology in the field.
Optionally, a purification step is further included after at least one of the steps of linker ligation, termination of the TET enzyme reaction, and cytosine deaminase treatment; purification reagents such as Agencour AMPure XP Beads.
Based on the database building method, the initial amount of DNA of the nucleic acid sample is low, and compared with the traditional bisulfite methylation database building method, the library quality and the sequencing quality are obviously improved.
The invention also provides a methylation library construction kit, which comprises:
end repairing and adding A reaction reagent, methylated joint connecting reagent, enzymatic methylation treatment reagent and PCR amplification reagent;
the reagents are as defined in the methylation library construction method of the invention.
Optionally, the kit further comprises a purification reagent.
The invention also provides a DNA ligase enhancing solution as defined above.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
1. End repair and addition of A
The solution containing nucleic acid was used as a template DNA, and the end repair and A addition reaction were carried out.
Reagent: ER/dA enzyme mixture (containing T4 polynucleotide kinase, Taq DNA polymerase, T4 DNA polymerase), 5XER/dA reaction buffer (containing dNTP, Tris-HCl, MgCl2、DTT)。
The amount of template DNA added to a 50. mu.l reaction system is at least 1ng, the concentration of T4 polynucleotide kinase is 0.6U/. mu.l, the concentration of Taq DNA polymerase is 0.3U/. mu.l, the concentration of T4 DNA polymerase is 0.5U/. mu.l, the concentration of dATP is 0.15 nmol/. mu.l, the concentration of dNTP is 0.3 nmol/. mu.l, the concentration of Tris-HCl is 80mM, MgCl2The concentration was 15mM and the concentration of DTT was 7 mM.
Reaction conditions are as follows: incubation was performed at 30 ℃ for 30min and then at 72 ℃ for 30 min.
The specific reaction system is as follows:
| reagent | Volume μ l |
| Template DNA | X |
| Nuclease-free water | 32.5-X |
| 5XER/dA reaction buffer | 10 |
| ER/dA enzyme mixture | 7.5 |
2. Methylated linker ligation
And taking the product of the end repair and the addition of A for carrying out methylation joint connection.
Reagent: t4 DNA ligase, T4 DNA ligase buffer solution and methylated linker.
80 μ l reaction: the concentration of T4 DNA ligase is 30U/. mu.l; the linker was formed by annealing linker 1 and linker 2 at a concentration of 15 pmol/. mu.l in T4 DNA ligase buffer including 50mM Tris-HCl, 10mM MgCl2、5mM DTT、1mMATP。
The sequence of the joint 1 is as follows: GATCGGAAGAGCACACGTCTGAACTCCAGTCAC
The sequence of the linker 2 is: TACACTCTTTCCCTACACGACGCTCTTCCGATC T
The C base in the linker needs to be methylated.
Reaction conditions are as follows: incubate at 25 ℃ for 30 min.
The specific reaction system is as follows:
| reagent | Volume μ l |
| End-repair plus A product | 50 |
| Nuclease-free water | 16.75 |
| T4 DNA Ligase | 4 |
| T4 DNA Ligation Buffer | 8 |
| Joint | 1.25 |
After the reaction is finished, the ligation product can be obtained by purifying the obtained product by using Agencour AMPure XP Beads.
3. Enzymatic methylation process
The ligation product was methylated using the enzyme Methyl-seq Kit.
(1) TET enzyme treatment
TET enzyme treatment reagent: TET2 buffer solution, oxidation supplement solution, oxidation enhancement solution, TET2 and ferrous solution.
The reaction conditions are as follows: incubate at 37 ℃ for 1 hour.
The specific reaction system is as follows:
| reagent | Volume μ l |
| Ligation product | 29 |
| TET2 buffer solution | 10 |
| Oxidation supplementary liquid | 1 |
| Oxidation enhancing liquid | 1 |
| TET2 | 4 |
| Ferrous solution | 5 |
(2) Termination of the reaction
To the TET enzyme treated product was added 1ul of a reaction terminator.
The reaction conditions are as follows: incubate at 37 ℃ for 30 min.
After the reaction is finished, 16 mu l of termination reaction product can be obtained by purifying the product by using Agencour AMPure XP Beads.
(3) Denaturation of DNA
To 16ul of the quenched reaction product was added 4ul of 0.1mol NaOH solution.
The reaction conditions are as follows: incubate at 50 ℃ for 10 min.
(4) Cytosine deamination
Cytosine deaminating agent: cytosine deaminase reaction buffer, BSA, cytosine deaminase.
The reaction conditions are as follows: incubate at 37 ℃ for 3 h.
The specific reaction system is as follows:
| reagent | Volume μ l |
| DNA denaturation product | 20 |
| Cytosine deaminase reaction buffer | 10 |
| BSA | 1 |
| Cytosine deaminase | 1 |
| Nuclease-free water | 68 |
After the reaction is finished, the methylation treatment product can be obtained by purifying the product by using Agencour AMPure XP Beads.
4. PCR amplification
PCR amplification reagents: i5 Index Primer, Universal Primer, KAPA HiFi HotStartUracil + ReadyMix.
The concentrations of i5 Index Primer and Universal Primer in the reaction system were both 10 pmol; the input volume of KAPA HiFiHotStart Uracil + ReadyMix was 15 ul. Wherein i5 Index Primer is the tag sequence.
The reaction conditions are as follows: 1min at 98 ℃; 15S at 98 ℃, 30S at 60 ℃ and 30S at 72 ℃ for a plurality of cycles; 72 ℃ for 1 min. Wherein the cycle number is determined by the initial input amount of the DNA for establishing a library, and when the nucleic acid sample is 1-10ng, the cycle number is 18 cycles; the nucleic acid sample is 10-50ng, and the cycle number is 12-15 cycles; nucleic acid samples > 50ng, cycle number 8-10 cycles.
The specific reaction system is as follows:
after the reaction is finished, the Agencour AMPure XP Beads are used for purification, and a library building product can be obtained.
Using the method of this example, samples of DNA content of 1ng and 10ng were tested (two replicates), respectively, with the following results:
comparative example 1
Methylation of step (3) with conventional bisulfite was performed, and the rest of the procedure was the same as in example 1.
Using the method of this example, samples of DNA content of 1ng and 10ng were tested (two replicates), respectively, with the following results:
example 2
The invention designs a DNA ligase enhancement solution, and tests the effect of the DNA ligase enhancement solution on the methylation library construction.
(1) The effect of cobalt hexaammine chloride on the methylation library construction of the invention was tested, taking 10ng of DNA input as an example, and the results were as follows:
(2) testing the Effect of betaine on the methylation library construction of the invention, taking 10ng of DNA input as an example
The results show that the cobalt hexaammine chloride can improve the quality of the library; further adding betaine can improve the quality of the library to a certain degree.
Test example
The library constructed by the embodiment and the comparative example is subjected to on-machine sequencing: the library was quantitated by performing a fragment quality check using an Agilent Bioanalyzer 2100 in conjunction with a High sensitivity DNA kit and qpcr after passage (main peak at 400-500 bp). The quantified library was mixed in the quantitative ratio of the sequencing data and diluted to 2nM, and the diluted library was denatured. After the denaturation treatment is finished, the mixture is subjected to gradient dilution to the concentration on the machine. And adding the diluted library into a corresponding sample hole of the sequencing kit and sequencing according to the operation protocol of a corresponding sequencer.
Sequencing data are shown as follows, and it can be seen that the reagent and the method can improve the sequencing quality greatly:
Claims (10)
1. a methylation library building method, comprising the steps of:
(1) simultaneously carrying out end repair and A addition reaction on a nucleic acid sample;
(2) carrying out methylation joint connection on the product obtained in the step (1);
(3) carrying out enzymatic methylation treatment on the product obtained in the step (2), wherein the enzymatic methylation treatment comprises TET enzyme treatment, TET enzyme reaction termination, DNA denaturation treatment and cytosine deaminase treatment in sequence;
(4) and (4) carrying out PCR amplification on the product obtained in the step (3).
2. The method of claim 1, wherein: in step (1), the DNA content in the nucleic acid sample is as low as 1 ng.
3. The method of claim 1, wherein: the methylated joint connecting reagent comprises T4 DNA ligase, T4 DNA ligase buffer solution and methylated joints;
optionally, the T4 DNA ligase buffer comprises 40-80mM Tris-HCl, 8-15mM MgCl2、3-7mM DTT、0.8-1.5mM ATP。
4. The method of claim 3, wherein: the methylated linker ligation reagent further comprises a DNA ligase enhancement solution comprising the following composition of a) or b):
a) hexaammine cobalt chloride
b) Hexaammine cobalt chloride and betaine;
optionally, the initial concentration of the hexaammine cobalt chloride in the reaction system is 0.1-1 mM;
optionally, the initial concentration of betaine in the reaction system is 0.5-3M.
5. The method of claim 1, wherein: the end repairing and A adding reaction are one-step reactions;
the reagents of the one-step reaction comprise: ER/dA enzyme mixture, ER/dA reaction buffer;
optionally, the ER/dA enzyme mixture comprises T4 polynucleotide kinase, Taq DNA polymerase, T4 DNA polymerase;
alternatively, the initial concentration in the reaction system: the T4 polynucleotide kinase is 0.2-1U/mul, the Taq DNA polymerase is 0.01-0.8U/mul, the T4 DNA polymerase is 0.1-1U/mul;
alternatively, the ER/dA reaction buffer comprises: dATP, dNTP, Tris-HCl, MgCl2 and DTT;
optionally, the initial concentration in the reaction system is 0.1-1nmol/μ l of dATP, 0.1-1nmol/μ l of dNTP, 50-100mM of Tris-HCl, 5-20mM of MgCl2, and 2-10mM of DTT.
6. The method of claim 1, wherein:
optionally, the Enzymatic methylation treatment reagent of step (3) comprises an enzymic Methyl-seq Kit;
optionally, the PCR amplification reagent of step (4) comprises i5 Index Primer, Universal Primer and KAPAHiFi HotStart Uracil + ReadyMix.
7. The method of claim 1, wherein:
optionally, a purification step is further included after at least one of the steps of linker ligation, termination of the TET enzyme reaction, and cytosine deaminase treatment.
8. A methylation library building kit comprising:
end repairing and adding A reaction reagent, methylated joint connecting reagent, enzymatic methylation treatment reagent and PCR amplification reagent;
the agent is as defined in any one of claims 3 to 6.
9. The kit of claim 8, wherein: the kit also includes a purification reagent.
A DNA ligase enhancing fluid comprising the following composition of a) or b):
a) hexaammine cobalt chloride
b) Hexaammine cobalt chloride and betaine;
optionally, the initial concentration of the hexaammine cobalt chloride in the reaction system is 0.1-1 mM;
optionally, the initial concentration of betaine in the reaction system is 0.5-3M.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910963720.0A CN110669824A (en) | 2019-10-11 | 2019-10-11 | Kit and method for methylation library construction of low-initial-amount plasma free DNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910963720.0A CN110669824A (en) | 2019-10-11 | 2019-10-11 | Kit and method for methylation library construction of low-initial-amount plasma free DNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110669824A true CN110669824A (en) | 2020-01-10 |
Family
ID=69081652
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910963720.0A Pending CN110669824A (en) | 2019-10-11 | 2019-10-11 | Kit and method for methylation library construction of low-initial-amount plasma free DNA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110669824A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725438A (en) * | 2021-02-05 | 2021-04-30 | 深圳市宝安区妇幼保健院 | Endometrial polyp methylation marker combination, detection kit and application |
| CN113881739A (en) * | 2021-09-28 | 2022-01-04 | 深圳吉因加医学检验实验室 | Method for oxidizing nucleic acid molecules containing jagged ends, reduction method and library construction method |
| CN113943763A (en) * | 2021-10-28 | 2022-01-18 | 深圳吉因加医学检验实验室 | Method for reducing nucleic acid and detection method thereof |
| WO2023193357A1 (en) * | 2022-04-08 | 2023-10-12 | 昌平国家实验室 | Method for constructing free dna methylation sequencing library and use thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120283144A1 (en) * | 2011-05-06 | 2012-11-08 | New England Biolabs, Inc. | Ligation Enhancement |
| CN103088433A (en) * | 2011-11-02 | 2013-05-08 | 深圳华大基因科技有限公司 | Construction method and application of genome-wide methylation high-throughput sequencing library and |
| CN104532360B (en) * | 2014-12-17 | 2017-02-22 | 北京诺禾致源科技股份有限公司 | Whole-genome methylation sequencing library and construction method thereof |
| CN107541791A (en) * | 2017-10-26 | 2018-01-05 | 中国科学院北京基因组研究所 | Construction method, kit and the application in plasma DNA DNA methylation assay library |
| WO2019136413A1 (en) * | 2018-01-08 | 2019-07-11 | Ludwig Institute For Cancer Research Ltd | Bisulfite-free, base-resolution identification of cytosine modifications |
| CN110305940A (en) * | 2019-07-31 | 2019-10-08 | 益善生物技术股份有限公司 | A kind of method and kit of the detection of 9 gene methylation of Septin |
-
2019
- 2019-10-11 CN CN201910963720.0A patent/CN110669824A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120283144A1 (en) * | 2011-05-06 | 2012-11-08 | New England Biolabs, Inc. | Ligation Enhancement |
| CN103088433A (en) * | 2011-11-02 | 2013-05-08 | 深圳华大基因科技有限公司 | Construction method and application of genome-wide methylation high-throughput sequencing library and |
| CN104532360B (en) * | 2014-12-17 | 2017-02-22 | 北京诺禾致源科技股份有限公司 | Whole-genome methylation sequencing library and construction method thereof |
| CN107541791A (en) * | 2017-10-26 | 2018-01-05 | 中国科学院北京基因组研究所 | Construction method, kit and the application in plasma DNA DNA methylation assay library |
| WO2019136413A1 (en) * | 2018-01-08 | 2019-07-11 | Ludwig Institute For Cancer Research Ltd | Bisulfite-free, base-resolution identification of cytosine modifications |
| CN110305940A (en) * | 2019-07-31 | 2019-10-08 | 益善生物技术股份有限公司 | A kind of method and kit of the detection of 9 gene methylation of Septin |
Non-Patent Citations (2)
| Title |
|---|
| J R RUSCHE等: "Hexamine cobalt chloride promotes intermolecular ligation of blunt end DNA fragments by T4 DNA ligase", 《NUCLEIC ACIDS RES.》 * |
| NEB: "NEBNext® Enzymatic Methyl-seq Kit", 《HTTPS://WWW.NEB.COM/PRODUCTS/E7120-NEBNEXT-ENZYMATIC-METHYL-SEQ-KIT#PRODUCT%20INFORMATION》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725438A (en) * | 2021-02-05 | 2021-04-30 | 深圳市宝安区妇幼保健院 | Endometrial polyp methylation marker combination, detection kit and application |
| CN112725438B (en) * | 2021-02-05 | 2023-08-22 | 深圳市宝安区妇幼保健院 | Endometrial polyp methylation marker combination, detection kit and application |
| CN113881739A (en) * | 2021-09-28 | 2022-01-04 | 深圳吉因加医学检验实验室 | Method for oxidizing nucleic acid molecules containing jagged ends, reduction method and library construction method |
| CN113943763A (en) * | 2021-10-28 | 2022-01-18 | 深圳吉因加医学检验实验室 | Method for reducing nucleic acid and detection method thereof |
| WO2023193357A1 (en) * | 2022-04-08 | 2023-10-12 | 昌平国家实验室 | Method for constructing free dna methylation sequencing library and use thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110669824A (en) | Kit and method for methylation library construction of low-initial-amount plasma free DNA | |
| JP6968894B2 (en) | Multiple detection method for methylated DNA | |
| EP2195449B1 (en) | Method for selectively amplifying, detecting or quantifying hypomethylated target dna | |
| JP7535611B2 (en) | Library preparation methods and compositions and uses therefor | |
| CN113106145B (en) | Compositions and methods for preparing nucleic acid libraries | |
| JP2008515447A (en) | Method for carryover protection in DNA amplification systems targeting methylation analysis achieved by modified pretreatment of nucleic acids | |
| CN114438184B (en) | Free DNA methylation sequencing library construction method and application | |
| CN110607353B (en) | Method and kit for rapidly preparing DNA sequencing library by utilizing efficient ligation technology | |
| JP2017525369A (en) | Preparation of adapter-connected amplicon | |
| US20200277654A1 (en) | Method for Detecting multiple DNA Mutations and Copy Number Variations | |
| WO2021253372A1 (en) | High-compatibility pcr-free library building and sequencing method | |
| CN113593636A (en) | Sequencing result analysis method and system, computer readable storage medium and electronic equipment | |
| CN110669823A (en) | ctDNA library construction and sequencing data analysis method for simultaneously detecting multiple liver cancer common mutations | |
| WO2019085546A1 (en) | Method for constructing microbial 16s rdna single-molecule level sequencing library | |
| CN110699425B (en) | Enrichment method and system of gene target region | |
| CN113943763B (en) | Method for reducing nucleic acid and detection method thereof | |
| CN113322523B (en) | RNA rapid library construction method and application thereof | |
| WO2018121634A1 (en) | Method and kit for nonspecific replication of dna fragment | |
| CN117844884A (en) | Method for repairing cfDNA tail end and establishing library | |
| WO2020073748A1 (en) | Method for constructing sequencing library | |
| US20180100180A1 (en) | Methods of single dna/rna molecule counting | |
| CN106701949B (en) | A kind of detection method of gene mutation and reagent reducing amplification bias | |
| WO2020118046A1 (en) | Quantifying foreign dna in low-volume blood samples using snp profiling | |
| CN113593637B (en) | Sequencing method, analysis method and analysis system thereof, computer-readable storage medium and electronic device | |
| CN115354067A (en) | Method for establishing free DNA methylation library |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200110 |