CN104152437A - Methylated DNA enrichment method used in methyl binding protein sequencing - Google Patents
Methylated DNA enrichment method used in methyl binding protein sequencing Download PDFInfo
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- CN104152437A CN104152437A CN201410394587.9A CN201410394587A CN104152437A CN 104152437 A CN104152437 A CN 104152437A CN 201410394587 A CN201410394587 A CN 201410394587A CN 104152437 A CN104152437 A CN 104152437A
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Abstract
The invention relates to the field of molecular biology, and discloses a methylated DNA enrichment method used in methyl binding protein sequencing; the method comprises the following steps: (1) magnetic bead activation and methyl binding protein cross-linking reaction; (2) hatching of a methyl binding protein-magnetic bead complex and fragmented DNA; (3) removal of non-methylated DNA; (4) eluting of methylated DNA; and (5) ethanol precipitation to obtain enriched methylated DNA. Compared with the prior art, in the step of removal of the non-methylated DNA, 160mM NaCl and 200mM NaCl are successively used for eluting of a mixture of the fragmented DNA and the methyl binding protein-magnetic bead complex for more complete removal of the non-methylated DNA and retaining of the methylated DNA, the proportion of the methylated DNA in the final enriched methylated DNA is increased, and the sequencing depth of a methylated region can be improved.
Description
Technical field
The present invention relates to methyl in conjunction with protein sequencing technical field, particularly a kind of methylate DNA enriching method using in conjunction with protein sequencing process at methyl.
Background technology
DNA methylation is important epigenetics label information, obtains the methylation level data in all C site in full genome range, significant for the research of epigenetics.The order-checking that methylates belongs to epigenetics research.Methyl is a kind of sequence measurement that methylate DNA enrichment and high-flux sequence are combined in conjunction with protein sequencing.
The method of order-checking of methylating has a variety of, as the order-checking of full genomic methylation, simplifies the order-checking that methylates, and methylate DNA co-immunoprecipitation checks order, and methyl is in conjunction with protein sequencing etc.Full genomic methylation order-checking is that bisulf iotate-treated is combined with high throughput sequencing technologies, thereby draws the DNA methylation collection of illustrative plates of single base resolving power.Simplifying the order-checking that methylates is to utilize restriction enzyme to carry out enzyme to genome to cut, and can improve very significantly the order-checking degree of depth in CpG region, has reduced a large amount of order-checking amounts compared with full genomic methylation order-checking.The order-checking of methylate DNA co-immunoprecipitation is the full genomic methylation detection technique checking order based on antibody enrichment principle, adopt methylate DNA Immunoprecipitation, by there is methylated DNA fragmentation on 5'-methylcystein antibodies specific enrichment genome, then can in full genomic level, carry out the hyper-methylation regional study that high-precision CpG is intensive by high-flux sequence.
Methyl in conjunction with the principle of protein sequencing is: generally occur in owing to methylating in Mammals on 5 carbon atoms of cytosine(Cyt) of CpG, so can be by the DNA fragmentation of the albumen MBD enrichment hyper-methylation of specific binding methylate DNA, and in conjunction with s-generation high-flux sequence, the DNA fragmentation being enriched to is checked order, thereby detect the site that methylates in full genome range.
In process at methyl in conjunction with protein sequencing, utilize the specific binding of MBD albumen and methylate DNA, remove non-methylate DNA, methylate DNA enrichment is specifically got off, then go to build library by this part of methylate DNA and also check order.In the Insert Fragment in library, the shared ratio of non-methylate DNA is less, and in the final data that order-checking obtains, the shared data volume of methylate DNA part is larger, and fraction of coverage and the overburden depth in the region that correspondingly methylates are also larger, and the order-checking degree of depth is also larger.If, after non-methylate DNA wash-out, residual non-methylate DNA is more, will cause non-methylate DNA in library to increase in experiment, in final data, the partial data quantitative change of methylate DNA is little.So in the situation that obtaining same quantity of data, it is cleaner that non-methylate DNA is removed, the data volume in the region that methylates is larger, and the order-checking degree of depth is darker, and correspondingly cost also can reduce.
Summary of the invention
The object of the present invention is to provide a kind of enriching method of methylate DNA, the method is applied to methyl in conjunction with in protein sequencing, can improve methylate DNA in enrichment DNA ratio, reduce the ratio of non-methylate DNA, thereby improve the order-checking degree of depth in the region that methylates.
For solving the problems of the technologies described above, the invention provides a kind of methyl that is applied in conjunction with the methylate DNA enriching method in protein sequencing, comprise following steps:
(1) activation of magnetic bead is combined protein-crosslinking and is reacted with methyl
Magnetic bead is activated, then make the magnetic bead generation crosslinking reaction of biotin labeled methyl in conjunction with albumen and after activating, obtain methyl in conjunction with albumen-magnetic bead mixture;
(2) methyl is in conjunction with the hatching of albumen-magnetic bead mixture and fragmentation DNA
Add methyl that step (1) obtains in conjunction with in albumen-magnetic bead mixture fragmentation DNA, hatch, methylate DNA is combined specifically with mixture;
(3) remove non-methylate DNA
To being combined the mixture of albumen-magnetic bead mixture with methyl through the fragmentation DNA of hatching in step (2), use 160mM NaCl is magnetic bead wash-out 4 times, then uses 200mM NaCl wash-out 3 times, removes non-methylate DNA;
(4) methylate DNA wash-out
To the removal obtaining in step (3) mixture of non-methylate DNA, use 2000mM NaCl to carry out wash-out to magnetic bead, obtain methylate DNA component;
(5) ethanol precipitation
The methylate DNA component that adopts ethanol precipitation to obtain wash-out in step (4) precipitates, and after being dried, dissolves with TE damping fluid, obtains the methylate DNA of enrichment.
Methylate DNA enriching method provided by the present invention, point five steps are carried out, and first make magnetic bead after activation be combined albumen with biotin labeled methyl and occur crosslinkedly, and acquisition methyl is in conjunction with albumen-magnetic bead mixture; Next hatches this mixture and fragmentation DNA; Then carry out wash-out with 160mM NaCl and 200mM NaCl successively, to remove non-methylate DNA; After removing non-methylate DNA, carry out again the wash-out of methylate DNA with 2000mM NaCl; Finally adopt ethanol precipitation to obtain the methylate DNA of enrichment, be further used for follow-up methylating in conjunction with protein sequencing.
In the time that the NaCl that utilizes different concns carries out gradient elution, along with increasing of NaCl concentration, the DNA methylation degree eluting is higher.The present invention, in to non-this step of methylate DNA wash-out, has been combined with two kinds of elutriants of 160mM NaCl and 200mM NaCl; First with 160mM NaCl to the fragmentation DNA after hatching be combined with methyl albumen-magnetic bead mixture mixture wash-out 4 times, to reach the object of the non-methylate DNA of removal; And then with 200mM NaCl wash-out 3 times, further non-methylate DNA is removed totally as much as possible.By above-mentioned two kinds of elution step that elutriant completes successively, remove more completely non-methylate DNA, retain methylate DNA, improved the ratio of methylate DNA in the DNA being finally enriched to, then the DNA obtaining with enrichment goes to build library order-checking.In the Insert Fragment in library, the shared ratio of non-methylate DNA is less, and in the final data that order-checking obtains, the shared data volume of methylate DNA part is larger, and fraction of coverage and the overburden depth in the region that correspondingly methylates are also larger, and the order-checking degree of depth is also larger.If, after non-methylate DNA wash-out, residual non-methylate DNA is more, will cause non-methylate DNA in library to increase in experiment, in final data, the partial data quantitative change of methylate DNA is little.So in the situation that obtaining same quantity of data, it is cleaner that non-methylate DNA is removed, the data volume in the region that methylates is larger, and the order-checking degree of depth is darker, and correspondingly cost also can reduce.
Preferably, in methylate DNA enriching method provided by the present invention, while carrying out the activation of magnetic bead in step (1), the amount that adds magnetic bead in DNA sample is 1 μ g DNA/10 μ l magnetic bead.
Preferably, in methylate DNA enriching method provided by the present invention, in step (1), make biotin labeled methyl in conjunction with albumen with activation after magnetic bead generation crosslinking reaction time, the protein-bonded amount of biotin labeled methyl using is that the biotin labeled methyl of 1 μ gDNA/7 μ l is in conjunction with albumen.
Preferably, in methylate DNA enriching method provided by the present invention, make fragmentation DNA be combined albumen-magnetic bead mixture with methyl while hatching in step (2), at room temperature hatch, incubation period is 1 hour.
Preferably, in methylate DNA enriching method provided by the present invention, in step (3), during with 200mMNaCl wash-out, the amount that at every turn adds 200mM NaCl is 200 microlitres.
Preferably, in methylate DNA enriching method provided by the present invention, in step (4) with the NaCl wash-out of 2000mM 3 times.
Preferably, in methylate DNA enriching method provided by the present invention, in step (5), the volumetric concentration of the ethanol using in ethanol precipitation is 70%, is dried and at room temperature carries out, and is less than 5 minutes time of drying.
Brief description of the drawings
Fig. 1 is the electrophorogram obtaining with the non-methylate DNA of the non-primer amplification that methylates, and wherein, on swimming lane, target numeral is the concentration of NaCl in elutriant, and unit is mM;
Fig. 2 is the electrophorogram obtaining with the primer amplification methylate DNA that methylates, and wherein, on swimming lane, target numeral is the concentration of NaCl in elutriant, and unit is mM.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, the embodiments of the present invention are explained in detail.But, persons of ordinary skill in the art may appreciate that in the each embodiment of the present invention, in order to make reader understand the application better, many ins and outs are proposed.But, even without these ins and outs and the many variations based on following embodiment and amendment, also can realize the each claim of the application technical scheme required for protection.
Embodiment 1 methylate DNA enrichment experiment example
Magnetic bead (
) preparation
Attention: magnetic bead
can not concuss, mixed with the soft method of beating of inhaling up and down with rifle, in order to use.
1.1Beads activation
1) using without the water of enzyme (is 800mM NaCl by 5X Bind/Wash Buffer, below use 5XBind/Wash Buffer be all 800mM NaCl)) be diluted to 1X Bind/Wash Buffer (be 160mM NaCl, below use 1X Bind/Wash Buffer be all 160mM NaCl).
2) mix with the soft method of beating of inhaling up and down with rifle
3) after adding and mix in the ratio of 1 μ gDNA/10 μ l Beads
in 1.5ml centrifuge tube.
4) in the 3rd step, add 1X Bind/Wash Buffer to make its cumulative volume reach 100 μ l, suction is beaten and is mixed up and down.
5) centrifuge tube is placed on to 1min on magnetic frame, in the time that all Beads are adsorbed on tube wall, abandons supernatant.
6) centrifuge tube is lifted down from magnetic frame, added 100 μ l1X Bind/Wash Buffer, suction is beaten and is mixed up and down.
7) repeat 5-6 and walk at least one times, this mixed solution will be used for the biotin labeled methyl of next step in conjunction with albumen (MBD-Biotin albumen) and
crosslinked.
1.2MBD-Biotin albumen with
crosslinked action
1) in 1 μ g DNA/7 μ l, (3.5 μ are the ratio of MBD-Biotin albumen g), and MBD-Biotin albumen is added to new 1.5ml centrifuge tube.
2) add 1X Bind/Wash Buffer to make its cumulative volume reach 200 μ l at 1.5ml centrifuge tube, suction is beaten and is mixed up and down.
3) the MBD-Biotin albumen of dilution is added the 7th step above activate
in, suction is beaten and is mixed up and down.
4), under room temperature condition, mixture is the soft 1h that rotates on blood mixer.
The wash-out of 1.3MBD-Beads
1) after mixed at room temperature 1h, centrifuge tube is positioned over to magnetic frame 1min, when all
while being adsorbed on tube wall, abandon supernatant.
2) centrifuge tube is taken out to magnetic frame, add 100 μ l1X Bind/Wash Buffer, beat and mix with rifle suction.
3) under room temperature condition, the soft 5min that rotates on blood mixer.
4) repeat 1-3 step at least twice.
5) centrifuge tube is positioned over to magnetic frame 1min, then abandons supernatant.
6) centrifuge tube is taken out to magnetic frame, add 100 μ l1XBind/Wash Buffer, suction is beaten and is mixed up and down, for experiment below.
The hatching of 2.MBD-Beads and fragmentation DNA
1) add 100 μ l5X Bind/Wash Buffer at new 1.5ml centrifuge tube.
2) by fragmentation DNA, (>1-10 μ g, l) in the centrifuge tube of upper step, (cumulative volume may have 40-400 μ l) to concentration=25ng/ μ.
3) add the water without enzyme, make its cumulative volume reach 500 μ l.
4) mixture of DNA and Buffer is transferred to above and included in MBD-Beads centrifuge tube in the 6th step, suction is beaten and is mixed up and down.
5), under room temperature condition, mixture is the soft 1h that rotates on blood mixer.
3. remove non-methylate DNA (Non-Captured DNA)
Prepare the Elution Buffer of 200mM, with High-Salt Elution Buffer (being 2000mM NaCl): Low-Salt Elution Buffer (containing NaCl)=1:9 mixing.
1) by being housed, MBD-Beads and the fragmentation DNA mixture centrifuge tube of having hatched be placed on 1min on magnetic frame, when all
while being adsorbed on tube wall, abandon supernatant.
2) centrifuge tube is taken off from magnetic frame, add the 160mM NaCl of 200 μ l, suction is beaten and is mixed up and down.
3), under room temperature condition, centrifuge tube is the soft 3min that rotates on blood mixer.
4) centrifuge tube is placed on to 1min on magnetic frame, in the time that all Beads are adsorbed on tube wall, abandons supernatant.
5) repeat 3-5 step three times.
6) centrifuge tube is taken off from magnetic frame, add 200 μ l200mM NaCl, suction is beaten and is mixed up and down.
7), under room temperature condition, centrifuge tube is the soft 3min that rotates on blood mixer.
8) centrifuge tube is placed on to 1min on magnetic frame, in the time that all Beads are adsorbed on tube wall, abandons supernatant.
9) repeat twice of 7-9 step.
Attention: when wash-out, MBD-Beads can not be dried
4. methylate DNA (Captured DNA) wash-out
1) after wash-out Non-Captured DNA, add 400 μ l HighSalt Elution Buffer (2000mMNaCl), suction is beaten and is mixed up and down.
2), under room temperature condition, centrifuge tube is placed on to the soft 3min of rotation on blood mixer.
3) centrifuge tube is placed on to 1min on magnetic frame, in the time that all Beads are adsorbed on tube wall, supernatant is proceeded in the centrifuge tube of a clean 1.5ml.
4) repeat to walk twice of 1-3 step.
Can guarantee that like this 95% DNA can be eluted.
5. ethanol precipitation
1), by the elutriant of the 1.2ml collecting, point install in the centrifuge tube that two new 1.5ml are clean every pipe.
2) in every pipe, add 1 μ l glycogen (Glycogen) (20 μ g/ μ l), 1/10 volume of3M NaAc, 2 times of volume 100% ethanol ethanol.
3) mix 80 DEG C of rear Fang Zhi Yu –, 2h.
4) under 4 DEG C or room temperature condition, >=12, the centrifugal 15min of 000 × g
5) abandon supernatant.
6) add 70% the ethanol that 500 μ l are cold
7) under 4 DEG C or room temperature condition, >=12, the centrifugal 5min of 000 × g
8) abandon supernatant
9) repeat 6-8 step
10) be at room temperature dried～5min (noting: not too dry)
11) add 25 μ l TE buffer wash-outs.Two pipe solution are merged into a pipe.
Elutriant can Chu Cun – 20 DEG C.
Embodiment 2 methylate DNA concentration effect confirmatory experiments:
Adopt methylate DNA and non-methylate DNA proportion in the qualitative wash-out part of method of following PCR:
What the length first K-562DNA of 1 μ g fragmentation being provided with test kit was 80bp methylate mixes with the non-double-stranded DNA that methylates (each 10pg), it is carried out to methylate DNA enrichment by the method described in embodiment 1, collecting is 160mM by concentration respectively, 200mM, 350mM, 450mM, 600mM, 1000mM, the DNA part that the NaCl of 1500mM and 2000mM elutes.
Then respectively taking the several DNA part that is enriched to as template, what provide with test kit increases with the corresponding primer of the double-stranded DNA that methylates (Forward Primer for Methylated DNA Control and Reverse Primer for Methylated DNA Control) and with the corresponding primer of non-methylate DNA (Forward Primer for Non-Methylated Control and Reverse Primer for Non-Methylated Control).
PCR system is as follows:
PCR program is as follows:
●94℃?2min
● 26 circulations:
—98℃?10?s
—60℃?30?s
—72℃?30?s
●72℃?5?min
●10℃?∞
After PCR finishes, from PCR product, get 5 μ l, for agarose gel electrophoresis.
Accompanying drawing 1 is for using and the corresponding primer of the non-methylate DNA electrophorogram that several DNA parts of being enriched to obtain that increases respectively, and Fig. 2 uses and the corresponding primer of the methylate DNA electrophorogram that several DNA parts of being enriched to obtain that increases respectively.
As can be seen from the figure: the main methylated DNA of right and wrong in the DNA eluting with the NaCl of 200mM, although also have residual non-methylate DNA in the DNA eluting with the NaCl of 350mM, but methylated DNA content sharply rises, and has accounted for larger ratio.And in test kit operation instructions originally, the DNA that the NaCl of 200mM is eluted also goes enrichment as methylated DNA, will cause non-methylate DNA in library to increase, in final data, the partial data quantitative change of methylate DNA is little.Correspondingly, the data volume in the region that methylates diminishes, and the order-checking degree of depth reduces, and cost also can raise.
Persons of ordinary skill in the art may appreciate that the respective embodiments described above are to realize specific embodiments of the invention, and in actual applications, can do various changes to it in the form and details, and without departing from the spirit and scope of the present invention.
Claims (10)
1. be applied to methyl in conjunction with the methylate DNA enriching method in protein sequencing, adopt methylate DNA enrichment test kit to carry out, it is characterized in that, comprise following step:
(1) activation of magnetic bead is combined protein-crosslinking and is reacted with methyl
Magnetic bead is activated, then make the magnetic bead generation crosslinking reaction of biotin labeled methyl in conjunction with albumen and after activating, obtain methyl in conjunction with albumen-magnetic bead mixture;
(2) methyl is in conjunction with the hatching of albumen-magnetic bead mixture and fragmentation DNA
Add methyl that above-mentioned steps (1) obtains in conjunction with in albumen-magnetic bead mixture fragmentation DNA, hatch, methylate DNA is combined specifically with mixture;
(3) remove non-methylate DNA
To being combined the mixture of albumen-magnetic bead mixture with methyl through the fragmentation DNA of hatching in above-mentioned steps (2), use 160mM NaCl is magnetic bead wash-out 4 times, then uses 200mM NaCl wash-out 3 times, removes non-methylate DNA;
(4) methylate DNA wash-out
To the removal obtaining in above-mentioned steps (3) mixture of non-methylate DNA, use 2000mMNaCl magnetic bead is carried out to wash-out, obtain methylate DNA component;
(5) ethanol precipitation
The methylate DNA component that adopts ethanol precipitation to obtain wash-out in above-mentioned steps (4) precipitates, and after being dried, dissolves with TE damping fluid, obtains the methylate DNA of enrichment.
2. methylate DNA enriching method according to claim 1, is characterized in that, while carrying out the activation of magnetic bead in described step (1), the amount that adds magnetic bead in DNA sample is 1 μ g DNA/10 μ l magnetic bead.
3. methylate DNA enriching method according to claim 1, it is characterized in that, in described step (1), make biotin labeled methyl in conjunction with albumen with activation after magnetic bead generation crosslinking reaction time, the protein-bonded amount of biotin labeled methyl using is that the biotin labeled methyl of 1 μ gDNA/7 μ l is in conjunction with albumen.
4. methylate DNA enriching method according to claim 1, is characterized in that, makes fragmentation DNA be combined albumen-magnetic bead mixture with methyl while hatching in described step (2), at room temperature hatches.
5. methylate DNA enriching method according to claim 1, is characterized in that, makes fragmentation DNA be combined albumen-magnetic bead mixture with methyl while hatching in described step (2), and incubation period is 1 hour.
6. methylate DNA enriching method according to claim 1, is characterized in that, in described step (3), during with 200mM NaCl wash-out, the amount that at every turn adds 200mM NaCl is 200 microlitres.
7. methylate DNA enriching method according to claim 1, is characterized in that, in described step (4) with the NaCl wash-out of 2000mM 3 times.
8. methylate DNA enriching method according to claim 1, is characterized in that, in described step (5), the volumetric concentration of the ethanol using in ethanol precipitation is 70%.
9. methylate DNA enriching method according to claim 1, is characterized in that, being dried in described step (5) at room temperature carried out.
10. methylate DNA enriching method according to claim 1, is characterized in that, be less than 5 minutes the time of drying in described step (5).
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| CN110734907A (en) * | 2019-09-17 | 2020-01-31 | 上海凌恩生物科技有限公司 | chloroplast DNA enrichment method |
| CN110791499A (en) * | 2019-09-17 | 2020-02-14 | 上海凌恩生物科技有限公司 | Enrichment method of mitochondrial DNA |
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| CN110791499A (en) * | 2019-09-17 | 2020-02-14 | 上海凌恩生物科技有限公司 | Enrichment method of mitochondrial DNA |
| CN115595372A (en) * | 2022-12-16 | 2023-01-13 | 南京世和基因生物技术股份有限公司（Cn） | Methylation detection method of plasma free DNA source, lung cancer diagnosis marker and kit |
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Application publication date: 20141119 |