CN109321647A - The construction method of marking composition and methylolation nucleic acid library - Google Patents
The construction method of marking composition and methylolation nucleic acid library Download PDFInfo
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- CN109321647A CN109321647A CN201811256087.3A CN201811256087A CN109321647A CN 109321647 A CN109321647 A CN 109321647A CN 201811256087 A CN201811256087 A CN 201811256087A CN 109321647 A CN109321647 A CN 109321647A
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- 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
- C12Q1/6869—Methods for sequencing
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- 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
Abstract
The present invention relates to field of biotechnology, in particular to marking composition and the construction method of methylolation nucleic acid library.The present invention carries out double labeling to genomic DNA using UDP-6-N3-Glu and β GT；Furthermore the present invention also carries out specific marker using biotin, and the DNA fragmentation with 5hmC modification is enabled to be captured entirely as far as possible；The DNA of capture carries out winkler ground using NEB related reagent, can guarantee that the quality in library is more excellent, and sequencing result is made to become reliable and stable.
Description
Technical field
The present invention relates to field of biotechnology, in particular to the building side of marking composition and methylolation nucleic acid library
Method.
Background technique
5-hydroxymethyl cytosine (5hmC) is a kind of modification for being present in cytimidine in bacteriophage early in nineteen fifty-two discovery
Form, and what is detected in the neuron and embryonic stem cell of mouse simultaneously recently are present in mammalian genome
Another modification mode.Then, a large amount of research concentrate on disclose 5hmC and TET protein family oxidizing ferment genomic organization with
And the role that may be undertaken in murine stem cell differentiation, and prove that TET protease family can convert 5mC by oxidation
For 5hmC.And the development of Tet family enzyme and disease is related with regulation.It is many different to have reported that TET albumen and 5hmC are present in
In tissue, and their expression/activity is strictly regulated and controled during ES cell differentiation.TET1 and TET2 is and cancer
Disease in relation to: TET1 is a ligand of MLL in Acute Meyloid sample leukaemia (AML) and acute lymphocytic leukemia (ALL),
And the missing of TET2 function also with Acute Meyloid sample leukaemia (AML) and various bone marrow cell dysfunction syndromes and marrow
Proliferative disease is closely related.TET albumen and hmC cause DNA methylation to be lacked of proper care, in embryonic stem cell multipotency, carcinogenicity
May play a significant role in the function of conversion and neuron.
5-hydroxymethyl cytosine (5hmC) is a kind of new epigenetics modification for being referred to as the 6th kind of base, it is
5- methyl is aoxidized under the conditions of iron ion is with existing for α-ketoglutaric acid as Tet (Ten-Eleven-Translocation) family
Cytimidine (5mC).The discovery of research detection at present, in fertilized eggs, embryonic stem cell and brain tissue, the content of 5hmC is all
It is quite abundant, and almost ten times of its hetero-organization in brain tissue and embryonic stem cell, therefore further to study its biology
It learns function and provides clue.
Being used to detect the main method of 5hmC at present is sodium bisulfite method and β glycosyl transferase (β-
Glucosyltransferase, β GT) method.Researcher is reported in after handling by sodium hydrogensulfite, 5- methylol born of the same parents
Pyrimidine (5hmC) can be changed into 5- methylene sulfonate form cytimidine (cytosine5-methylenesulfonate,
CMS), the deamination speed ratio methylcystein of the cytimidine of this sulfonate form is slower.By PCR amplification of signal it
Afterwards, the sequencing result of 5-hydroxymethyl cytosine position can also be shown as cytimidine.Therefore, traditional sodium bisulfite method is sequenced simultaneously
It cannot completely reflect the true distribution situation of various bases in genome, there is an urgent need to find new method to solve 5- by people
The problem of hydroxymethyl cytosine detects.
The prior art handles genomic DNA by β GT, is cut using MSPI enzyme, jointing, BST enzymatic treatment is repaired
Multiple, EcoP15I digestion, Piece Selection connects P7 connector, and PCR amplification establishes library.But above-mentioned experimental procedure is cumbersome, needs
A variety of enzymes carry out cutting connection；And it is easily lost the DNA fragmentation of 5hmC modification, cause sequencing result inaccurate.
Therefore it provides a kind of detection method of 5-hydroxymethyl cytosine has important practical significance.
Summary of the invention
In view of this, the present invention provides the construction method of a kind of marking composition and methylolation nucleic acid library.It utilizes
UDP-6-N3-Glu and β GT carries out double labeling to genomic DNA；Specific marker additionally, which is carried out, using biotin makes band
The DNA fragmentation for having 5hmC to modify can be captured entirely as far as possible；The DNA of capture we utilize NEB related reagent carry out
Winkler ground can guarantee that the quality in library is more excellent, and sequencing result is made to become reliable and stable.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of marking compositions, including UDP-6-N3-Glu and β GT.
In some specific embodiments of the invention, the marking composition further includes biotin.
On this basis, the answering in detection nucleic acid methylolation modification the present invention also provides the marking composition
With.
The present invention also provides the marking compositions in detection preparation and/or the detection for preparing the modification of nucleic acid methylolation
Application in kit.
In some specific embodiments of the invention, the nucleic acid is genomic DNA.
The present invention also provides the detection preparations of nucleic acid methylolation modification, including the marking composition.
The present invention also provides the detection kits of nucleic acid methylolation modification, including the marking composition.
It further include NEB reagent commonly used in the art in some specific embodiments of the invention.
The present invention also provides the construction methods of methylolation nucleic acid library, include the following steps:
Step 1: nucleic acid fragment is obtained, using marking composition as described in claim 1 to the 5- hydroxyl in nucleic acid fragment
Methylcystein (5hmC) modification is marked, and uses biotin labeling after purification, obtains after purification phonetic containing 5- methylol born of the same parents
The nucleic acid of pyridine (5hmC) modification；
Step 2: using being connected after the pretreatment of NEB reagent, purifying, purified again after amplification, obtain methylolation nucleic acid text
Library.
The present invention interrupts method, 5hmC label, capture, until the foundation of 5hmC DNA library by DNA ultrasound.Pass through this
The current existing method of 5hmC DNA library ratio that method obtains, substantially increases the capture rate of 5hmC, Library Quality, so that sample
Product result is relatively reliable, accurate and better authenticity.First, it is determined that ultrasound interrupts the condition of DNA fragmentation, can be with from Fig. 1
Find out 25 watts 10 minutes 5 minutes to 100 watts, DNA can be made to be broken into the range of 200-500bp segment, but in these conditions
In again with 50 watts 5 minutes to be optimal.Secondly as the enrichment of 5hmC needs the DNA of high concentration, the method for optimizing DNA concentration,
Present invention combination glycogen, ethyl alcohol and sodium acetate joint carry out DNA concentration, compared with conventional method (ethyl alcohol and sodium acetate are used in conjunction) concentration
Efficiency has obvious raising (Fig. 2, P < 0.05), and the DNA loss amount made is preferably minimized, and ensures that subsequent experimental
It goes on smoothly, while ensure that the accuracy and integrality of experimental result to greatest extent.Finally using UDP-6-N3-Glu and
Biotin is used in combination, and the DNA fragmentation of 5hmC is had for the label of specificity, this step is particularly important, if biological
Element label is unsuccessful, then directly results in that following dynabeads enrichment is unsuccessful, i.e., capture rate is low.The use of this method makes
5hmC modification becomes easier to and more efficiently, lay a solid foundation for the foundation in following library.
In some specific embodiments of the invention, the nucleic acid fragment is the segment of 200~500bp.
In some specific embodiments of the invention, purification process used in the present invention is Ampure-XP magnetic
Pearl method, this method can specifically be shown in Table 1 according to the number of magnetic bead come the selective different size of DNA fragmentation of purifying:
Table 1
| Clip size | 150bp | 200bp | 250bp | 300bp | 400bp | 500bp | 700bp |
| Pearl ratio | 0.9X | 0.8X | 0.7X | 0.6X | 0.55X | 0.5X | 0.45X |
Therefore we can by control AMPure-XP magnetic bead number come DNA fragmentation size required for screening.
In some specific embodiments of the invention, the construction method in the library specifically comprises the following steps:
One, the DNA fragmentation with 5hmC modification is obtained
The ultrasonication (to be broken into the segment of 200~500bp) of 1.gDNA
It carries out DNA using ultrasonic cell disrupte machine (Ningbo Xin Yi ultrasonic device Co., Ltd) to be crushed, volume 200ul.
Ultrasonic power selects 100W, tri- power of 140W, 180W, 2,5 minutes two time points of ultrasonic time, between ultrasonic
It is the endless form that ultrasound stops 5 seconds for 5 seconds every the time, sample whole process is placed on when ultrasonic operates on ice.
100W~180W is concluded that according to result (Fig. 1), can get satisfactory DNA fragmentation within 2~5 minutes.
2. concentration of DNA sample
If DNA concentration is relatively low after ultrasound, can be concentrated with method once.
1) 40 μ L 3M NaAC pH 5.1 are added in the DNA solution (200ul system) after ultrasound, flick mixing.
2) 2 μ L 5mg/mL Glycogen are added and flick mixing, brief centrifugation 2-3s.
3) 800 μ L dehydrated alcohol, after mixing well ,@- 80 DEG C, overnight.
4) 4 DEG C, maximum speed, 20min abandons supernatant.
5) 70% ethanol washing precipitating is primary, and 4 DEG C, maximum speed, 10min abandons supernatant.
6) 4 DEG C, maximum speed, 5min, as far as possible exhaustion liquid.
7)27μL Millipore H2O, dissolution precipitating, surveys concentration.
Note: H2The volume of O is depending on the amount of DNA, final concentration≤1 μ g/ μ L.
VH2OThe volume of DNA in=V1+V2V1, β GT reaction；V2 surveys volume required when concentration.
DNA total amount * 100% before DNA total amount/concentration after enrichment factor=concentration
3. with UDP-6-N3-Glu and β GT in DNA fragmentation 5hmC modification be marked (design 3 experimental groups,
10ugDNA group, 20ugDNA group and 40ugDNA group)
Reaction mixture is configured according to the reagent information in following table, uses the PCR pipe of 0.2ml.
PCR pipe is placed in PCR instrument after mixing and is incubated for, condition is 37 degree, and 2-2.5 hours, Gai Wen was set as 50 degree.
4. being purified using AMPure-XP pearl
1) 54ul AMPure-XP pearl (Beckman, A63881) is added in reaction solution one step up, it is mixed with whirlpool instrument
Incubation at room temperature 5 minutes after even.
2) PCR pipe is placed on magnetic frame 5 minutes, separates magnetic bead；Supernatant moves on to in new PCR pipe (supernatant can be first
Retain).
3) ethyl alcohol (concentration 80%) of the fresh configuration of 200ul is added into precipitating, abandons supernatant after placing 30 seconds at room temperature.
(this process operates on magnetic frame).Repetitive operation is primary, is then air-dried on magnetic frame 5 minutes.It should not overdrying
Pearl, this may cause rate of recovery reduction.
4) 35ul deionization aqua sterilisa is added into PCR pipe, is placed at room temperature after mixing well 2 minutes, elution of bound
DNA on pearl.
5) PCR pipe is placed on magnetic frame until careful 30ul supernatant of drawing is to new PCR pipe after solution change clarification
In, concentration is surveyed with Nano-Drop and is recorded.
5. biotinylation reacts
1) 2ul, the disulfide-biotin linker (Click of 2.5mM are added in the supernatant obtained one step up
Chemistry tools, 1168-10), make final concentration of 150uM.
2) PCR pipe is placed in PCR instrument after mixing and is incubated for 2 hours, condition is 37 degree, 50 degree of lid temperature.
6. being purified using AMPure-XP pearl, consistent with step 4, difference is that finally eluting deionization used goes out
The volume of bacterium water is 55ul, and transfer supernatant is 50ul.
7. obtaining the DNA modified containing 5hmC
1) it usesUnder (Invitrogen, 65305) streptavidin C1 comes
Draw biotinylated DNA.
2) 2X buffer configures
3) it takes 25ul Dynabeads into new PCR pipe, is washed twice with 1X buffer, abandoned after magnetic frame beads spun
Supernatant.
4) in the 50ulDNA sample obtained into step 6 be added 50ul 2X buffer, transferred them to after mixing containing
In the PCR pipe of Dynabeads, room temperature, which rotates, mixes 30 minutes (being 15 minutes on specification here), after magnetic frame beads spun
Abandon supernatant.
5) it is washed pearl 3 times with 1X buffer, room temperature rotation mixes 5 minutes when last time is washed.(in order to preferably remove
Unbonded DNA fragmentation).
6) eluted dna from pearl, and addition 50ul 100mM DTT (Invitrogen, 15508013, deionization aqua sterilisa
Configuration) solution, it is incubated at room temperature 2 hours.
8. being purified using AMPure-XP pearl to remove DTT
1) after making bead pellet on magnetic frame, carefully supernatant is transferred in new centrifuge tube (50ul).
2) it is added after 50ul AMPure-XP pearl is mixed with whirlpool instrument and is incubated at room temperature 5 minutes into the supernatant of transfer.
Consistent described in remaining step and step 4, the volume that difference is finally to elute deionization aqua sterilisa used is 15ul,
Transfer supernatant is 10-11ul.
9.Nano-Drop and Qubit quantifies the DNA of acquisition
10. calculating capture rate, it is shown in Table 3.
Two, the foundation of the DNA library with 5hmC modification is obtained
1. being attached preceding preparation using NEBNext Ultra II End Prep (E6000L) reagent
1) solution allocation is carried out according to information listed below
2) 10 mixings are gently blown and beaten
3) be incubated in PCR instrument, actual conditions are Gai Wen >=75 DEG C, setting program be 20 degree 30 minutes；65 degree 30 points
Clock；Last 4 degree of heat preservations.
Note: it not stop, directly be attached
2.Adaptor connection
1) determine whether Adaptor needs to dilute, referring to following table:
| Input amount of DNA | Adaptor dilution ratio | Adaptor final concentration |
| 101ng-1ug | It does not dilute | 15mM |
| 5ng-100ng | 1:10 dilution | 1.5mM |
| 1-2.5ng | 1:20 dilution | 0.75mM |
2) required reagent is added in new centrifuge tube by following message
3) 10 mixings are gently blown and beaten
4) be incubated in PCR instrument, 20 degree 15 minutes
5) 3ul USERTM enzyme (red) is added, is incubated for 15 minutes for 37 degree in PCR instrument, Gai Wen >=47 DEG C
3. being purified using AMPure-XP pearl, consistent with step 1-4, difference is finally to elute deionization used
The volume of aqua sterilisa is 25ul, and transfer supernatant is 23ul.
4.PCR expands the DNA of connector connection, uses NEBNext Multiplex Oligos for Illumina reagent
Box (includes index primer)
1) by following message addition reagent into new PCR pipe
2) gently 10 mixings of piping and druming are placed in PCR instrument and carry out PCR amplification.
3) PCR program setting see the table below.
5. carrying out purified pcr product using AMPure-XP pearl, consistent with step 1-4, difference is finally to elute institute
Volume with deionization aqua sterilisa is 16ul, and transfer supernatant is 14-15ul.
6. being detected using Qubit to Library Quality.The present invention also provides the detection sides of nucleic acid methylolation modification
Method is sequenced the methylolation nucleic acid library that the construction method obtains, analytical sequence information, obtains the modification of nucleic acid methylolation
Information.The information includes site, the quantity of modification etc. of modification.
By the way that saccharide ring is transferred on 5 methylols of 5-hydroxymethyl cytosine using β glycosyl transferase, by 5- methylol
Cytimidine protects, and 5-methylcytosine is then oxidized to 5- carboxyl cytimidine using excessive TET oxidizing ferment.Passing through
After β-glycosyl transferase and TET oxidation enzymatic treatment, in final sodium bisulfite method sequencing result, at 5-methylcytosine
It is shown as thymidine T, and 5-hydroxymethyl cytosine is shown as cytimidine C.The sodium bisulfite method of this TET collaboration is surveyed
Sequence (Tet-assisted bisulfite sequencing, TAB-Seq), which efficiently solves conventional method, cannot be distinguished 5- methyl
The problem of cytimidine and 5-hydroxymethyl cytosine, and reached the level that 5-hydroxymethyl cytosine is sequenced in genome, it is practical
The detection of 5-hydroxymethyl cytosine provides a kind of very reliable method in sample.Saccharide ring is transferred to DNA by β glycosyl transferase
On the hydroxyl of 5-hydroxymethyl cytosine in sequence, and then increase the steric hindrance of 5 one hydroxymethyl cytosines, can succeed area
Divide 5-hydroxymethyl cytosine and 5-methylcytosine.After glycosyl transferase acts on 5-hydroxymethyl cytosine, on 5
Hydroxyl by being protected by saccharide ring, steric hindrance increase lead to that bromine father-in-law's ionic intermediate can not be formed in subsequent reaction.
This method is difficult to the problem of distinguishing to 5-hydroxymethyl cytosine in DNA chain and 5-methylcytosine, effectively eliminates 5- first
The interference of 5-hydroxymethyl cytosine in the detection of base cytimidine.Whole process includes the cutting of N- glycosidic bond and opening for base reparation
It is dynamic.And UDP-6-N3-Glu can be glycosylated with specific recognition, and can be further by biotin labeling.
The present invention also provides the concentrate formulations of DNA, including marking composition of the present invention.
On this basis, the present invention also provides the method for concentration of DNA, take marking composition and DNA of the present invention
Mixing, concentration.
The present invention carries out double labeling to genomic DNA using UDP-6-N3-Glu and β GT；Additionally using biotin into
Row specific marker enables the DNA fragmentation with 5hmC modification to be captured entirely as far as possible；The DNA of capture we benefit
Winkler ground is carried out with NEB related reagent, can guarantee that the quality in library is more excellent, sequencing result is made to become reliable and stable.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows the effect picture that different ultrasound conditions are crushed DNA；
Fig. 2 shows DNA enrichment factor comparison diagram；
Fig. 3 shows techniqueflow chart；
Fig. 4 shows that Library Quality concentration compares；
Fig. 5 shows library effect picture.
Specific embodiment
The invention discloses a kind of marking composition and the construction method of methylolation nucleic acid library, those skilled in the art
Present disclosure can be used for reference, realization of process parameters is suitably modified.In particular, it should be pointed out that all similar substitutions and modifications pair
It is it will be apparent that they are considered as being included in the present invention for those skilled in the art.Method and application of the invention is
Through being described by preferred embodiment, related personnel obviously can not depart from the content of present invention, in spirit and scope to this
Methods and applications described in text are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
Raw materials used, reagent in the construction method of marking composition and methylolation nucleic acid library provided by the invention
It is bought by market.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1 obtains the DNA fragmentation with 5hmC modification
The ultrasonication (to be broken into the segment of 200-500bp) of 1.gDNA
It carries out DNA using ultrasonic cell disrupte machine (Ningbo Xin Yi ultrasonic device Co., Ltd) to be crushed, volume 200ul,
Ultrasonic power selects 100W, and tri- power of 140W, 180W, at 2,5 minutes two time points of ultrasonic time, ultrasonic interval time is
Ultrasound stops 5 seconds endless form for 5 seconds, and sample whole process is placed on when ultrasonic operates on ice.It is concluded that according to result (Fig. 1)
100W~180W can get satisfactory DNA fragmentation in 2~5 minutes.
2. concentration of DNA sample
If DNA concentration is relatively low after ultrasound, can be concentrated with method once.
8) 40 μ L 3M NaAC pH 5.1 are added in the DNA solution (200ul system) after ultrasound, flick mixing.
9) 2 μ L 5mg/mL Glycogen are added and flick mixing, brief centrifugation 2-3s.
10) 800 μ L dehydrated alcohol, after mixing well ,@- 80 DEG C, overnight.
11) 4 DEG C, maximum speed, 20min abandons supernatant.
12) 70% ethanol washing precipitating is primary, and 4 DEG C, maximum speed, 10min abandons supernatant.
13) 4 DEG C, maximum speed, 5min, as far as possible exhaustion liquid.
14)27μL Millipore H2O, dissolution precipitating, surveys concentration.
Note: H2The volume of O is depending on the amount of DNA, final concentration≤1 μ g/ μ L.
VH2OThe volume of DNA in=V1+V2V1, β GT reaction；V2 surveys volume required when concentration.
DNA total amount * 100% before DNA total amount/concentration after enrichment factor=concentration
3. with UDP-6-N3-Glu and β GT in DNA fragmentation 5hmC modification be marked (design 3 experimental groups,
10ugDNA group, 20ugDNA group and 40ugDNA group)
Reaction mixture is configured according to the reagent information in table 2, uses the PCR pipe of 0.2ml.
Table 2
| Volume | Sample ID | Final concentration | Reagent source |
| 3μL | 10X βGT Reaction Buffer | 1X | NEB, M0357L |
| 23μL | 20μg gDNA | 1μg/μL | |
| 1.5μL | UDP-6-N3-Glu | 100μM | SinvoA, SL-07793 |
| 2.5μL | βGT | NEB, M0357L | |
| 30μL | Total volume |
PCR pipe is placed in PCR instrument after mixing and is incubated for, condition is 37 degree, and 2-2.5 hours, Gai Wen was set as 50 degree.
4. being purified using AMPure-XP pearl
6) 54ul AMPure-XP pearl (Beckman, A63881) is added in reaction solution one step up, it is mixed with whirlpool instrument
Incubation at room temperature 5 minutes after even.
7) PCR pipe is placed on magnetic frame 5 minutes, separates magnetic bead；Supernatant moves on to in new PCR pipe (supernatant can be first
Retain).
8) ethyl alcohol (concentration 80%) of the fresh configuration of 200ul is added into precipitating, abandons supernatant after placing 30 seconds at room temperature.
(this process operates on magnetic frame).Repetitive operation is primary, is then air-dried on magnetic frame 5 minutes.It should not overdrying
Pearl, this may cause rate of recovery reduction.
9) 35ul deionization aqua sterilisa is added into PCR pipe, is placed at room temperature after mixing well 2 minutes, elution of bound
DNA on pearl.
10) PCR pipe is placed on magnetic frame until careful 30ul supernatant of drawing is to new PCR pipe after solution change clarification
In, concentration is surveyed with Nano-Drop and is recorded.
5. biotinylation reacts
3) 2ul, the disulfide-biotin linker (Click of 2.5mM are added in the supernatant obtained one step up
Chemistry tools, 1168-10), make final concentration of 150uM.
4) PCR pipe is placed in PCR instrument after mixing and is incubated for 2 hours, condition is 37 degree, 50 degree of lid temperature.
6. being purified using AMPure-XP pearl, consistent with step 4, difference is that finally eluting deionization used goes out
The volume of bacterium water is 55ul, and transfer supernatant is 50ul.
7. obtaining the DNA modified containing 5hmC
7) it usesUnder (Invitrogen, 65305) streptavidin C1 comes
Draw biotinylated DNA.
8) 2X buffer configures
9) it takes 25ul Dynabeads into new PCR pipe, is washed twice with 1X buffer, abandoned after magnetic frame beads spun
Supernatant.
10) in the 50ulDNA sample obtained into step 6 be added 50ul 2X buffer, transferred them to after mixing containing
In the PCR pipe of Dynabeads, room temperature, which rotates, mixes 30 minutes (being 15 minutes on specification here), after magnetic frame beads spun
Abandon supernatant.
11) it is washed pearl 3 times with 1X buffer, room temperature rotation mixes 5 minutes when last time is washed.(in order to preferably go
Except unbonded DNA fragmentation).
12) eluted dna from pearl, 50ul 100mM DTT is added, and (Invitrogen, 15508013, deionization sterilizes
Water configuration) solution, it is incubated at room temperature 2 hours.
8. being purified using AMPure-XP pearl to remove DTT
3) after making bead pellet on magnetic frame, carefully supernatant is transferred in new centrifuge tube (50ul).
4) it is added after 50ulAMPure-XP pearl is mixed with whirlpool instrument and is incubated at room temperature 5 minutes into the supernatant of transfer.
Consistent described in remaining step and step 4, the volume that difference is finally to elute deionization aqua sterilisa used is 15ul,
Transfer supernatant is 10-11ul.
9.Nano-Drop and Qubit quantifies the DNA of acquisition
10. calculating capture rate, it is shown in Table 3.
3 three groups of capture rates of table
| Group | 10ugDNA group | 20ugDNA group | 40ugDNA group |
| InputDNA amount | 8300ng | 16160ng | 32290ng |
| Capture dna amount | 167.2ng | 323.2ng | 356ng |
| Capture rate | 2.01% | 2.00% | 1.10% |
Embodiment 2 obtains the foundation of the DNA library with 5hmC modification
7. being attached preceding preparation using NEBNext Ultra II End Prep (E6000L) reagent
4) solution allocation is carried out according to information listed below
5) 10 mixings are gently blown and beaten
6) be incubated in PCR instrument, actual conditions are Gai Wen >=75 DEG C, setting program be 20 degree 30 minutes；65 degree 30 points
Clock；Last 4 degree of heat preservations.
Note: it not stop, directly be attached
8.Adaptor connection
6) determine whether Adaptor needs to dilute, referring to table 4:
Table 4
| Input amount of DNA | Adaptor dilution ratio | Adaptor final concentration |
| 101ng-1ug | It does not dilute | 15mM |
| 5ng-100ng | 1:10 dilution | 1.5mM |
| 1-2.5ng | 1:20 dilution | 0.75mM |
7) required reagent is added in new centrifuge tube by following message
8) 10 mixings are gently blown and beaten
9) be incubated in PCR instrument, 20 degree 15 minutes
10) 3ul USER is addedTMEnzyme (red) is incubated for 15 minutes for 37 degree in PCR instrument, Gai Wen >=47 DEG C
9. being purified using AMPure-XP pearl, consistent with step 1-4, difference is finally to elute deionization used
The volume of aqua sterilisa is 25ul, and transfer supernatant is 23ul.
10.PCR expands the DNA of connector connection, uses NEBNext Multiplex Oligos for Illumina reagent
Box (includes index primer)
4) by following message addition reagent into new PCR pipe
5) gently 10 mixings of piping and druming are placed in PCR instrument and carry out PCR amplification.
6) PCR program setting is shown in Table 5.
Table 5
11. carrying out purified pcr product using AMPure-XP pearl, consistent with step 1-4, difference is finally to elute institute
Volume with deionization aqua sterilisa is 16ul, and transfer supernatant is 14-15ul.
12. being detected using Qubit to Library Quality.As a result as shown in table 6, Fig. 2~Fig. 5.
DNA concentration and volume after 6 three groups of library constructions of table
| Group | 10ugDNA group | 20ugDNA group | 40ugDNA group |
| DNA concentration | 17.4ng/ul | 21.7ng/ul | 27.2ng/ul |
| Volume | 16ul | 16ul | 16ul |
| Total amount | 278.4ng | 347.2ng | 435.2ng |
Capture rate according to capture rate 20ug capture rate ratio 40ug is high, and the capture total amount than 10ug is again much higher,
Therefore 20ug is relatively preferable.
Comparative example
Step 1: three groups of reactions of setting, wherein one group carries out glycosylation processing to nucleic acid, remaining two groups nucleic acid is without sugar
Baseization processing；
Step 2: by step 1 through glycosylating the one of one group of treated nucleic acid and the nucleic acid without glycosylation processing
Group difference carries out MspI endonuclease reaction in parallel；One group of nucleic acid without glycosylation processing remaining in step 1 is carried out simultaneously
HpaII endonuclease reaction；
Step 3: every group of endonuclease bamhi in step 2 three groups is separately connected containing different Index and is wrapped simultaneously
The connector A of the restriction enzyme site containing Ecop15I or MmeI, the nucleic acid that will be connected with each group of different connector A is admixed together, obtains
One includes the nucleic acid library without modification, methylation modification and methylolation modification；
Step 4: a chain jaggy in the double-stranded DNA connector in nucleic acid library is carried out with Bst archaeal dna polymerase
It repairs；
Step 5: Ecop15I or MmeI digestion is carried out to nucleic acid obtained in the previous step, generates short segment DNA sequence；
Step 6: the short dna segment of recycling jointing A；
Step 7: by one section, there are two the short dna segments of the P7 connector of base cohesive ends and the jointing A of step 6
It is attached；
Step 8: with the DNA sequence dna of connector A and P7 connector design universal primer to step 7 obtain be connected with P7 connector and
The short dna segment of connector A carries out PCR amplification, and recovery purifying PCR product establishes sequencing library；
Step 9: being sequenced the sequencing library that step 8 obtains, and sequence information is compared in analysis, obtains nucleic acid methylation
The information of modification and methylolation modification.
Effect example
The corresponding data of Fig. 2 are as shown in table 7.
The comparison of 7 DNA enrichment factor of table
| Comparative example | This method |
| 89.22118 | 96.54517* |
| 86.99367 | 93.73115* |
| 82.49336 | 97.72246* |
Note:*Show that compared with comparative example, there is significant difference (P < 0.05)；#Show has extremely significant difference compared with comparative example
(P < 0.01).
The corresponding data of Fig. 4 are as shown in table 8.
8 Library Quality concentration of table compares
| Comparative example | This method |
| 75 | 140# |
| 80 | 138# |
| 85 | 201# |
Note:*Show that compared with comparative example, there is significant difference (P < 0.05)；#Show has extremely significant difference compared with comparative example
(P < 0.01).
The library concentration constructed through the invention is shown compared with the conventional method by above data, is found after statistical analysis,
Construction method provided by the invention significantly improves library concentration (Fig. 4, P < 0.05).
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of marking composition, which is characterized in that including UDP-6-N3-Glu and β GT.
2. marking composition as described in claim 1, which is characterized in that further include biotin.
3. application of the marking composition as claimed in claim 1 or 2 in the modification of detection nucleic acid methylolation or concentration of DNA.
4. marking composition as claimed in claim 1 or 2 is in detection preparation and/or the detection for preparing the modification of nucleic acid methylolation
Application in kit or DNA concentrate formulation.
5. application as described in claim 3 or 4, which is characterized in that the nucleic acid is genomic DNA.
6. the detection preparation of nucleic acid methylolation modification, which is characterized in that including marking combination as claimed in claim 1 or 2
Object.
7. the detection kit of nucleic acid methylolation modification, which is characterized in that including mark group as claimed in claim 1 or 2
Close object.
8. detection kit as claimed in claim 6, which is characterized in that further include NEB reagent.
9. the construction method of methylolation nucleic acid library, which comprises the steps of:
Step 1: nucleic acid fragment is obtained, using marking composition as described in claim 1 to the 5- methylol in nucleic acid fragment
Cytimidine modification is marked, and uses biotin labeling after purification, obtains the core containing 5-hydroxymethyl cytosine modification after purification
Acid；
Step 2: using being connected after the pretreatment of NEB reagent, purifying, purified again after amplification, obtain methylolation nucleic acid library.
The concentrate formulation of 10.DNA, which is characterized in that including marking composition as claimed in claim 1 or 2.
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