CN110331446A - DNA methylation marker kit for screening and method - Google Patents
DNA methylation marker kit for screening and method Download PDFInfo
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Abstract
The present invention relates to DNA methylation marker kit for screening and methods.The method of the present invention includes digestion, the connection of methylation connector, methylation processing and index primer amplification, wherein, the digestion is carried out to DNA sequence dna interested using to insensitive restriction enzyme is methylated, obtain digestion products, wherein, the digestion products are directly connect with the connector with methylation modification without end reparation and polishing, and wherein, the digestion products include digestion products and the optional digestion products with flat end with cohesive end.The present invention also provides corresponding joint sequence, index primer sequence, by preferred reagent, kit and relevant application.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to DNA methylation marker kit for screening and method.
Background technique
DNA methylation occurs mainly in the island CpG, is important epigenetic modification.Known dna methylation participates in gene and turns
Record regulation process, and, formation including cancer related to numerous biological processes.Screen DNA first relevant to biological process
The method of base marker includes full-length genome weight bisulfite sequencing (WGBS) and simplified methylation sequencing (RRBS) etc..
Although WGBS detection is comprehensively, higher cost, a large amount of non-DNA methylation modified regions are sequenced.It is compared to WGBS, RRBS energy
It is enough enriched with the site CpG, greatly reduces sequencing cost.Traditional RRBS is enriched with the site CpG through digestion, carries out blunt end, passes through
TA connection type adds connector, then carries out amplification reaction.It is larger that the shortcomings that this method, is that DNA builds library original amounts, to starting
Sub-district and the island the CpG region site CpG enrichment degree are low (especially for the second-rate FFPE sample of DNA), different samples it
Between site reproducibility it is not high.It would therefore be desirable to have the site CpG enrichment degree is higher, reproducibility preferably methylates marker between sample
Screening method.
Summary of the invention
The present invention provides a kind of methylation marker screening method of optimization, experiment stream of this method to traditional detection method
Journey is optimized, and builds library reagent and Library development flow using preferred.
The present invention provides a kind of method for constructing DNA methylation library, and the method includes digestion, methylation connectors to connect
It connects, methylate processing and index primer amplification, wherein use to the insensitive restriction enzyme that methylates to DNA interested
Sequence carries out the digestion, obtains digestion products, wherein the digestion products repaired without end and polishing and directly with have
The connector connection of methylation modification, and wherein, the digestion products include the digestion products with cohesive end.
In one or more embodiments, the digestion products further include the digestion products with flat end.
In one or more embodiments, the methylation connector includes having to match with digestion products cohesive end
Cohesive end methylation connector.
In one or more embodiments, the methylation connector further includes and the digestion without digestion and polishing processing
The methylation connector for the flat end that the flat end of product matches.
In one or more embodiments, the described pair of insensitive restriction enzyme of methylation be selected from MSP I,
One of HaeIII, BanII, HpyCH4V, AluI, SphI and BssSI or a variety of.
In one or more embodiments, methylation connector connection include using 5 ' App DNA/RNA ligases,
T4 RNA ligase, T4 DNA ligase, Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, CircLigase
Ligase, electricity turn one of ligase, flat end/TA ligase, instantaneous stickiness ligase, HiFi Taq DNA ligase or
It is a variety of that methylation connector is connected to digestion products.
In one or more embodiments, the methylation processing is carried out using bisulfite conversion kit.
In one or more embodiments, using contain respectively and the methylation connector in not complementary partial complementarity
The index primer of sequence be indexed primer amplification.
In one or more embodiments, using selected from Phusion U Hot Start archaeal dna polymerase, PfuTurbo
One of Cx Hotstart archaeal dna polymerase, Taq archaeal dna polymerase and LafU archaeal dna polymerase or a variety of archaeal dna polymerases into
The row index primer amplification.
In one or more embodiments, before digestion, magnetic bead or Column methods purified genomic dna are used.
In one or more embodiments, after methylation connector connection, the carrier DNA for interrupting or not interrupting is added.
In one or more embodiments, using Ago-Gel be tapped and recovered method, configuration glue be tapped and recovered method,
Magnetic beads for purifying segment screening technique or Blue Pippin method recycle connection product.
In one or more embodiments, the method successively includes:
Using magnetic bead or Column methods purified genomic dna, the DNA of purifying is obtained；
Using selected from one of MSP I, HaeIII, BanII, HpyCH4V, AluI, SphI and BssSI or a variety of to described pure
The DNA of change carries out digestion, obtains digestion products；
Methylation connector connection is carried out to digestion products obtained, wherein connect using selected from T4 DNA ligase, Taq DNA
It meets enzyme, T7 DNA ligase and the one or more of T3 DNA ligase to be attached, obtains connection product；
Method is tapped and recovered using configuration glue and purifies the connection product；
Methylation processing is carried out using connection product of the bisulfite conversion method to purifying；With
Using selected from Phusion U Hot Start archaeal dna polymerase, PfuTurbo Cx Hotstart archaeal dna polymerase and Taq
One of archaeal dna polymerase or a variety of archaeal dna polymerases are indexed primer amplification to the product through methylation processing.
In one or more embodiments, the methylation connector be selected from the group methylation one of joint sequence or
It is a variety of:
The methylation joint sequence that SEQ ID NO:1 and SEQ ID NO:2 is formed；
The methylation joint sequence that SEQ ID NO:3 and SEQ ID NO:4 is formed；
The methylation joint sequence that SEQ ID NO:5 and SEQ ID NO:6 is formed；
The methylation joint sequence that SEQ ID NO:7 and SEQ ID NO:8 is formed；
The methylation joint sequence that SEQ ID NO:9 and SEQ ID NO:10 is formed；
The methylation joint sequence that SEQ ID NO:11 and SEQ ID NO:12 is formed；
The methylation joint sequence that SEQ ID NO:13 and SEQ ID NO:14 is formed；
The methylation joint sequence that SEQ ID NO:15 and SEQ ID NO:16 is formed；
The methylation joint sequence that SEQ ID NO:17 and SEQ ID NO:18 is formed；
The methylation joint sequence that SEQ ID NO:19 and SEQ ID NO:20 is formed.
In one or more embodiments, the index primer is selected from: SEQ ID NO:21 and SEQ ID NO:22, and
SEQ ID NO:23 and SEQ ID NO:24.
The present invention also provides a kind of DNA methylation libraries to build library kit, and the kit includes insensitive to methylating
Restriction enzyme and joint sequence product, the joint sequence product include have with without end repair and polishing processing
Digestion products the joint sequence of cohesive end that matches of cohesive end.
In one or more embodiments, the DNA methylation library builds library kit and further includes and have without end
The joint sequence for the flat end that end is repaired and the flat end of the digestion products of polishing processing matches.
It is described to have and repair and mend without end in the joint sequence product in one or more embodiments
The concentration of the joint sequence for the cohesive end that the cohesive end of the digestion products handled together matches is 0.1-1.5 μM.
In one or more embodiments, it further includes in following reagent that library kit is built in the DNA methylation library
It is one or more:
(1) primer containing special index sequence；Wherein, the primer includes forward and reverse primer, forward and reverse primer point
Not Bao Kuo respectively with the sequence of not complementary partial complementarity in the methylation connector；
(2) DNA ligase；With
(3) archaeal dna polymerase.
In one or more embodiments, the described pair of insensitive restriction enzyme of methylation be selected from MSP I,
One of HaeIII, BanII, HpyCH4V, AluI, SphI and BssSI or a variety of.
In one or more embodiments, DNA ligase be selected from 5 ' App DNA/RNA ligases, T4 RNA ligase,
T4 DNA ligase, Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, CircLigase ligase, electricity turn company
Connect one of enzyme, flat end/TA ligase, instantaneous stickiness ligase, HiFi Taq DNA ligase or a variety of.
In one or more embodiments, the archaeal dna polymerase polymerize selected from Phusion U Hot Start DNA
One of enzyme, PfuTurbo Cx Hotstart archaeal dna polymerase, Taq archaeal dna polymerase and LafU archaeal dna polymerase are more
Kind.
In one or more embodiments, the kit further includes one of following reagent or a variety of:
(a) reagent needed for carrying out digestion；
(b) reagent needed for carrying out the connection of methylation connector；
(c) reagent needed for carrying out methylation conversion；
(d) reagent needed for carrying out DNA cloning；
(e) reagent needed for carrying out DNA purifying；With
(f) fragmentation used or the carrier DNA of un-segmentedization required for product purification or segment sorting are attached.
In one or more embodiments, reagent needed for the progress digestion includes buffer and non-methylation λ
DNA。
In one or more embodiments, it is described carry out methylation connector connection needed for reagent include buffer and
ATP。
In one or more embodiments, the required reagent of methylation conversion that carries out is bisulfite conversion examination
Agent.
In one or more embodiments, reagent needed for the progress DNA cloning includes amplification buffer and 4 kinds
dNTP。
In one or more embodiments, the required reagent of DNA purifying that carries out includes magnetic bead and/or purification column.
In one or more embodiments, the methylation connector be selected from the group methylation one of joint sequence or
It is a variety of:
The methylation joint sequence that SEQ ID NO:1 and SEQ ID NO:2 is formed；
The methylation joint sequence that SEQ ID NO:3 and SEQ ID NO:4 is formed；
The methylation joint sequence that SEQ ID NO:5 and SEQ ID NO:6 is formed；
The methylation joint sequence that SEQ ID NO:7 and SEQ ID NO:8 is formed；
The methylation joint sequence that SEQ ID NO:9 and SEQ ID NO:10 is formed；
The methylation joint sequence that SEQ ID NO:11 and SEQ ID NO:12 is formed；
The methylation joint sequence that SEQ ID NO:13 and SEQ ID NO:14 is formed；
The methylation joint sequence that SEQ ID NO:15 and SEQ ID NO:16 is formed；
The methylation joint sequence that SEQ ID NO:17 and SEQ ID NO:18 is formed；With
The methylation joint sequence that SEQ ID NO:19 and SEQ ID NO:20 is formed.
In one or more embodiments, the index primer is selected from: SEQ ID NO:21 and SEQ ID NO:22, and
SEQ ID NO:23 and SEQ ID NO:24.
The present invention also provides a kind of DNA sequencing methods, which is characterized in that the method includes using any implementation of the present invention
The step of method described in scheme constructs DNA methylation library and the DNA sequence dna in library is sequenced.
The present invention also provides a kind of joint sequence product, the joint sequence product include have with without end repair and
The connector for the cohesive end that the cohesive end of the digestion products of polishing processing matches.
In one or more embodiments, in the joint sequence product, the concentration of connector is 0.1-1.5 μM.
In one or more embodiments, the cohesive end repaired without end with the digestion products of polishing processing
It is viscous caused by one of MSP I, BanII, SphI and BssSI restriction enzyme or a variety of progress digestion using being selected from
Property end.
In one or more embodiments, the joint sequence is selected from the group one of joint sequence or a variety of:
The joint sequence that SEQ ID NO:1 and SEQ ID NO:2 is formed；
The joint sequence that SEQ ID NO:3 and SEQ ID NO:4 is formed；
The joint sequence that SEQ ID NO:7 and SEQ ID NO:8 is formed；
The joint sequence that SEQ ID NO:9 and SEQ ID NO:10 is formed；
The joint sequence that SEQ ID NO:11 and SEQ ID NO:12 is formed；
The joint sequence that SEQ ID NO:13 and SEQ ID NO:14 is formed；
The joint sequence that SEQ ID NO:17 and SEQ ID NO:18 is formed；With
The joint sequence that SEQ ID NO:19 and SEQ ID NO:20 is formed.
In one or more embodiments, the joint sequence is methylation joint sequence.
The present invention also provides a kind of joint sequence product, containing joint sequence described in any embodiment of the present invention and
The optional joint sequence with the flat end to match with the flat end of the digestion products without end reparation and polishing processing.
It is described with flat with the digestion products without end reparation and polishing processing in one or more embodiments
The joint sequence for the flat end that end matches is selected from: the joint sequence and SEQ ID NO:15 that SEQ ID NO:5 and 6 is formed
The joint sequence formed with 16.
The present invention also provides described in the restriction enzyme and/or any embodiment of the present invention insensitive methylation
The application of joint sequence or joint sequence product in building DNA methylation library, or in preparation building DNA methylation library
Application in kit.
In one or more embodiments, the described pair of insensitive restriction enzyme of methylation be selected from MSP I,
One of HaeIII, BanII, HpyCH4V, AluI, SphI and BssSI or a variety of.
Detailed description of the invention
Fig. 1: the basic schematic diagram of banking process of the present invention.
Fig. 2: the DNA methylation library fragments of one of sample analyze result in embodiment 1.
Specific embodiment
It should be understood that in the scope of the invention, above-mentioned each technical characteristic of the invention and in below (eg embodiment) specifically
It can be combined with each other between each technical characteristic of description, to constitute preferred technical solution.
The method that the present invention methylates to traditional detection is optimized, provide a kind of site CpG enrichment degree it is higher,
Reproducibility preferably methylates marker screening method between sample.
Fig. 1 gives the schematic diagram of the method for the present invention.Enzyme is carried out to interested DNA sequence dna using restriction enzyme
It cuts, so that the digestion products and the optional digestion products with flat end with cohesive end are obtained, later using methylation
Connector is attached, then carries out methylation conversion, indexes primer amplification, so that building obtains DNA methylation library, and can be to this
Library is sequenced.In method of the invention, after obtaining digestion products, does not need to carry out digestion products end reparation, mend
It is neat and generate cohesive end (as plus A), and directly carry out methylation connector using the digestion products and connect, or by the digestion products
Methylation connector connection is carried out after purification.
In the present invention, restriction enzyme be can be to the insensitive restriction enzyme that methylates, including but not limited to
MSP I, HaeIII, BanII, HpyCH4V, AluI, SphI and BssSI.Preferably, this kind of restriction enzyme specific recognition
Sequence have higher distribution in the high GC sequence area of genome.Thus, it can be real using restriction enzyme of the invention
Existing promoter region and the higher bioaccumulation efficiency in the island the CpG region site CpG.
In the present invention, it a kind of restriction enzyme insensitive to methylation can be used to carry out digestion, also can be used two
Kind is two or more to the restriction enzyme for methylating insensitive progress digestion.In some embodiments, being used only can produce
One of restriction enzyme of raw cohesive end or a variety of carry out digestions；In other embodiments, viscous using that can generate
Property end one of restriction enzyme or a variety of and one of restriction enzyme of flat end or a variety of can be generated
Carry out digestion.It should be understood that it is insensitive to methylating in view of used restriction enzyme, and the sequence of its specific recognition
The high GC sequence area for being listed in genome has higher distribution, therefore, even if a kind of restriction enzyme as described herein is used only
Enzyme carries out digestion, can also generate similar even preferably to the mixed enzyme progress digestion using two or more restriction enzymes
Effect, such as similar or higher bioaccumulation efficiency.
Herein, can generate cohesive end restriction enzyme include but is not limited to MSP I, BanII, SphI and
BssSI etc.；It includes but is not limited to HaeIII, HpyCH4V and AluI that the restriction enzyme of flat end, which can be generated,.Ability can be used
The restriction enzyme of domain common vendor carries out specific digestion to DNA interested, such as genomic DNA.It is suitable restricted interior
Enzyme cutting manufacturer includes but is not limited to Thermo, NEB and Takara etc..
Specific limitation is had no to digestion condition, is carried out usually under the optimum reaction conditions of used restriction endonuclease.
For example, the final concentration of restriction enzyme can be in the range of 0.1-1U/ microlitres in reaction system.The endonuclease reaction time can be with
It is 1-14 hours.Endonuclease reaction temperature can be the optimal reaction temperature of selected enzyme.For example, illustrative endonuclease reaction condition packet
About 37 DEG C are included, reaction time 1-5 hour after reaction, in about 65-80 DEG C progress enzyme-deactivating, finally keeps the temperature at about 4 DEG C.Thus
Obtain digestion products and the optional digestion products with flat end with cohesive end.
Subsequent connection reaction can be directly carried out after endonuclease reaction, can also first be purified, then be carried out again subsequent
Connection reaction does not need the conventional end reparation of progress, polishing and generates cohesive end (as added A).This field week can be used
The technology known is purified, such as magnetic bead well known in the art can be used to be purified.
Before endonuclease reaction, the method purified genomic dna of magnetic bead or column purification can be used.Preferred magnetic bead can be
Selected from Backman Ampure Xp beads and Vazyme VAHTS DNA Clean Beads.In general, purifying magnetic bead and sample
Volume ratio can be in the range of 0.8 ~ 2 times.When using column purification, the common DNA product purification kit of Tiangeng can be used
(DP204) column purification was carried out.
Reagent commonly used in the art can be used, joint sequence is connected to digestion products both ends.Suitable ligase brand packet
Include 5 ' App DNA/RNA ligases, T4 RNA ligase, T4 DNA ligase, Taq DNA ligase, T7 DNA ligase, T3
DNA ligase, CircLigase ligase, electricity turn ligase, flat end/TA ligase, instantaneous stickiness ligase, HiFi Taq
DNA ligase is any one or more of.Commercially available ligase can be used to be attached, Takara(2011A such as can be used),
ThermoFisher(EL0013), NEB(M0202T) and KAPA(KK6010, KK6110) in any one carry out connector company
It connects.
According to the enzyme connected, connect reaction condition can appropriate adjustment, to be carried out under its optimum reaction conditions.Example
Property reaction temperature can in the range of 15-20 DEG C, connect the reaction time can be in the range of 15-30 hours.Connection reaction knot
Shu Hou, can for example, about 65 DEG C at a temperature of carry out enzyme-deactivating.It can finally be kept the temperature at about 4 DEG C.
Herein, term " methylation connector " refers to the joint sequence that its all C is methylated.Therefore, of the invention
In some embodiments, the method for the present invention also includes the method for providing butt joint and implementing methylation modification.Use methylation connector
It can effectively avoid sequence measuring joints to the operation bring interference such as subsequent bisulf iotate-treated, such as at bisulf iotate-treated
Joint sequence may be changed during reason.It will be understood to those skilled in the art that butt joint carries out methylation modification
Method is not particularly limited, and be can use any method butt joint known in the art and is carried out methylation modification.
In some embodiments, it can also include label in methylation connector, thus be convenient to construct simultaneously a variety of
The high-throughput sequencing library of the specific genome area of sample, and high-flux sequence platform can be effectively applied to, thus
After carrying out data analysis to sequencing result, the sequence information based on label, it will be able to accurately distinguish the genome of a variety of samples
The methylation information of the specific genome area of the sequence information and sample of the high-throughput sequencing library of specific region.Label
Length is usually in 5-10bp or so, such as 6bp or 8bp.
In the present invention, the final concentration of the connector in reaction system with cohesive end is preferably 0.01-0.10 μM, such as
0.01-0.06 μM or 0.02-0.05 μM.Preferably, when using the connector with flat end, end in the reaction system is dense
Degree is also within above range.Above-mentioned concentration range taken into account target digestion products it is abundant connection and as far as possible reduction connector it
Between form dimer.When using two or more joint sequences, the sum of final concentration in the reaction system is also in above range
It is interior, the ratio of two or more connector is had no specifically limited, such as can be equal proportion.
Exemplary adapter sequence of the invention can be selected from: the methylation connector that SEQ ID NO:1 and SEQ ID NO:2 is formed
Sequence；The methylation joint sequence that SEQ ID NO:3 and SEQ ID NO:4 is formed；SEQ ID NO:5 and SEQ ID NO:6 shape
At methylation joint sequence；The methylation joint sequence that SEQ ID NO:7 and SEQ ID NO:8 is formed；SEQ ID NO:9 with
The methylation joint sequence that SEQ ID NO:10 is formed；The methylation connector sequence that SEQ ID NO:11 and SEQ ID NO:12 is formed
Column；The methylation joint sequence that SEQ ID NO:13 and SEQ ID NO:14 is formed；SEQ ID NO:15 and SEQ ID NO:16
The methylation joint sequence of formation；The methylation joint sequence that SEQ ID NO:17 and SEQ ID NO:18 is formed；SEQ ID
The methylation joint sequence that NO:19 and SEQ ID NO:20 is formed.It should be understood that based on produced by used restriction enzyme
Digestion products, may be selected suitable joint sequence carry out methylation connector connection.For example, when digestion products have cohesive end
When, the joint sequence with the cohesive end complementary with digestion products cohesive end may be selected；When digestion products both include having
The digestion products of cohesive end also include when having the digestion products of flat end, then using with mutual with digestion products cohesive end
The mixture of the joint sequence of the cohesive end of benefit and the joint sequence with flat end.The cohesive end of inventive joint sequence
Match with the cohesive end of digestion products, it is intended that meet base pair complementarity principle.
In some case study on implementation of the invention, the carrier of appropriate fragmentation or non-fragmentation can be added after connection reaction
DNA, it is possible thereby to improve the recovery efficiency during subsequent connection product purifying or weight bisulfite conversion.
After connection, can be used Ago-Gel be tapped and recovered method, configuration glue be tapped and recovered method, magnetic beads for purifying piece
Section screening technique or Blue Pippin method recycle connection product, to remove the dimer being likely to form between connector and connector.It
After can use nuclease-free water eluted dna.Thus obtained DNA molecular can be used for weight bisulfite conversion.In some embodiment party
In case, method recycling connection product is tapped and recovered using configuration glue.
In the present invention, heavy bisulfite conversion method well known in the art can be used, DNA interested is handled, obtain
The DNA molecular of bisulfite conversion must be weighed.For example, the weight interested DNA sequence dna of bisulf iotate-treated can be used, make to own
The cytimidine (C) not methylated is converted into uracil (U), and the cytimidine to methylate is constant.This field can be used
The reagent known is converted.For example, in certain embodiments, using the MethylCodeTM of ThermoFisher company
Bisulfite Conversion Kit(MECOV50) or ZYMO research company EZ DNA methylation-
Lighting Kit (D5030) or EZ DNA Methylation-Gold Kit(D5006) it is converted.Alternatively, can also
Voluntarily prepare bisulfite conversion reagent.The condition of conversion is known in the art, for example, the sample that will joined conversion reagent
Be placed in PCR instrument, run following procedure: 98 DEG C 10 minutes, 64 DEG C 30 minutes 2 hours, finally in 4 DEG C keep the temperature.For different
Conversion reagent, Transformation Program can be different.This can select by those skilled in the art according to practical conversion situation and true
It is fixed.After conversion, the DNA of inverted processing can be recycled, and elution DNA can be used.
After conversion, can be used containing special index sequence primer (herein also referred to as index primer, including it is positive and
Reverse primer) amplification converted product.When multiple samples are sequenced simultaneously, need to add the index of particular sequence to each sample.
Meanwhile to make DNA that can grow up to the cluster that can be sequenced on sequence testing chip, it is necessary to while adding at sample DNA both ends can be with chip
The sequence that upper oligomerization sequence (oligo) combines.Therefore, in addition to containing special index sequence, the primer further includes can be with chip
The sequence that upper contained oligomerization sequence-specific combines.Forward and reverse primer respectively further comprises the connector with amplified production respectively
The sequence of not complementary partial complementarity in sequence.Special index sequence commonly used in the art can be used.Illustrative index primer
To as shown in this paper SEQ ID NO:21 and 22 and SEQ ID NO:23 and 24.
In order to be suitable for different microarray datasets, joint sequence, the specification table B other than specification Table A also can be used
Index primer sequence in addition, the change of these connectors and index primer is so that finally building library can be adapted for Thermo
The sequencing of the platform of Fisher Ion series microarray dataset or other reading DNA sequence dnas.
Amplification PCR reagent used includes reagent, including but not limited to amplification buffer, 4 needed for conventional implementation PCR
Kind dNTP and archaeal dna polymerase etc., wherein usually contain Mg2+ ion in amplification buffer.Using poly- to dUTP insensitive DNA
Synthase is expanded.The polymerase that can be used includes Phusion U Hot Start archaeal dna polymerase (Thermo
Scientific, F555L), PfuTurbo Cx Hotstart archaeal dna polymerase (Agilent Technologies,
600410), Taq archaeal dna polymerase (Takara, R007Q), LafU archaeal dna polymerase (Cvent, LafU DNA
Polymerase), Taq archaeal dna polymerase (NEB, E5000S) etc..After reaction, amplified production is purified, so that building obtains
Library of the present invention.Purifying can be the purification process of this field routine, for example, magnetic beads for purifying or column purification side can be used
Method is purified.
PerkinElmer company labChip GX Touch fragment analyser can be used, be also possible to Agilent 2100
Bioanalyzer, agarose gel electrophoresis or other DNA fragmentation analysis methods carry out library analysis.When sequencing, it can be used
The platform of Illumina microarray dataset, Thermo Fisher Ion series microarray dataset or other reading DNA sequence dnas.Due to
When methylation processing, the cytimidine (C) not methylated in DNA sequence dna interested is converted into uracil (U), and methylates
Cytimidine it is constant, therefore, can determine the methylation in the site CpG in DNA molecular interested according to sequencing result.
Therefore, the feature of building DNA methylation library provided by the present application or DNA sequencing method is included at least using process
Restriction enzyme after it is preferred that carries out digestion processing to genomic DNA；It is repaired not carrying out conventional end to digestion products
Multiple, polishing and and in the case where generating cohesive end, directly make the digestion products and the preferred connector of process matched with its into
Row connection；The methylation conversion and index primer amplification of this field routine are carried out again, thus building obtains DNA methylation library,
And it can get sequencing result.Preferably, model of the final concentration of the connector in reaction system with cohesive end at 0.01-0.10 μM
In enclosing.
The present invention includes joint sequence as described herein, which matches with cohesive end caused by digestion.
In some embodiments, which can be methylation joint sequence.Joint sequence of the invention can be in routine
Increase base on the basis of joint sequence on a chain at its 5 ' end or 3 ' ends to form cohesive end and obtain, viscosity end
The viscosity at end and the original generation of the digestion with restriction enzyme product without end reparation, polishing and generation cohesive end processing
Termini-complementary.Therefore, unless otherwise stated, joint sequence as described herein is in addition to cohesive end, rest part is usually equal
For the sequence of conventional use of connector in DNA library and sequencing.Illustrative joint sequence is shown in sequence shown in specification Table A.This
Invention also provides a kind of joint sequence product, contains the joint sequence as described herein with cohesive end and optional tool
There is the joint sequence of flat end.
The invention also includes a kind of kits, contain joint sequence as described herein or joint sequence product.The reagent
Box can be DNA methylation and build library kit.Therefore, in kit can also containing building DNA methylation library needed for its
Its reagent, one of including but not limited to following reagents or a variety of: reagent needed for carrying out digestion carries out methylation connector
Reagent needed for connection carries out reagent needed for methylation converts required reagent, carries out DNA cloning and carries out DNA purifying
Required reagent.Preferably, in the joint sequence product in kit, the concentration of the connector with cohesive end is 0.1-1.5 μ
M, preferably 0.3-1.0 μM.When in the product containing the connector with flat end, concentration can also be in above-mentioned concentration range.
Reagent needed for carrying out digestion includes enzyme needed for buffer, digestion and non-methylation λ DNA.Illustrative buffering
Liquid can be Tango buffer or Cutsmart buffer or the buffer of other autogamys.Illustrative enzyme includes MSP
I, HaeIII, BanII, HpyCH4V, AluI, SphI, BssSI etc. mix restriction enzyme.Preferred restriction enzyme factory
Quotient is Thermo, NEB and Takara.It is non-used in library that non-methylation λ DNA can be such that this field builds conventionally used for DNA methylation
Methylate λ DNA.
Reagent needed for carrying out the connection of methylation connector includes buffer, DNA ligase and ATP.Suitable ligase
Including 5 ' App DNA/RNA ligases, T4 RNA ligase, T4 DNA ligase, Taq DNA ligase, T7 DNA ligase,
T3 DNA ligase, CircLigase ligase, electricity turn ligase, flat end/TA ligase, instantaneous stickiness ligase, HiFi
Taq DNA ligase is any one or more of.Commercially available ligase can be used to be attached, Takara(2011A such as can be used),
ThermoFisher(EL0013), NEB(M0202T) and KAPA(KK6010, KK6110) in any one carry out connector company
It connects.
Reagent needed for carrying out methylation conversion is bisulfite conversion reagent.Illustratively conversion reagent includes
The MethylCodeTM Bisulfite Conversion Kit(MECOV50 of ThermoFisher company) or ZYMO
The EZ DNA methylation-Lighting Kit (D5030) or EZ DNA Methylation- of research company
Gold Kit(D5006).
Reagent needed for DNA cloning includes amplification buffer, 4 kinds of dNTP, index primer and archaeal dna polymerase.Illustrative DNA
Polymerase includes Phusion U Hot Start DNA Polymerase(Thermo Scientific, F555L),
PfuTurbo Cx Hotstart DNA Polymerase(Agilent Technologies, 600410), Taq DNA
Polymerase(Takara, R007Q), LafU DNA Polymerase(Cvent, LafU DNA Polymerase), Taq
DNA Polymerase(NEB, E5000S).Illustrative index primer includes primer sequence shown in specification table B.
The purified reagent for including in kit can the reagent of purified genomic dna and/or the reagent of purifying digestion products.It can
Using magnetic bead or the method purified genomic dna of column purification.Preferred magnetic bead can be selected from Backman Ampure Xp
Beads and Vazyme VAHTS DNA Clean Beads.In general, purifying magnetic bead and sample volume ratio can be in 0.8 ~ 2 times of models
In enclosing.When using column purification, the common DNA product purification kit (DP204) of Tiangeng can be used to carry out column purification.For enzyme
Product is cut, magnetic bead can be used and purified.It therefore, may include the magnetic bead and/or column of purifying in kit of the invention.
The kit can also be DNA sequencing kit.Therefore, reagent needed for may also include DNA tests in kit.
In some embodiments, kit of the invention contains the restriction enzyme and matched connector
Sequence.Preferably, this kind of kit can also contain T4 DNA ligase and index primer.
The invention also includes restriction enzymes, such as MSP I, HaeIII, BanII, HpyCH4V, AluI, SphI, BssSI
One of restriction enzyme or a variety of and joint sequences with the end that there is the end of its digestion products to match are in structure
Build the application in DNA methylation library, or the application in the kit in preparation building DNA methylation library.
Advantage of the present invention includes but is not limited to: being purified before endonuclease reaction, guarantees abundant digestion, it is inclined to avoid the occurrence of digestion
Tropism, digestion are insufficient；Digestion is carried out using preferred restriction enzyme, uses the matched methyl after preferably
Change joint sequence to be attached, improves the bioaccumulation efficiency in the area the promoter region JiCpGDao site CpG in genome；After connection reaction
Carrier DNA is added and improves connection product purifying and weight bisulfite conversion recovery efficiency；Preferred segment method for separating guarantees
Experimental implementation stability and sample reproducibility.It is then preferred that reagent and experimental arrangement or method choice guarantee that experimental implementation is steady
It is qualitative, sample reproducibility.In addition, conventional method needs to carry out digestion products end reparation, polishing and adds A, in this way, all
Digestion products all have cohesive end A and without specificity, thus for second-rate DNA sample, such as FFPE DNA and
CfDNA, subsequent processing, which can amplify together impurity DNA sequence dna, to be come, to significantly reduce bioaccumulation efficiency；With existing method
Unlike, the present invention repairs without end, polishing and generates cohesive end, and therefore, the present invention can recycle only that both ends are all
The digestion products being digested.Therefore, even for second-rate DNA sample, such as FFPE DNA and cfDNA, the present invention can also
Obtain very high stable experiment and the better island CpG or promoter region bioaccumulation efficiency.
The present invention will be hereafter illustrated in a manner of specific embodiment.It should be understood that these embodiments are only illustrative, and
Not intended to limit protection scope of the present invention.Used method and reagent in embodiment, unless otherwise stated, being ability
The method and reagent of domain routine, for example, these reagents can be commercially available from commercially available approach.
Embodiment 1
One, carries out magnetic beads for purifying to the DNA of extraction
1. half an hour takes out AMPure magnetic bead in advance, it is placed at room temperature for.It is vortexed after mixing magnetic bead, 50 microlitres of magnetic beads is added to 50 microlitres
In DNA solution, is blown and beaten and mixed with liquid-transfering gun.It is placed at room temperature for 5 minutes.
2. being transferred on magnetic frame, until liquid is clarified completely.
3. being maintained on magnetic frame, supernatant is discarded, with 80% ethanol washing magnetic bead of 150 microlitres of fresh configurations.
4. repeating step 3.
5. completely removing ethyl alcohol, uncap 5 minutes dry.
6. sample is removed from magnetic frame, add 32 microlitres of nuclease-free waters, magnetic bead is sufficiently resuspended with liquid-transfering gun, room temperature is put
It sets 5 minutes.
7. magnetic frame is transferred samples to, until clarification completely.
8. shifting 30 microlitres of eluents into new pipe.
9. it is quantitative that the DNA after pair purifying carries out Qubit.
Two, take the sample DNA of 100ng after purification.Following reaction system is configured, mixed enzyme endonuclease reaction is carried out:
Reaction temperature is as follows: 37 DEG C 3 hours, 80 DEG C 20 minutes, 4 DEG C preservation.Wherein 37 DEG C be endonuclease reaction temperature, 80 DEG C
For enzyme-deactivating temperature.
Mixed enzyme include 7 kinds of restriction enzymes: MSP I(NEB, R0106L), HaeIII(NEB, R0108L), BanII
(Thermo Scientific, ER0281), HpyCH4V(NEB, R0620L), AluI(Thermo Scientific,
ER0011), SphI(NEB, R0182L) and BssSI(Thermo Scientific, ER1841)
Three, carry out the connection of methylation connector to above-mentioned digestion products
Reaction temperature is as follows: 16 DEG C 22 hours, 65 DEG C 20 minutes, 4 DEG C preservation.Wherein 16 DEG C are to connect reaction temperature, 65 DEG C
For enzyme-deactivating temperature.
The joint sequence information that methylates uses combination 1 or 2 referring to lower Table A, this experiment.
Table A: mixed methyl joint sequence information
All C have been carried out methylation conversion processing in joint sequence.S, W, R and Y be degeneracy base, respectively indicate g or
C, a or t, g or a and t or c.
Four, carry out magnetic beads for purifying to above-mentioned connection product
1. half an hour takes out AMPure magnetic bead in advance, it is placed at room temperature for.It is vortexed after mixing magnetic bead, is added 50 microlitres and arrives connection product
In, 1 microlitre of carrier DNA is added, is blown and beaten and is mixed with liquid-transfering gun.It is placed at room temperature for 5 minutes.
2. being transferred on magnetic frame, until liquid is clarified completely.
3. being maintained on magnetic frame, supernatant is discarded, with 80% ethanol washing magnetic bead of 150 microlitres of fresh configurations.
4. repeating step 3.
5. completely removing ethyl alcohol, uncap 5 minutes dry.
6. sample is removed from magnetic frame, add 32 microlitres of nuclease-free waters, magnetic bead is sufficiently resuspended with liquid-transfering gun, room temperature is put
It sets 5 minutes.
7. magnetic frame is transferred samples to, until clarification completely.
8. shifting 30 microlitres of eluents into new pipe.
Five, carry out bisulfites using the EZ DNA Methylation-Gold Kit of ZYMO research company
Conversion processing.
1. pressing following proportional arrangement conversion reagent
2. adding the above-mentioned conversion reagent of 120 ul into step 3 product.
3. putting sample to PCR instrument, run following procedure: 98 DEG C 10 minutes, 64 DEG C 30 minutes 2 hours, 4 DEG C of holdings.
According to the DNA after the recovery processing of reagent specification, finally with 42 microlitres of elution DNA, 41.25 microlitres are shifted
Into next step reaction.
Six, index primer amplifications
Reaction temperature is as follows: 98 DEG C 30 seconds, 98 DEG C 10 seconds plus 65 DEG C 75 seconds 20 circulation, 65 degrees Celsius 5 minutes, 4 DEG C guarantor
It holds.
It indexes primer sequence information and participates in following table B, this experiment uses index primer pair 1 or 2.
Table B: index primer sequence information
Note: if joint sequence is indexed primer and used index primer pair 1 using combination 1；If joint sequence uses combination 2, rope
Primer uses index primer pair 2.N indicates a, c, t or g.
Seven, carry out magnetic beads for purifying to above-mentioned amplified production
1. half an hour takes out AMPure magnetic bead in advance, it is placed at room temperature for.It is vortexed after mixing magnetic bead, is added 50 microlitres and arrives connection product
In, it is blown and beaten and is mixed with liquid-transfering gun.It is placed at room temperature for 5 minutes.
2. being transferred on magnetic frame, until liquid is clarified completely.
3. being maintained on magnetic frame, supernatant is discarded, with 80% ethanol washing magnetic bead of 150 microlitres of fresh configurations.
4. repeating step 3.
5. completely removing ethyl alcohol, uncap 5 minutes dry.
6. sample is removed from magnetic frame, add 42 microlitres of nuclease-free waters, magnetic bead is sufficiently resuspended with liquid-transfering gun, room temperature is put
It sets 5 minutes.
7. magnetic frame is transferred samples to, until clarification completely.
8. shifting 40 microlitres of eluents into new pipe.
Eight library Quality Controls: library concentration is measured with two methods of Qubit and qPCR, and with PerkinElmer company
LabChip fragment analyser analyzes library fragments size.As a result as shown in Figure 2.
Nine, sequencing: it is sequenced with Illumina platform instrument Hiseq X Ten.
Ten, results
As a result as shown in table 1 below.Table 1 is shown using conventional method (Genome-scale DNA methylation
Mapping of clinical samples at single-nucleotide resolution, Nat Methods. 2010
February；7 (2): 133-136. doi:10.1038/nmeth.1414) and hereinbefore method obtain high-flux sequence
Read assessment result.The results show that detecting that the site CpG is more concentrated on the island CpG region or promoter after improvement
Region, realization are preferably enriched with the two regions.
Table 1: methylation libraries high throughput sequencing reading assessment result
Sequence table
<110>the remote Bioisystech Co., Ltd of Shang Hai Kun
<120>DNA methylation marker kit for screening and method
<130> 190396
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
acactctttc cctacacgac gctcttccga tc 32
<210> 2
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(2)
<223>s is g or c
<400> 2
ssgatcggaa gagcacacgt ctgaactcca gtc 33
<210> 3
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
acactctttc cctacacgac gctcttccga tc 32
<210> 4
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(1)
<223>w is a or t
<220>
<221> misc_feature
<222> (2)..(3)
<223>s is g or c
<220>
<221> misc_feature
<222> (4)..(4)
<223>w is a or t
<400> 4
wsswgatcgg aagagcacac gtctgaactc cagtc 35
<210> 5
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
acactctttc cctacacgac gctcttccga tc 32
<210> 6
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gatcggaaga gcacacgtct gaactccagt c 31
<210> 7
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (33)..(33)
<223>r is g or a
<220>
<221> misc_feature
<222> (36)..(36)
<223>y is t or c
<400> 7
acactctttc cctacacgac gctcttccga tcrgcy 36
<210> 8
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gatcggaaga gcacacgtct gaactccagt c 31
<210> 9
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (33)..(33)
<223>s is g or c
<220>
<221> misc_feature
<222> (34)..(35)
<223>w is a or t
<220>
<221> misc_feature
<222> (36)..(36)
<223>s is g or c
<400> 9
acactctttc cctacacgac gctcttccga tcswws 36
<210> 10
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gatcggaaga gcacacgtct gaactccagt c 31
<210> 11
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tcgtcggcag cgtcagatgt gtataagaga cag 33
<210> 12
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(2)
<223>s is g or c
<400> 12
ssctgtctct tatacacatc tccgagccca cgagac 36
<210> 13
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
tcgtcggcag cgtcagatgt gtataagaga cag 33
<210> 14
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(1)
<223>w is a or t
<220>
<221> misc_feature
<222> (2)..(3)
<223>s is g or c
<220>
<221> misc_feature
<222> (4)..(4)
<223>w is a or t
<400> 14
wsswctgtct cttatacaca tctccgagcc cacgagac 38
<210> 15
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
tcgtcggcag cgtcagatgt gtataagaga cag 33
<210> 16
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
ctgtctctta tacacatctc cgagcccacg agac 34
<210> 17
<211> 37
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (34)..(34)
<223>r is g or a
<220>
<221> misc_feature
<222> (37)..(37)
<223>y is t or c
<400> 17
tcgtcggcag cgtcagatgt gtataagaga cagrgcy 37
<210> 18
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
ctgtctctta tacacatctc cgagcccacg agac 34
<210> 19
<211> 37
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (34)..(34)
<223>s is g or c
<220>
<221> misc_feature
<222> (35)..(36)
<223>w is a or t
<220>
<221> misc_feature
<222> (37)..(37)
<223>s is g or c
<400> 19
tcgtcggcag cgtcagatgt gtataagaga cagswws 37
<210> 20
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ctgtctctta tacacatctc cgagcccacg agac 34
<210> 21
<211> 57
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (30)..(37)
<223>n is a, c, g or t
<400> 21
aatgatacgg cgaccaccga gatctacacn nnnnnnnaca ctctttccct acacgac 57
<210> 22
<211> 53
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (25)..(32)
<223>n is a, g, c or t
<400> 22
caagcagaag acggcatacg agatnnnnnn nngtgactgg agttcagacg tgt 53
<210> 23
<211> 51
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (30)..(37)
<223>n is a, g, c or t
<400> 23
aatgatacgg cgaccaccga gatctacacn nnnnnnntcg tcggcagcgt c 51
<210> 24
<211> 47
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (25)..(32)
<223>n is a, g, c or t
<400> 24
caagcagaag acggcatacg agatnnnnnn nngtctcgtg ggctcgg 47
Claims (24)
1. a kind of method for constructing DNA methylation library, which is characterized in that the method includes digestion, methylation connectors to connect,
Methylation processing and index primer amplification；
Wherein, the digestion is carried out to DNA sequence dna interested using to insensitive restriction enzyme is methylated, obtains digestion
Product；
Wherein, the digestion products are directly connect with the connector with methylation modification without end reparation and polishing；And
Wherein, the digestion products include the digestion products with cohesive end, the reaction system of the methylation connector connection
In include the methylation connector with the cohesive end to match with the cohesive ends of digestion products, and methyl in reaction system
Final concentration of 0.01-0.1 μM for changing connector.
2. the method as described in claim 1, which is characterized in that the digestion products further include that there is the digestion of flat end to produce
Object further includes with the flat end to match with the flat ends of the digestion products in the reaction system of the methylation connector connection
Methylation connector.
3. the method as described in claim 1, which is characterized in that
The insensitive restriction enzyme of described pair of methylation is selected from MSP I, HaeIII, BanII, HpyCH4V, AluI, SphI
With one of BssSI or a variety of；
Methylation connector connection include using 5 ' App DNA/RNA ligases, T4 RNA ligase, T4 DNA ligase,
Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, CircLigase ligase, electricity turn ligase, flat end/TA
One of ligase, instantaneous stickiness ligase, HiFi Taq DNA ligase or a variety of connectors that will methylate are connected to digestion
Product；
The methylation processing is carried out using bisulfite conversion kit；
Drawn using containing to be indexed with the index primer of the sequence of partial complementarity not complementary in the methylation connector respectively
Object amplification.
4. the method as described in claim 1, which is characterized in that using selected from Phusion U Hot Start archaeal dna polymerase,
One of PfuTurbo Cx Hotstart archaeal dna polymerase, Taq archaeal dna polymerase and LafU archaeal dna polymerase or a variety of DNA
Polymerase carries out the index primer amplification.
5. the method as described in claim 1, which is characterized in that the method also includes using magnetic bead or column purification before digestion
Method purified genomic dna；And/or after methylation connector connection, the carrier DNA for interrupting or not interrupting is added.
6. the method as described in claim 1, which is characterized in that after methylation connector connection, tapped rubber using Ago-Gel
Recovery method, configuration glue are tapped and recovered method, magnetic beads for purifying segment screening technique or Blue Pippin method recycling connection product.
7. the method as described in claim 1, which is characterized in that the method successively includes:
Using magnetic bead or Column methods purified genomic dna, the DNA of purifying is obtained；
Using selected from one of MSP I, HaeIII, BanII, HpyCH4V, AluI, SphI and BssSI or a variety of to described pure
The DNA of change carries out digestion, obtains digestion products；
Methylation connector connection is carried out to digestion products obtained, wherein connect using selected from T4 DNA ligase, Taq DNA
It meets enzyme, T7 DNA ligase and the one or more of T3 DNA ligase to be attached, obtains connection product；
Method is tapped and recovered using configuration glue and purifies the connection product；
Methylation processing is carried out using connection product of the bisulfite conversion method to purifying；With
Using selected from Phusion U Hot Start archaeal dna polymerase, PfuTurbo Cx Hotstart archaeal dna polymerase and Taq
One of archaeal dna polymerase or a variety of archaeal dna polymerases are indexed primer amplification to the product through methylation processing.
8. such as method of any of claims 1-7, which is characterized in that
The methylation connector is selected from the group one of methylation joint sequence or a variety of:
The methylation joint sequence that SEQ ID NO:1 and SEQ ID NO:2 is formed；
The methylation joint sequence that SEQ ID NO:3 and SEQ ID NO:4 is formed；
The methylation joint sequence that SEQ ID NO:5 and SEQ ID NO:6 is formed；
The methylation joint sequence that SEQ ID NO:7 and SEQ ID NO:8 is formed；
The methylation joint sequence that SEQ ID NO:9 and SEQ ID NO:10 is formed；
The methylation joint sequence that SEQ ID NO:11 and SEQ ID NO:12 is formed；
The methylation joint sequence that SEQ ID NO:13 and SEQ ID NO:14 is formed；
The methylation joint sequence that SEQ ID NO:15 and SEQ ID NO:16 is formed；
The methylation joint sequence that SEQ ID NO:17 and SEQ ID NO:18 is formed；
The methylation joint sequence that SEQ ID NO:19 and SEQ ID NO:20 is formed；
The index primer is selected from: SEQ ID NO:21 and SEQ ID NO:22 and SEQ ID NO:23 and SEQ ID NO:24.
9. library kit is built in a kind of DNA methylation library, which is characterized in that the kit includes:
To the insensitive restriction enzyme that methylates；
Joint sequence product, the joint sequence product contain concentration be 0.1-1.5 μM have with without end repair and polishing
The connector for the cohesive end that the cohesive end of the digestion products of processing matches；With
The joint sequence of the optional flat end to match with the flat end of the digestion products without end reparation and polishing processing；
Wherein, the digestion products are obtained using the described pair of insensitive digestion with restriction enzyme of methylation.
10. library kit is built in DNA methylation library as claimed in claim 9, which is characterized in that the kit also include with
One of lower reagent is a variety of:
(1) primer containing special index sequence；Wherein, the primer includes forward and reverse primer, forward and reverse primer point
Not Bao Kuo respectively with the sequence of not complementary partial complementarity in the methylation connector；
(2) DNA ligase；With
(3) archaeal dna polymerase.
11. library kit is built in DNA methylation library as claimed in claim 9, which is characterized in that described pair of methylation is insensitive
Restriction enzyme be selected from one of MSP I, HaeIII, BanII, HpyCH4V, AluI, SphI and BssSI or a variety of.
12. library kit is built in DNA methylation library as claimed in claim 10, which is characterized in that
DNA ligase be selected from 5 ' App DNA/RNA ligases, T4 RNA ligase, T4 DNA ligase, Taq DNA ligase,
T7 DNA ligase, T3 DNA ligase, CircLigase ligase, electricity turn ligase, flat end/TA ligase, instantaneously stick
One of property ligase, HiFi Taq DNA ligase are a variety of；
It is poly- that the archaeal dna polymerase is selected from Phusion U Hot Start archaeal dna polymerase, PfuTurbo Cx Hotstart DNA
One of synthase, Taq archaeal dna polymerase and LafU archaeal dna polymerase are a variety of.
13. library kit is built in DNA methylation library as claimed in claim 9, which is characterized in that the kit further include with
One of lower reagent is a variety of:
(a) reagent needed for carrying out digestion；
(b) reagent needed for carrying out the connection of methylation connector；
(c) reagent needed for carrying out methylation conversion；
(d) reagent needed for carrying out DNA cloning；
(e) reagent needed for carrying out DNA purifying；With
(f) fragmentation used or the carrier DNA of un-segmentedization required for product purification or segment sorting are attached.
14. library kit is built in DNA methylation library as claimed in claim 13, it is characterised in that:
Reagent needed for the carry out digestion includes buffer and non-methylation λ DNA；
Reagent needed for the connection for carrying out methylation connector includes buffer and ATP；
The required reagent of methylation conversion that carries out is bisulfite conversion reagent；
Reagent needed for the carry out DNA cloning includes amplification buffer and 4 kinds of dNTP；
The required reagent of DNA purifying that carries out includes magnetic bead and/or purification column.
15. library kit is built in DNA methylation library as claimed in claim 9, which is characterized in that
The methylation connector is selected from the group one of methylation joint sequence or a variety of:
The methylation joint sequence that SEQ ID NO:1 and SEQ ID NO:2 is formed；
The methylation joint sequence that SEQ ID NO:3 and SEQ ID NO:4 is formed；
The methylation joint sequence that SEQ ID NO:5 and SEQ ID NO:6 is formed；
The methylation joint sequence that SEQ ID NO:7 and SEQ ID NO:8 is formed；
The methylation joint sequence that SEQ ID NO:9 and SEQ ID NO:10 is formed；
The methylation joint sequence that SEQ ID NO:11 and SEQ ID NO:12 is formed；
The methylation joint sequence that SEQ ID NO:13 and SEQ ID NO:14 is formed；
The methylation joint sequence that SEQ ID NO:15 and SEQ ID NO:16 is formed；
The methylation joint sequence that SEQ ID NO:17 and SEQ ID NO:18 is formed；
The methylation joint sequence that SEQ ID NO:19 and SEQ ID NO:20 is formed；
The index primer is selected from: SEQ ID NO:21 and SEQ ID NO:22 and SEQ ID NO:23 and SEQ ID NO:24.
16. a kind of DNA sequencing method, which is characterized in that the method includes using side of any of claims 1-8
The step of method constructs DNA methylation library and the DNA sequence dna in library is sequenced.
17. a kind of joint sequence product, which is characterized in that the joint sequence product includes to have and repair and mend without end
The connector for the cohesive end that the cohesive end of the digestion products handled together matches, wherein connector described in the product it is dense
Degree is 0.1-1.5 μM.
18. joint sequence product as claimed in claim 17, which is characterized in that described to be repaired and polishing processing without end
The cohesive end of digestion products is using selected from one of MSP I, BanII, SphI and BssSI restriction enzyme or a variety of
Carry out cohesive end caused by digestion.
19. joint sequence product as claimed in claim 17, which is characterized in that the joint sequence is selected from the group joint sequence
One of or it is a variety of:
The joint sequence that SEQ ID NO:1 and SEQ ID NO:2 is formed；
The joint sequence that SEQ ID NO:3 and SEQ ID NO:4 is formed；
The joint sequence that SEQ ID NO:7 and SEQ ID NO:8 is formed；
The joint sequence that SEQ ID NO:9 and SEQ ID NO:10 is formed；
The joint sequence that SEQ ID NO:11 and SEQ ID NO:12 is formed；
The joint sequence that SEQ ID NO:13 and SEQ ID NO:14 is formed；
The joint sequence that SEQ ID NO:17 and SEQ ID NO:18 is formed；With
The joint sequence that SEQ ID NO:19 and SEQ ID NO:20 is formed.
20. joint sequence product as claimed in claim 17, which is characterized in that the joint sequence product also contain have with
The joint sequence of the flat end to match with the flat end of the digestion products of polishing processing is repaired without end.
21. joint sequence product as claimed in claim 20, which is characterized in that it is described have with without end repair and polishing
The joint sequence for the flat end that the flat end of the digestion products of processing matches is selected from: the connector sequence that SEQ ID NO:5 and 6 is formed
The joint sequence of column and SEQ ID NO:15 and 16 formation.
22. the joint sequence product as described in any one of claim 16-21, which is characterized in that the joint sequence is first
Base joint sequence.
23. joint sequence product described in any one of claim 17-22 and optional insensitive restricted to methylating
Application of the restriction endonuclease in building DNA methylation library, or the application in the kit in preparation building DNA methylation library.
24. application as claimed in claim 23, which is characterized in that the described pair of insensitive restriction enzyme of methylation is selected from
One of MSP I, HaeIII, BanII, HpyCH4V, AluI, SphI and BssSI or a variety of.
Priority Applications (2)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111235638A (en) * | 2020-02-20 | 2020-06-05 | 北京良芯生物科技发展有限公司 | Method and kit for constructing sequencing library |
| CN111945232A (en) * | 2020-08-27 | 2020-11-17 | 天津诺禾医学检验所有限公司 | Y-shaped joint, kit and method for constructing RRBS library |
| CN112080555A (en) * | 2019-06-14 | 2020-12-15 | 上海鹍远健康科技有限公司 | DNA methylation detection kit and detection method |
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| CN108103169A (en) * | 2017-12-11 | 2018-06-01 | 西北大学 | A kind of PCR method of the connector mediation based on hot asymmetric reaction |
| CN109735611A (en) * | 2018-12-19 | 2019-05-10 | 珠海铂华生物工程有限公司 | A kind of assortment of genes for detecting bone marrow failure syndrome, primer library, the method and its application for constructing high-throughput sequencing library |
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| WO2012019320A1 (en) * | 2010-08-11 | 2012-02-16 | 中国科学院心理研究所 | High-throughput sequencing method for methylated dna and use thereof |
| 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 |
| CN108103169A (en) * | 2017-12-11 | 2018-06-01 | 西北大学 | A kind of PCR method of the connector mediation based on hot asymmetric reaction |
| CN109735611A (en) * | 2018-12-19 | 2019-05-10 | 珠海铂华生物工程有限公司 | A kind of assortment of genes for detecting bone marrow failure syndrome, primer library, the method and its application for constructing high-throughput sequencing library |
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| CN112080555A (en) * | 2019-06-14 | 2020-12-15 | 上海鹍远健康科技有限公司 | DNA methylation detection kit and detection method |
| CN111235638A (en) * | 2020-02-20 | 2020-06-05 | 北京良芯生物科技发展有限公司 | Method and kit for constructing sequencing library |
| CN111945232A (en) * | 2020-08-27 | 2020-11-17 | 天津诺禾医学检验所有限公司 | Y-shaped joint, kit and method for constructing RRBS library |
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