CN105296655A - Method for detecting whether cis-acting element has methylated modification or not and method for detecting methylated modification sites - Google Patents
Method for detecting whether cis-acting element has methylated modification or not and method for detecting methylated modification sites Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, particularly to a method for detecting whether a cis-acting element has methylated modification or not and a method for detecting methylated modification sites. The starting points of the two methods belong to an integral inventive concept which comprises the following steps: performing targeted enrichment on the cis-acting element; performing enzyme digestion through using restriction enzyme of special recognition methylated sites; selecting a template for designing a primer according to the difference characteristics before and after enzyme digestion; amplifying the DNA segments which are subjected to enzyme digestion and are not subjected to enzyme digestion, so as to amplify the difference signal; comparing the difference information of two groups, so as to know the methylated modification sites of the cis-acting element and whether the cis-acting element has methylated modification or not. According to the method for detecting the methylated modification sites, the use amount of a used sample is less, the problems that sulfite interferes follow-up data analysis and the experimental error is relatively large can be reduced or avoided, and the experimental result is reliable; the method for detecting whether the cis-acting element has methylated modification or not is easy and feasible, and is high in accuracy.
Description
Technical field
The present invention relates to biology field, in particular to detecting the method for cis-acting elements with or without methylate modification or its decorating site.
Background technology
Transcription factor (transcriptionfactor) is the group energy trans-acting factor that particular sequence specificity is combined on DNA cis-acting elements, can ensure that goal gene is expressed in the specific time and space with specific intensity.Cis-acting elements (cis-actingelement) is present in the sequence that gene non-coding region can affect genetic expression.Eukaryotic cis-acting elements comprises promotor, enhanser, separaant etc., and their effect is the regulation and control participating in genetic expression.Cis-acting elements not coded protein itself, it provides an action site, carrys out regulate gene expression by interacting with trans-acting factor.
Cis-acting elements usually occurs DNA methylation (DNAmethylation), and DNA methylation is that a kind of epigenetic the most common changes.Large quantity research shows, DNA methylation can cause the change of chromatin Structure, DNA conformation, DNA stability and DNA and protein interaction mode, thus controlling gene is expressed.In human cell, the DNA base of nearly 1% there occurs and methylates.In ripe somatic tissue, DNA methylation generally betides CpG dinucleotide (CpGdinucleotide) position.Its function mainly can be summed up as following 4 aspects: the stability maintaining genome genetic material, the expression of regulatory gene, sets up epigenetic pattern and participates in differentiation, the growth course of cell and embryo.DNA methylation is expressed correct controlling gene and cell identity---the cell making cell have identical genetic material becomes neurocyte, muscle cell or skin cells, plays vital effect.
Some disease and the DNA methylation pattern that comprise cancer change relevant, and methylating on research cis-acting elements, can help us to understand transcription factor and associate with methylated, and help to develop some new therapys.
Carry out for the control region of known specific gene at present that high-throughout methylation analysis methods mainly sets up based on ChIP-sequencing method.But the initial ChIP-DNA of the method at least needs 100ng, if sample is comparatively rare, be usually not easy to obtain abundant amount.In addition; ChIP-sequencing also usually can apply to full-length genome bisulfite sequencing technologies when follow-up order-checking; U (methylated C occurring unaffected) is become by there is not methylated C base transition in genome; T is become after carrying out pcr amplification; make a distinction with the C base originally with the decorating site that methylates; again in conjunction with high throughput sequencing technologies, the complete genome DNA methylation profiles of single base discrimination rate can be drawn.But the method can introduce a large amount of sodium bisulfites, although can through desalting treatment, also can inevitably have sodium bisulfite to remain, for follow-up data analysis brings a lot of interference.
In addition, employing methylates chip technology or sequencing technologies, the particular combination of the DNA methylation decorating site on genome, transcription factor is measured respectively, utilizing information biology means to analyze relevant range, is also one of current control region common technology means of carrying out high-throughout methylation analysis for known specific gene.But its shortcoming is, this method is not cover all flow processs with same group of sample, and distribute in heterogeneous cell colony in an epigenetic modification site, the DNA methylation observed and the direct correlation of transcription factor have comparatively big error.
In view of this, special proposition the present invention.
Summary of the invention
The first object of the present invention is to provide a kind of cis-acting elements that detects to methylate the method for decorating site, can solve that sample consumption in traditional method is many, data analysis difficulty is large after sulfiting problem, certain cis functional element can be identified high-throughput and to methylate the site, place of modification.
The second object of the present invention is to provide a kind of cis-acting elements that detects with or without the method for modifying that methylates, and the method easy handling is cost-saving.
The starting point of two kinds of methods of the application all belongs to a total inventive concept, i.e. target enrichment cis-acting elements, carry out enzyme with the restriction enzyme of specific recognition methylation sites again to cut, difference characteristic before and after cutting with enzyme subsequently chooses template design primer, enzyme is cut the DNA fragmentation cut with non-enzyme to carry out respectively increasing to amplify difference signal, namely the different information comparing two groups is known that cis-acting elements methylates and is modified decorating site or it is with or without the modification that methylates.
In order to realize above object, spy of the present invention by the following technical solutions:
Detect cis-acting elements to methylate the method for decorating site, described method comprises the steps:
Target enrichment cis-acting elements, the small pieces segment DNA of known array is connected at the two ends of described cis-acting elements, carry out enzyme with the restriction enzyme of specific recognition methylation sites again to cut, subsequently with the primer that the small pieces segment DNA connected is stencil design, enzyme is cut the DNA fragmentation cut with non-enzyme to increase respectively, to amplify difference signal, namely the different information comparing two groups knows that cis-acting elements methylates decorating site.
Due to all unknown to the information of institute's enrichment cis-acting elements methylation sites, and usually need through interrupting ultrasonic for DNA at random in the experimentation of enrichment cis-acting elements, in order to obtain the information of all methylation sites on this cis-acting elements, need the small pieces segment DNA all two ends by the fragment interrupted all being connected known array, enzyme is cut the DNA fragmentation cut with non-enzyme and is increased respectively by the primer being stencil design with the small pieces segment DNA connected again, to ensure that all information comprising the fragment of methylation sites is all embodied.
The method, through DNA cloning Product management model signal, thus requires less to the consumption of initial DNA; The method, from the enrichment of DNA, is cut to connection, enzyme, is increased, until upper chip hybridization, same lot sample originally in used always being, thus respectively organizes experiment condition consistent, good stability, repeatable strong; This law needn't with the coupling of full-length genome bisulfite sequencing technologies, do not have sulphite remain.
Preferably, the as above cis-acting elements that detects methylates the method for decorating site, and described method specifically comprises the following steps:
1), target enrichment cis-acting elements from complete genome DNA;
2), at described complete genome DNA be connected the small pieces segment DNA of known array with the cis-acting elements two ends after enrichment, obtain Input-DNA and DNA-S respectively;
3), the restriction enzyme of DNA-S specific recognition methylation sites is carried out enzyme cut, obtain digestion products DNA-E;
4), with step 2) in the small pieces segment DNA of known array be template design primer, Input-DNA, DNA-S, DNA-E are increased;
5), by step 4) in the increase DNA that obtains carry out marking and hybridize with DNA chip respectively;
6), scan chip and add up the signal value of three core assembly sheets, find the strength of signal in Input-DNA group, DNA-S group all than the point that the strength of signal in DNA-E group has significance to raise, namely obtain the DNA element that specific transcription factor combines and to methylate decorating site information.
For convenience of description, what above-mentioned Input-DNA referred to is the product obtained after complete genome DNA two ends connect the small pieces segment DNA of known array; The product that the certain cis functional element two ends that what DNA-S referred to is after enrichment obtain after connecting the small pieces segment DNA of known array; What DNA-E referred to is the digestion products being carried out by the restriction enzyme of DNA-S specific recognition methylation sites obtaining after enzyme is cut.
According to the difference of selected chip type, the chip that methylates can cover the CpG site information that quantity does not wait, be exactly to determine that certain cis functional element methylates decorating site with the main purpose of the chip hybridization that methylates.Wherein, when analyzing methylation sites, the main of data carries out in DNA-S group and DNA-E group, and DNA-E group is cut due to enzyme, cannot increase, so signal can not or much more weak than DNA-S group.Input-DNA group is mainly used in doing positive control.
Because methylated DNA fragmentation is digested, make step 2) in the small pieces segment DNA of known array that inserts of two ends be also separated, follow-up amplification just cannot be carried out.That is in the chip hybridization process of follow-up DNA methylation DNA-E group no signal or signal very weak, can judge that certain cis functional element methylates decorating site thus.Because the specific DNA cis-acting elements of enrichment uses the antibody of associated transcription factor to carry out immunoprecipitation often, thus the present invention can be used to study DNA regulating and controlling sequence in different cell model, modulin (often for transcription factor) or histone modification and DNA methylation modify between relation.
Preferably, the method for described target enrichment cis-acting elements comprises chromatin immune chemical coprecipitation technique, the formaldehyde of controlling element assists isolation technique.
Chromatin immune chemical coprecipitation technique (ChromatinImmunoprecipitation, ChIP) ultimate principle is fixing protein-DNA mixture under viable cell state, and the chromatin small segment it is cut at random within the scope of certain length, then this complex body is precipitated by immunological method, the DNA fragmentation of enrichment target protein combination specifically, by to the purifying of object segment and detection, thus obtain protein and the interactional information of DNA.ChIP technology is widely adopted at present, is the method for the DNA fragmentation that the specific transcription factor of a kind of comparatively ripe target enrichment combines.
The formaldehyde of controlling element assists isolation technique (Formaldehyde-AssistedIsolationofRegulatoryElements, FAIRE) to be the new technology just set up in recent years, is applied to yeast cell at first, after be used to many cells.FAIRE experimentation comprises: cell or tissue cultivation, formaldehyde crosslinking, lysis, ultrasonic wave interrupt chromatin, then carries out phenol chloroform and prepares aqueous phase DNA.In FAIRE phenol chloroform process, the uncrosslinked DNA of albumen is soluble in the aqueous phase, and protein binding type DNA then stays two-phase interface.
Preferably, the as above cis-acting elements that detects methylates the method for decorating site:
In step 2) in, the concentration of the cis-acting elements after described complete genome DNA and enrichment is 1 ~ 10ng/ μ l, and consumption is 30 ~ 100ng;
In step 3) in, the concentration of described DNA-S is 2 ~ 10ng/ μ l, and consumption is 20 ~ 100ng;
In step 5) in, the DNA concentration for marking is 4 ~ 20ng/ μ l, and consumption is 1 ~ 5 μ g, and for the concentration of three groups of DNA that marks and consumption identical.
The method is through DNA cloning Product management model signal, thus require less to the consumption of initial DNA, traditional C hIP-sequencing method needs initial DNA usually to need more than 100ng, the minimum initial DNA of 30ng that only needs of this law can test, greatly reduce enrichment difficulty, save cost, be priorly reduction of experiment difficulty, be more suitable for the analysis and treament of rare sample.
Step 5) amount of DNA used determined by the follow-up specifically used chip type that methylates, rigorous for guaranteeing experiment, should be consistent for three groups of DNA concentration marking and consumption.
Preferred further, in step 3) in, described restriction enzyme is McrBC.
McrBC is a kind of endonuclease, and no matter methylcystein is that McrBC can cut on a side chain of DNA or on side chain.To unmethylated DNA, McrBC without effect.The recognition site of the upper McrBC of DNA is 5 ' ... Pu
mc (N
40-3000) Pu
mc ... 3 ', by two (G/A)
mthe half site composition of C form, the distance between these two half sites can reach 3kb, but the distance of the best is 55-103bp.The cutting of McrBC needs GTP, and when there is the analogue of GTP of non-hydrolysable, this enzyme can be attached on methylate DNA specifically, but can not cut.McrBC acts on a pair Pu
mcG sequential element, detects methylated CpGs at high proportion with this, but can not identify the HpaII/MspI site (CCGG) that internal cytosine has methylated.
It should be noted that the restriction enzyme that relates in the present invention is just preferably McrBC, this is because this enzyme has certain broad spectrum for the identification of methylation sites with shearing.This area researchist can choose different more special restriction enzymes according to concrete experiment purpose.
Preferably, in step 5) in, the method for described marker DNA is isotopic labeling or fluorescent substance mark.
The method of usual mark is method mark above radioactivity 32P/33P or the fluorescent substance through pcr amplification.Comparatively conventional by the method for fluorescent substance mark at present, advantage is reproducible, simple and safe operation.Common fluorescent substance is mainly Cy3/Cy5, and the two all belongs to 3H-indoles cyanine type dye, and Cy3 marks fluoresced green, and Cy5 mark sends out red fluorescence.
Preferably, the as above cis-acting elements that detects methylates the method for decorating site, and described DNA chip comprises full-length genome and to methylate chip, promotor chip, CpG island chip or enhanser chip.
The selection of chip experimentally object is determined, except commercialization chip, also can be used for the chip etc. that methylates of personalized customization.
Detect cis-acting elements with or without the method for modifying that methylates, described method comprises the steps:
Target enrichment cis-acting elements, carry out enzyme with the restriction enzyme of specific recognition methylation sites to cut, for template design primer, enzyme is cut the DNA fragmentation cut with non-enzyme with the DNA sequence dna of described cis-acting elements subsequently to increase respectively, to amplify difference signal, namely the different information comparing two groups knows that this cis acting is with or without the modification that methylates.
Preferably, the cis-acting elements that detects as above is with or without the method for modifying that methylates, and described method specifically comprises the following steps:
1), from complete genome DNA target enrichment cis-acting elements, obtain DNA-S ';
2), DNA-S ' is carried out enzyme with the restriction enzyme of specific recognition methylation sites cut, obtain digestion products DNA-E ';
3), with step 1) described in the DNA sequence dna of cis-acting elements be template design primer, DNA-S ', DNA-E ' are increased;
4), by step 3) in the increase DNA that obtains of DNA-S ', DNA-E ' carry out agarose gel electrophoresis and/or whether Real-time PCR Analysis exists the modification that methylates;
5), scan and add up gray-scale value and/or the Δ CT value of two groups of tracks, in strength of signal relatively in DNA-S ' group and DNA-E ' group, strength of signal has there was no significant difference, judges that DNA element that this specific transcription factor combines is with or without the modification that methylates thus.
For convenience of description, the certain cis functional element that what above-mentioned DNA-S ' referred to is after enrichment; What DNA-E ' referred to is the restriction enzyme of DNA-S ' specific recognition methylation sites is carried out enzyme to cut the digestion products carrying out obtaining after enzyme is cut.
Concrete, if known to the methylation sites information in studied cis-acting elements, then in step 3) in step 1) described in the DNA sequence dna of cis-acting elements when being template design primer, only need choose the DNA sequence dna design primer at the methylation sites two ends that will study;
If unknown to the methylation sites information in studied cis-acting elements, in step 3) in step 1) described in the DNA sequence dna of cis-acting elements when being template design primer, then need with the primers of this cis-acting elements 3 ' and 5 ' end (or design multipair primer cover this cis-acting elements total length), to ensure that the methylation state on all sites can detect.
Preferably, the cis-acting elements that detects as above is with or without the method for modifying that methylates:
The method of described target enrichment cis-acting elements comprises chromatin immune chemical coprecipitation technique, the formaldehyde of controlling element assists isolation technique;
Described restriction enzyme comprises McrBC, Msp1, HpaII, DpnII, or its corresponding isozyme coupling of Msp1, HpaII, DpnII.
Wherein, McrBC is general enzyme, no matter studied the known the unknown of methylation sites information in cis-acting elements all can adopt, cannot increase owing to there is methylated fragment, thus corresponding result determination methods is: have significance to raise if the strength of signal in DNA-S ' group compares with strength of signal in DNA-E ' group, then the DNA element of this specific transcription factor combination methylates modification; As two groups of indifferences, then the DNA element of this specific transcription factor combination is without the modification that methylates.
Msp1, HpaII, DpnII can only be used for the known situation of site information, and these enzyme identifications be known methylation sites not methylated time particular sequence, there is not methylated fragment cannot increase owing to only having, thus corresponding result determination methods is: have significance to raise if the strength of signal in DNA-S ' group compares with strength of signal in DNA-E ' group, then the DNA element of this specific transcription factor combination is without the modification that methylates; As two groups of indifferences, then the DNA element that this specific transcription factor combines methylates modification.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention and chip technology coupling, once experiment can determine the multiple methylation sites on cis-acting elements; Require less, cost-saving to the consumption of initial DNA, and be applicable to the analysis of rare sample; From the enrichment of DNA, cut to connection, enzyme, increase, until upper chip hybridization, same lot sample originally in used always being, thus respectively organizes experiment condition consistent, good stability, repeatable strong; Can distribute in heterogeneous cell colony in an epigenetic modification site, the DNA methylation observed and the direct correlation of transcription factor; Needn't with the coupling of full-length genome bisulfite sequencing technologies, do not have sulphite to remain, to the analysis of experimental result more accurately and reliably.
(2) the present invention can be used as detecting certain cis functional element with or without the method for modifying that methylates, and operation is simple and reliable, and repeatability is strong.
(3) because the specific DNA cis-acting elements of enrichment uses the antibody of associated transcription factor to carry out immunoprecipitation often, thus the present invention can be used to study DNA regulating and controlling sequence in different cell model, modulin (often for transcription factor) or histone modification and DNA methylation modify between relation.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 is ultrasonic rear DNA fragmentation size in embodiment 7 step 1.3, and wherein left several first swimming lane is marker, left several second and the 3rd swimming lane be ultrasonic after fragment;
Fig. 2 is the result of the DNA methylation of the method detection specific site that embodiment 6 step 5 enzyme is cut, result is presented in selected clone, HOXA9, LHX5, NKX2.5, the site modified that methylates such as PAX7 is digested disconnected, and the upper integrity still maintaining fragment because not methylating modification of the active house-keeping gene GAPDH expressed.
Fig. 3 adopts the method for ChIP-realtimePCR to verify ChIP result in embodiment 7 step 1.7; Wherein, IgG/NoAb is negative control, can see that DLL4, LHX5, PAX2 and PAX7 of combining with SUZ12 are higher relative to enrichment degree, and on the active house-keeping gene GAPDH expressed, the enrichment degree of SUZ12 is very low;
Fig. 4 adopts the method for agarose gel electrophoresis to verify ChIP result in embodiment 7 step 1.7; Shown band is HOXA9, LHX5, NKX2.5, PAX7DNA, wherein; Input is positive control, the DNA that IP precipitates for ChIP; NAB is negative control;
Fig. 5 is embodiment 7 step 6 chips results of hybridization figure, and wherein left figure is the ChIP-chip result of SUZ12, and right figure is the chip results of modifying and detecting that methylates of SUZ12 rich segment; Black part represents the positive signal site of P<0.01, and Dark grey part represents the positive signal site of P<0.05;
Fig. 6 is the checking to DNA methylation result in embodiment 7; Hollow dots represents the base methylating and modify, and solid dot represents the base methylating and modify.Wherein, 5-AZA is DNA methylation inhibitor, in contrast, uses the modification that methylates of this inhibitor rear section cell to be reversed.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, be and can buy by commercially available the conventional products obtained.
Embodiment 1
Detect the method for cis-acting elements methylation sites, described method comprises the steps:
1), target enrichment cis-acting elements from complete genome DNA;
2), at described complete genome DNA be connected the small pieces segment DNA of known array with the cis-acting elements two ends after enrichment, obtain Input-DNA and DNA-S respectively;
3), the restriction enzyme of DNA-S specific recognition methylation sites is carried out enzyme cut, obtain digestion products DNA-E;
4), with step 2) in the small pieces segment DNA of known array be template design primer, Input-DNA, DNA-S, DNA-E are increased;
5), by step 4) in the increase DNA that obtains carry out marking and hybridize with DNA chip respectively;
6), scan chip and add up the signal value of three core assembly sheets, find the strength of signal in Input-DNA group, DNA-S group all than the point that the strength of signal in DNA-E group has significance to raise, namely obtain the DNA element that specific transcription factor combines and to methylate decorating site information.
Embodiment 2
Detect cis-acting elements with or without the method for modifying that methylates, described method specifically comprises the following steps:
1), from complete genome DNA target enrichment cis-acting elements, obtain DNA-S ';
2), DNA-S ' is carried out enzyme with the restriction enzyme of specific recognition methylation sites cut, obtain digestion products DNA-E ';
3), with step 1) described in the DNA sequence dna of cis-acting elements be template design primer, DNA-S ', DNA-E ' are increased;
4), by step 3) in the increase DNA that obtains of DNA-S ', DNA-E ' carry out agarose gel electrophoresis and whether analysis exists the modification that methylates;
5), scan and add up the gray-scale value of two groups of tracks, if the strength of signal in DNA-S ' group compares with strength of signal in DNA-E ' group significant difference, then the DNA element that this specific transcription factor combines methylates modification; As two groups of no difference of science of statistics, then the DNA element of this specific transcription factor combination is without the modification that methylates;
Embodiment 3
A kind of method detecting CRE (cAMPresponseelement, cAMP response element) gene methylation decorating site in hippocampus of rats, described method comprises the steps:
1), anti-CREB antibody is utilized, with chromatin immune technology target enrichment CRE-DNA from hippocampus of rats genomic dna;
2), get hippocampus of rats genomic dna and enrichment after each 100ng of CRE-DNA (concentration is 10ng/ μ l), two ends connect the small pieces segment DNA of known array, obtain Input-DNA and DNA-S respectively;
3), the DNA-S McrBC that gets 100ng (concentration is 10ng/ μ l) carries out enzyme and cuts, and obtains digestion products DNA-E;
4), with step 2) in the small pieces segment DNA of known array be template design primer, Input-DNA, DNA-S, DNA-E are increased;
5), by step 4) in increase the DNA (each 10 μ g, concentration 20ng/ μ l) that obtains carry out fluorescent substance mark and respectively with promotor chip hybridization;
6), scanning chip add up the signal value of three core assembly sheets, find the strength of signal in Input-DNA group, DNA-S group all than the point that the strength of signal in DNA-E group has significance to raise, the DNA element namely obtained methylates decorating site information.
Described significance raises the setting of threshold value used because of concrete research object, and selected high throughput testing strategy and different.
Embodiment 4
A kind of method (this gene methylation site is unknown) detecting MEIS1 gene regulatory elements in the differentiation-inducing process of human monocytemacrophage and modify with or without methylating, described method comprises the steps:
1), in the differentiation-inducing front and back of human monocytemacrophage from genomic dna, assist the enrichment of isolation technique target to have the DNA element of potential adjusting function with the formaldehyde of controlling element, obtain DNA-S ';
2), getting the DNA-S ' of 100ng (concentration is 10ng/ μ l) with identifying that the McrBC of methylation sites carries out enzyme and cuts, obtaining digestion products DNA-E ';
3), with the sequence of aim sequence DNA3 ' and the 5 ' end on MEIS1 gene for template design primer, DNA-S ', DNA-E ' are increased;
4), by step 3) in the increase DNA that obtains of DNA-S ', DNA-E ' carry out agarose gel electrophoresis and whether analysis exists the modification that methylates; Analytical procedure is specially:
Add up the gray-scale value of two groups of tracks, have significance to raise if the strength of signal in DNA-S ' group compares with strength of signal in DNA-E ' group, then the DNA element that this specific transcription factor combines methylates modification; As two groups of indifferences, then the DNA element of this specific transcription factor combination is without the modification that methylates.
Embodiment 5
A kind ofly detect CD21 gene regulatory elements in the differentiation-inducing process of human myeloma with or without the method (this gene methylation site is known) of modifying that methylates, described method comprises the steps:
1), in the differentiation-inducing front and back of human monocytemacrophage from genomic dna, assist the enrichment of isolation technique target to have the DNA element of potential adjusting function with the formaldehyde of controlling element, obtain DNA-S ';
2), the DNA-S ' that gets 50ng (concentration is 5ng/ μ l) carries out enzyme respectively with the Msp1 and isozyme EcoR1 thereof that identify methylation sites and cuts, and obtains digestion products DNA-E ' 1 and DNA-E ' 2;
3), with the DNA sequence dna at the methylation sites two ends on CD21 gene for template design primer, DNA-E ' 1, DNA-E ' 2 are increased;
4), by step 3) in the increase DNA that obtains of DNA-E ' 1, DNA-E ' 2 carry out agarose gel electrophoresis and whether analysis exists the modification that methylates; Analytical procedure is specially:
Add up the gray-scale value of two groups of tracks, have significance to raise if the strength of signal in DNA-E ' is 1 group compares with strength of signal in DNA-E ' 2 groups, then this specific DNA element methylates modification; Otherwise as, then this specific DNA element is without the modification that methylates.
Embodiment 6
In a kind of people's of detection follicular lymphoma cell, LHX5 gene regulatory elements is with or without the method (this gene methylation site is known) of modifying that methylates, and described method comprises the steps:
1), extract people's follicular lymphoma cell genomic dna and obtain DNA-S ';
2), getting the DNA-S ' of 50ng (concentration is 5ng/ μ l) with identifying that the McrBC of methylation sites carries out enzyme and cuts, obtaining digestion products DNA-E ';
3), with the DNA sequence dna at the methylation sites two ends on LHX5 gene for template design primer, DNA-S ', DNA-E ' are increased;
4), by step 3) in DNA-S ', the DNA-E ' DNA obtained that increases carry out agarose gel electrophoresis, electrophoresis result is as described in Figure 2.
Analyze and whether there is the modification that methylates.Analytical procedure is specially: the gray-scale value adding up two groups of tracks, result is presented at strength of signal in DNA-S ' group (the second swimming lane) and compares with strength of signal in DNA-E ' group (the first swimming lane) and have significance to raise, then the DNA element of this specific transcription factor combination methylates modification;
Simultaneously by step 3) in DNA-S ', the DNA-E ' DNA obtained that increases carry out real-time quantitative PCR.
Analyze and whether there is the modification that methylates.Analytical procedure is specially: add up two groups of Δ CT values, the result strength of signal be presented in DNA-S ' group compares with strength of signal in DNA-E ' group has significance to raise, the DNA element that then this specific transcription factor combines methylates modification, and this result is consistent with electrophoresis the result.
Embodiment 7
The DNA methylation high throughput testing of follicular lymphoma cell SUZ12 binding site.
1, chromatin imrnunoprecipitation
1.1, cell is crosslinked
Get about 5 × 10
6follicular lymphoma cell (RL cell), the formaldehyde with 1% fixes 10 minutes, the glycine process of 0.125M 5 minutes, and centrifugal 5 minutes of 400g, PBS wash, centrifugal 5 minutes of 400g.
1.2, the cracking of cell
In Mute mixer, with 1ml cell pyrolysis liquid (containing proteinase inhibitor), 4 DEG C process 1 hour, centrifugal 5 minutes of 600g.250 μ l nucleus lysate (containing proteinase inhibitor), 4 DEG C process 1 hour.
1.3, ultrasonic
Utilizing Ultrasonic Cell Disruptor, being interrupted by chromatin dna to being less than 1000bp, master tape is at about 500bp, and the electrophoresis detection after fragmentation, detected result as shown in Figure 1.
1.4, the enrichment of differential protein-DNA complex
Get the chromatin dna of 1/200, preserve as Input.Hatch with chromatin dna in Mute mixer with anti-SUZ12 antibody, 4 DEG C are spent the night.Next day, with the DNA fragmentation that ProteinA/GAgarose enrichment is special.2000rpm is centrifugal.With the washing of gradient wash liquid, each 5 minutes.Be resuspended in TE damping fluid.With sodium bicarbonate buffer liquid process 2 times, each 30 minutes, the centrifugal absorption supernatant of 12000rpm.
1.5, solution is crosslinked
Respectively by the DNA of SUZ12 enrichment, InputDNA, IgGDNA, add pH6.5Tris, RNaseA, hatches 5 hours or spend the night in 65 DEG C of metal baths, then hatches 2 hours with Proteinase K and 37 DEG C.
1.6, the purifying of DNA
With the concentrated and purified test kit of the DNA of Zymo company (article No. D4003), purifying is carried out to DNA.
1.7, DNA quantitatively with checking
Priority NanoDrop and Picogreen carries out quantitatively to DNA;
Verify ChIP result by the method for ChIP-PCR and agarose gel electrophoresis, the result as shown in figures 2-3.
2, the connection of general junction fragment
Get the special enriched DNA fragments of InputDNA and 30ng of 10ng (concentration equal 1ng/ μ l) respectively, according to test kit (Sigma, article No. WGA2) illustrate, two ends are connected into DNA fragmentation, by product called after Input and the SUZ12-S respectively obtained.
3, enzyme is cut
Get 20ngSUZ12-S (concentration 2ng/ μ l), hatch 2 hours with restriction enzyme McrBC (NEB company) in 37 DEG C.Cross column purification DNA (Invitrogen, article No. K3100-01), by the product called after SUZ12-E obtained.
4, whole genome amplification
The whole genome amplification test kit of Sigma company (article No. WGA2) is utilized to be increased by three groups of DNA.
5, the fluorescent mark of DNA fragmentation and chip hybridization
Utilize Invitrogen test kit, for Input/SUZ12-S, and SUZ12-S/SUZ12-E, four samples, point two groups, work, gets the DNA (concentration 10ng/ μ l) of 5 μ g respectively, carries out the mark of Cy5, Cy3.The DNA marked is added in pairs the double-colored chip of Aglient (article No. G4492A), be placed in hybrid heater and spend the night, wash.
6, chip detection and analysis
By the chip scanning of having hybridized, and carry out statistical study by information biology means.
In this example, the result that chip obtains after overscanning, uses ChIP analysis software, according to central zone and the P (X being close to fragment
bar) and P (X) numerical value, at A) determine positive site, B in Input/SUZ12-S chip) SUZ12-S/SUZ12-E determines negative positions; SUZ12 is in conjunction with DNA be simultaneously at A with methylated site) positives and at B) in the fragment of feminine gender.To the analysis chart of chip hybridization results as shown in Figure 4.Because high throughput testing exists false positive results usually, also need the checking to DNA methylation result in embodiment 7, the result as shown in Figure 5.
The present invention requires less to the consumption of initial DNA, from the enrichment of DNA, cuts to connection, enzyme, increases, until upper chip hybridization, same lot sample originally in used always being, thus respectively organizes experiment condition consistent, good stability, repeatable strong; By with the chip technology coupling that methylates, once experiment can determine on cis-acting elements multiple methylation sites, and does not have sulphite to remain in experiment, does not affect follow-up analysis of experimental data; The present invention also can be used as detecting certain cis functional element with or without the method for modifying that methylates, and operation is simple and reliable, is the method for a kind of detection cis-acting elements be worthy to be popularized with or without methylate modification and decorating site thereof.
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1. detect cis-acting elements to methylate the method for decorating site, it is characterized in that, described method comprises the steps:
Target enrichment cis-acting elements, the small pieces segment DNA of known array is connected at the two ends of described cis-acting elements, carry out enzyme with the restriction enzyme of specific recognition methylation sites again to cut, subsequently with the primer that the small pieces segment DNA connected is stencil design, enzyme is cut the DNA fragmentation cut with non-enzyme to increase respectively, to amplify difference signal, namely the different information comparing two groups knows that cis-acting elements methylates decorating site.
2. the as claimed in claim 1 cis-acting elements that detects methylates the method for decorating site, and it is characterized in that, described method specifically comprises the following steps:
1), target enrichment cis-acting elements from complete genome DNA;
2), at described complete genome DNA be connected the small pieces segment DNA of known array with the cis-acting elements two ends after enrichment, obtain Input-DNA and DNA-S respectively;
3), the restriction enzyme of DNA-S specific recognition methylation sites is carried out enzyme cut, obtain digestion products DNA-E;
4), with step 2) in the small pieces segment DNA of known array be template design primer, Input-DNA, DNA-S, DNA-E are increased;
5), by step 4) in the increase DNA that obtains carry out marking and hybridize with DNA chip respectively;
6), scan chip and add up the signal value of three core assembly sheets, find the strength of signal in Input-DNA group, DNA-S group all than the point that the strength of signal in DNA-E group has significance to raise, namely obtain the DNA element that specific transcription factor combines and to methylate decorating site information.
3. the as claimed in claim 1 or 2 cis-acting elements that detects methylates the method for decorating site, it is characterized in that, the method for described target enrichment cis-acting elements comprises chromatin immune chemical coprecipitation technique, the formaldehyde of controlling element assists isolation technique.
4. the as claimed in claim 2 cis-acting elements that detects methylates the method for decorating site, it is characterized in that:
In step 2) in, the concentration of the cis-acting elements after described complete genome DNA and enrichment is 1 ~ 10ng/ μ l, and consumption is 30 ~ 100ng;
In step 3) in, the concentration of described DNA-S is 2 ~ 10ng/ μ l, and consumption is 20 ~ 100ng;
In step 5) in, the DNA concentration for marking is 4 ~ 20ng/ μ l, and consumption is 1 ~ 5 μ g, and for the concentration of three groups of DNA that marks and consumption identical.
5. the as claimed in claim 1 or 2 cis-acting elements that detects methylates the method for decorating site, and it is characterized in that, described restriction enzyme is McrBC.
6. the as claimed in claim 2 cis-acting elements that detects methylates the method for decorating site, it is characterized in that, in step 5) in, the method for described mark is isotopic labeling or fluorescent substance mark.
7. the as claimed in claim 2 cis-acting elements that detects methylates the method for decorating site, it is characterized in that, described DNA chip comprises full-length genome and to methylate chip, promotor chip, CpG island chip or enhanser chip.
8. detect cis-acting elements with or without the method for modifying that methylates, it is characterized in that, described method comprises the steps:
Target enrichment cis-acting elements, carry out enzyme with the restriction enzyme of specific recognition methylation sites to cut, for template design primer, enzyme is cut the DNA fragmentation cut with non-enzyme with the DNA sequence dna of described cis-acting elements subsequently to increase respectively, to amplify difference signal, namely the different information comparing two groups knows that this cis acting is with or without the modification that methylates.
9. the cis-acting elements that detects as claimed in claim 8 is with or without the method for modifying that methylates, and it is characterized in that, described method specifically comprises the following steps:
1), from complete genome DNA target enrichment cis-acting elements, obtain DNA-S ';
2), DNA-S ' is carried out enzyme with the restriction enzyme of specific recognition methylation sites cut, obtain digestion products DNA-E ';
3), with step 1) described in the DNA sequence dna of cis-acting elements be template design primer, DNA-S ', DNA-E ' are increased;
4), by step 3) in the increase DNA that obtains of DNA-S ', DNA-E ' carry out agarose gel electrophoresis and/or whether Real-time PCR Analysis exists the modification that methylates;
5), scan and add up gray-scale value and/or the Δ CT value of two groups of tracks, in strength of signal relatively in DNA-S ' group and DNA-E ' group, strength of signal has there was no significant difference, judges that DNA element that this specific transcription factor combines is with or without the modification that methylates thus.
10. detect cis-acting elements as claimed in claim 8 or 9 with or without the method for modifying that methylates, it is characterized in that:
The method of described target enrichment cis-acting elements comprises chromatin immune chemical coprecipitation technique, the formaldehyde of controlling element assists isolation technique;
Described restriction enzyme comprises McrBC, Msp1, HpaII, DpnII, or its corresponding isozyme coupling of Msp1, HpaII, DpnII.
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