CN103080335B

CN103080335B - Methods for selecting methylation markers

Info

Publication number: CN103080335B
Application number: CN201180034459.8A
Authority: CN
Inventors: A·P·米德; M·J·威尔金森; C·M·R·罗佩茨
Original assignee: Aberystwyth University
Current assignee: Aberystwyth University
Priority date: 2010-05-12
Filing date: 2011-05-12
Publication date: 2015-04-29
Anticipated expiration: 2031-05-12
Also published as: AU2011251767A1; GB201007944D0; EP2580347A1; CN103080335A; BR112012028957A2; JP2013526271A; WO2011141711A1

Abstract

The present invention relates to a method of identifying sets of DNA loci for identifying atypical growth conditions and for identifying the identity or source of cells of interest. The method can include identifying a plurality of sets of DNA loci that may identify a plurality of atypical growth conditions in cells of different origin and/or genotype. There is also provided sets of DNA loci and uses thereof.

Description

Select the method for methylation signature

The priority of the GB Patent Application No. 1007944.0 of application claims submission on May 12nd, 2010.The content of GB Patent Application No. 1007944.0 is included in herein by introducing in full.

The present invention relates to compilation and use the method being arranged in biological genome DNA target DNA sequence dna group, for diagnosing and the exposure of identification of organism sample experience and/or the kind or the source that relate to tissue or cell.

All biological tissues (the many cells eukaryote from individual cells to complexity and animal organism) continue to be exposed to the envirment factor affecting cell processes.These envirment factors can adopt much different exposure forms, comprise physics, chemistry, biology (as insect and disease), virus and radiation exposure.When these expose the effective efficiency reducing biological tissue, be called and invite sharp (stress).

In its most broad sense, invite to swash to be defined as and depart from to biological tissue or the individual biology of effective efficiency optimum and any of non-biological conditions.

Therefore, when for context of the present invention, one or more are invited to swash to comprise and abioticly invite sharp and biological and growth to invite sharp (example includes but not limited to be exposed to disease and endogenous " system decomposition " the disease such as cancer and aging of pathogen-mediated).

Such as, several author lists affect in a large number plant abiotic and invites sharp, and also illustrates and be exposed to these and invite the physiological reaction (as Orcutt and Nilsen2000) swashing and induce; Padmanabh2005).Similarly, the environment of the job description of being write by Bijlsma and Loeschcke (1997) broad range is invited and is swashed and have studied its impact on evolutionary process.Goh etc. (2007) list a large amount of human disease and invite sharp, and report its impact on gene expression.Hruz etc. (2008) provide in several animal and plant species (comprising people, mouse and arabidopsis (Arabidopsis)) at Genevestigator website (https: //www.genevestigator.com) and invite sharp extensive list.

Abioticly invite that sharp example includes but not limited to heat, infiltration, physical trauma, nutritional deficiency, chemical induction invite and to swash and physiology is invited and swashed as toxin, prevention, chemical fertilizer and treatment or recreational drug.

Biological and growth invites sharp example to include but not limited to be exposed to the disease of pathogen-mediated, infestation, endogenous " system decomposition " disease, such as cancer and aging.

Also there is tradition to invite and swash the example of giving some health biological or other benefits, as the positive is invited sharp.A lot of positive invites sharp example to comprise the research of (2002) such as Callaway, described research shows in the phytobiocoenose of Alps, body adjoins another and gives healthy advantage one by one, instead of invites to plant-plant competition and swash more often relevant cost.Dhabhar and McEwen (1998) shows duration short mortifier and significantly strengthens delayed allergy in application on human skin, and invites the leucocyte swashing induction can mediate to skin transport the Immune-enhancing effect observed in research described in supposition.Poss and Tonegawa (1997) have studied adjustment and the activity of mouse Heme oxygenases-1 (Hmox1); Hmox1 is inviting the classical gene raised in sharp mammalian cell.Provide evidence show this gene upregulation give cell to oxidation invite sharp protection and therefore give from invite swash expose positive benefit.

In view of the performance of impact any biosystem optimum quantity of parameters and almost exist the congenital variable properties of zoic environment, each bion run in its life one or another kind of form to invite sharp be inevitable.

Tolerate (inviting sharp for viable suboptimum) by combination and/or avoid (swashing with also sharp for much reducing inviting of operating efficiency for not tolerant all inviting), single biological energy source adapts to invite sharp impact.

When biology run into unescapable invite sharp time, it invites sharp mechanism to relate to biological adaptation described in overcoming, usually be considered as the change (Madlung etc., 2004) of metabolic pathway, general use system redundancy and introducing/strengthen showing better path under New Terms.This change can realize in many ways, comprise carry out regulatory gene by the difference cytosine methylation inner at regulatory region upstream and gene itself transcribe control (as Aceituno etc., 2008).

Any given inviting is swashed the adjustment that the physiological change that causes is not limited to affect one or more initial acceptor genes (invite described in detection sharp those) and is changed, and also comprises the cascade reaction of other genes (stress response gene and stress response gene path) changed as receptor change result.

Sizable redundancy is had by the different stress response gene/paths inviting sharp elicitor to introduce.Such as, jasmonic path participates in arabidopsis to biological and abioticly invite sharp reaction (this summary acted on see Devoto and Turner2005 and Wasternack, 2007).

DNA methylation seems extensively to cross over a lot of eucaryon group, comprises plant, mammal, bird and invertebrate as nematode (Finnegan etc., 1998; Suzuki S etc., 2007; Gupta S etc., 2006; DelGaudio R etc., 1997).In plant, DNA methylation is connected with gene silencing, such as, and hyper-methylation allele (Jacobson and Meyerowitz, 1997 of gene SUP, PAI and AG in arabidopsis (Arabidopsis thaliana); Melquist etc., 1999; Jacobson etc., 2000) LCYC (Cubas etc., 1999) and in toodflax (Linaria).But, show ectopic expression (Kinoshita etc., 2004) at the hyper-methylation of arabidopsis allelic AP3 and FWA.The effect (Finnegan etc., 2000) of several report prompting DNA methylation in development of plants and cell type regulate.

Also known dna methylates and to work in the mammiferous epigenetic controlling gene comprising people is expressed, and can be connected with some organization type.The methylation measuring specific gene group DNA targeting regions interested in the field of study in useful.

In people and other mammals, find that methylcystein almost accounts for leading in cytosine-guanine (CpG) dinucleotides.Genomic DNA methylation level plays an important role in Gene regulation.Observe in human malignant lesion is as cancer the earliest with in modal gene alteration, atypical the methylating of so-called CpG island (being particularly positioned at gene 5 ' regulatory region Nei CpG island) causes the change of this gene expression.

Contact between DNA methylation and Gene regulation causes sizable research interest, by the specificity state that the appearance and degree that define the DNA methylation in site in use genome are in progress to diagnose growth as diagnostic tool, the kind of cell, tissue or organ, disease invites beginning or the exposure of sharp form with other.

Described stress reaction path itself affects methylating of gene, and relevant with the regulatory region maintained or operate in path of running one's home.Relevant with these gene expressions any methylate to change unlikely diagnose separately accurately inviting of causing it activate sharp, because much difference is invited and sharply can be activated identical stress response gene.As visible herein, this site is not almost worth in the reaction induction of demethylation (methylate or) directly assignment cause (single invite sharp), because several different inviting sharply can cause change identical in DNA methylation.For this reason, use DNA methylation to diagnose the illness or other invite the effort of sharp form to focus on from the beginning inquiry separately or common diagnosis specified disease or invite sharp site, have each disease or invite the novel site swashing set.

There is the research example much establishing and contact between single or multiple sites DNA methylation and particular person disease in gene or associated adjustment district.These comprise leukaemia; Head and neck cancer; Hodgkin's disease; Cancer of the stomach; Prostate cancer; Kidney; Carcinoma of urinary bladder; Breast cancer; Burkitt lymphoma; Wilms' tumor; Handkerchief-Er Shi Wei (Prader-Willi)/Angleman (Angelman) syndrome; ICF syndrome; Histiocytoma; Hypertension; Paediatrics Neurobiology; Autism; Ulcerative colitis; Fragile X mental retardation and Huntington disease.See such as:

Also identify in other (inhuman) higher organism genomes and swash and the methylating of specific site of disease association with specific inviting.Example comprises:

In tobacco plant (tobacco (Nicotiana tabacum)), the silence of I type transmethylase NtMET1 causes methylated reduction in genome.Wada etc. (2004) show about 30 gene upregulation, exceed half and participate in directly biological or abioticly invite sharp response.In addition, Choi etc. (2007) also show in tobacco plant, the methylation patterns of pathogen responsive genes NtGPDL changes in 24 hours in artificial infection tobacco mosaic virus (TMV) (TMV), and this change is relevant with the hypersensitivity (HR) of plant.

In corn (corn (Zeamays)), low temperature is invited to swash and is seemed to reduce transcribing of transmethylase, and also impliedly reduces DNA methylation (Stewart etc., (2000)).In the latter's research, (Stewart etc., (2002) identical group use HPLC analyze with confirm low temperature invite sharp cause always to methylate decrease beyond 10%, but seem to be confined to Ac/Ds transposons.

The also participation high concentration heavy metal that methylates invites sharp tolerance.Such as, Lee etc. (1998) display list reveals methyl transferase activity increases the hamster cell system of (methylating with therefore) more and shows heavy metal resistance and improve.

In biological context with invite the summary of sharp relevant methylated genes seat see Peng and Zhang (2009) and Hruz etc.,, and Genevestigator website (https: //www.genevestigator.com) (2008).

With regard to diagnostic purpose, use the method for DNA methylation pattern to be the qualification of present level target in prior art and use locus to diagnose individual environmental exposure, namely specific diseases, growth are in progress or specifically invite sharp particular state.WO2007/132166 describes following discovery: in pregnant woman, be derived from some gene (such as RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB21P, PTPN6, THY1, TMEFF2 and PYCARD) high methylation of fetus, and the homologous genes of maternal source does not methylate.Described research describes how detect one or more these methylated fetus gene in from the biological sample of pregnant woman, as the general indicant occurring foetal DNA in described sample.These fetal methylations mark is described as useful especially positive control with regard to non-invasive analysis process, monitors quality and the quantity of foetal DNA in described process.

US2006-0183128 (being also disclosed as WO2005/019477) describes the full-length genome epigenetic figure of qualification differentiated tissue epigenetic mark; Described scheme comprises the correlation between CpG site (MVP) and genome DNA sample type methylating variable.Epigenetic figure is such as identifying sample type or is distinguishing in two or more sample types have extensive use.The epigenetic characteristic analyzing one or one group nucleotide sequence in epigenetic figure background only can measure the source of described nucleic acid, instead of environmental exposure/invite sharp overview.

US2007-0292866 relates to and detects human gene group DNA and to methylate overall to change and in human genome, specific region methylates the method changed.Described method can be diagnosed, the treatment process of prognosis and monitoring disease, and detects the change that methylates that diet and/or food supplement cause.Described invite swash only can separately identify.

US2004-0029117 describes the general inventive identifying differential methylation DNA locus, can be used as the mark measuring biological effect and/or pharmaceutically active, chemical substance and/or pharmaceutical composition.Described medicine, chemical substance and/or pharmaceutical composition only can use respective epigenetic to mark research separately respectively.

Therefore, use up to now DNA methylation qualification specific invite swash be confined to one next invite separately the mark swashing and use in research, or source material (such as, tissue/organ/cell type) described in other or set up physiological status.

Therefore need to study the more effective system of inviting sharp scope, and particularly multiplely in single analysis invite sharp research.Ideally, described same analysis also should be able to distinguish source material and physiological status.

The invention provides the system of energy identified gene group DNA internal labeling group, can be used for jointly and repeatedly identifying that individuality, tissue, organ or cell are in inviting of certain limit sharp, and/or diagnosis sample institute contact invite sharp extensively or special properties, and/or diagnose physiological status or source (the such as cell type or organ) of biological sample.

Marker DNA sequence of the present invention is (interchangeable) ' DNA locus hereinafter referred to as ', ' outpost (Sentinel) ' and ' invite sharp outpost (Sentinel) ', and can invite sharp (comprising single biology, organ, tissue or cell) one or more of identification of organism sample experience.Invite sharp outpost respectively to comprise sequence motifs, described motif changes its methylation state of DNA (usually but need not fixed limit in cytosine residues), such as biology experience some invite swash after but be not experience other invite sharp; And/or Cell Differentiation becomes some cell type but is not other; And/or according to obtaining the organ or tissue of sample, and/or the physiological age of the growth of cell type, tissue, organ or individuality or state.In this mode, when complete group of consideration invites sharp outpost, the common mode of DNA methylation can identify whether one or more invite swash under operate biological sample, or biological sample is in one or more and invites sharp, and/or can be used for diagnosing the source of sample or the physiological status of source material.Sample contacts invite swash can be that known (as above by inviting sharp outpost to identify) or (the inviting sharp outpost not identify) of the unknown are invited sharp.By inferring the physiological status that sample (tissue, organ or cell type) is originated or source (such as, relevant to from liver cell DNA DNA methylation overview mate imply that sample is from liver sample) with the ratio compared with normal of standard reference samples.

The determinant attribute of sharp outpost is invited to be that neither one outpost diagnoses separately any one to invite sharp, source material or physiological status, and outpost can use one to invite sharp (or source material or physiological status) to identify, but when be applied to be in difference invite sharp sample (or source material or physiological status) time, still remain diagnosis attribute.This attribute provides significant raising compared with seeking to identify the existing system of methylation signature, and described mark is according to the investigation of target state comparing device condition adjudgement for certain limit, and diagnosis invites sharp/tissue/developmental condition separately.It is easily impaired when more extensive sampling that the shortcoming of existing scheme is described scheme, causes finding that different state/tissue/cell types thinks the identical methylation state of diagnosis target state before having.On the contrary, outpost's mark comprises this possibility and is therefore very unlikely subject to the impact that more extensively samples, and (unlike existing system) can diagnose unidentified reaction to be above difference.

Outpost itself can from or not from the DNA relevant with code area, regulatory region or introne, and can be relevant with the known change in gene expression or irrelevant.And outpost site can be positioned at the noncoding region irrelevant with gene expression, prerequisite be described site invite swash between the change of methylation state be make peace reliably.

The present invention is by referring to this attribute of outpost's DNA methylation mark group (evidence that maybe can occur) example in arabidopsis, mouse and people, described mark is invited sharp differential methylation to select (or changing responsive differential gene expression to methylating and differential methylation of inferring according to known) based on response one group, and display can be identified to be exposed to and invites sharp and be not used in the sample (or organization type) of Marker selection process.

The establishment method measuring correlation or cluster according to the binary overview generated by these marks can be used for providing some guides to newly inviting extensively classification belonging to sharp (as sharp in inviting of not training).These include but not limited to multi-variables analysis, such as principal coordinate analysis or diagnostic decision tree or decision tree (analyzing ecological data see such as AnalysingEcological Data() (2007), New York Springer Verlag company (Springer), 15th chapter, 259-264 page, ISBN978-0-387-45967-7).

Also describe and control outpost's mark group of stress response gene regulatory region derived from methylating of arabidopsis, its expression pattern (with therefore methylation state) can be distinguished at least 78 kinds and different invite sharp type.Some providing about the potential sensitivity of described scheme by several inviting as mild as a dove sharp (the individual change of <5 in 78 genes investigate by induction institute) indicate.

Also carry out example outpost principle by training arabidopsis gene seat, use from 78 initial set 40 genetic marker subgroups series and use these marks with quantitative frequency, wherein identify that new inviting swashs for unique (namely different from training group), but different from contrast and existing invite sharp identical (namely finding in training group) or with contrast identical (false negative).Also show the ability that outpost uses the people's gene Zuo Lai appraisement organization type of one group of known differential methylation in different tissues.Show for restricted groups group selection and use outpost, still can diagnostic organization, and be not used in the described outpost of qualification.Within a context, diagnosis refers to that identification of cell, tissue, organ or biological interior physiology and phenotypic status or its change.

In this description, describe embodiment in the mode writing clear and accurate description, but be intended to and should be understood that embodiment can adopt multiple combination or separately not depart from the present invention.

In this description, term " about " refers to add deduct 20%, more preferably adds deduct 10%, even more preferably adds deduct 5%, most preferably adds deduct 2%.

In this description, term " growth " is used to refer to the living environment of discovery or placement or cultured cell or tissue or biology.Such as, described term can relate to growth, life, environment and/or developmental condition.Term " growth " and " growth " refer to survival and/or the propagation of cell or tissue or biology in stage interested or research.

In a first aspect of the present invention, provide the method for qualification one group of DNA locus (inviting sharp outpost) with regard to qualification atypical growth condition (inviting sharp), comprise step:

I. under contrast growth conditions, interested cell sample is cultivated;

Ii, in the environment that there is at least one atypical growth condition, cultivates identical cells of interest sample described with step (i);

Iii. DNA isolation from each cell sample of (i) and (ii);

Iv. the methylation state of the methylated locus of energy in the DNA be separated in authentication step (iii);

V. compare from the methylation state of step (i) with (ii) the middle DNA be separated.

Vi. select the locus subgroup with following characteristics, the feature of wherein said subgroup is: in the DNA be separated in step (ii), the methylation state of these locus is different from the DNA be separated in step (i).

Vii. the locus subgroup selected in authentication step (vi).

Viii. the methylation state of selected subgroup locus is associated with atypical growth condition and/or cells of interest sample.

Alternatively, described method comprises step:

Ix. by the methylation state of selected genes seat subgroup with notbe associated for the atypical growth condition of Select gene seat and/or tissue/cell type and/or its combination.

In a second aspect of the present invention, providing the method with regard to identifying qualification many groups DNA locus (inviting sharp outpost) with regard to specific atypical growth condition (inviting sharp), comprising step:

I. under contrast growth conditions, cultivate multiple interested cell sample, wherein interested various kinds of cell has different genotype;

Ii in the environment that there is at least one atypical growth condition, multiple cells of interest sample described in incubation step (i);

Iii. DNA isolation from each cell sample of (i) and (ii);

V. comparison step (i) and (ii) or (i) methylation state with the middle DNA be separated of (iii) or (ii) and (iii).

Vi. select the locus subgroup with following characteristics, the feature of wherein said subgroup is: in the DNA be separated in step (ii), the methylation state of these locus is different from the DNA be separated in step (i) and/or (iii); And/or methylation state difference compared with (i) of these locus in the DNA be wherein separated in step (iii).

The locus subgroup selected in vii authentication step (vi).

Viii. locus subgroup and its methylation state are associated with the atypical growth condition and/or cells of interest sample being used for Select gene seat.

Alternatively, also in steps:

In a third aspect of the present invention, providing the method with regard to identifying qualification one group of DNA locus with regard to multiple atypical growth condition, comprising step:

I. under contrast growth conditions, interested cell sample is cultivated;

Ii cultivates the multiple cells of interest sample identical with step (i) in the environment that there is at least one atypical growth condition, and wherein each sample grows respectively under different atypical growth conditions;

Iii. DNA isolation from each cell/sample of (i) and (ii);

V. compare from the methylation state of step (i) with (ii) the middle DNA be separated;

Vi. select the locus subgroup with following characteristics, the feature of wherein said subgroup is: in the DNA be separated in each sample step (ii), the methylation state of these locus is different from the DNA be separated in step (i);

The locus subgroup selected in vii authentication step (vi);

Viii. locus subgroup and its methylation state are associated from the different atypical growth conditions of (ii).

Alternatively, other steps are had:

Preferably, each locus in described locus subgroup does not diagnose single illness.

Preferably, the methylation state diagnosis of selected genes seat subgroup is not for the atypical growth condition of Select gene seat and/or tissue/cell type and/or its combination.

Preferably, the methylation state of selected genes seat subgroup diagnoses the atypical growth condition and/or tissue/cell type and/or its combination that are used for Select gene seat.

Preferably, methylation state diagnosis (i) of selected genes seat subgroup notfor the atypical growth condition of Select gene seat and/or tissue/cell type and/or its combination; (ii) the atypical growth condition that Select gene seat is used and/or tissue/cell type and/or its combination.

The center of the method for all aspects of the invention be for the identification of with the standard selecting methylation sites (outpost), described site can be used for diagnosing exceed selection course used invite sharp, source material and/or physiological status.For this reason, preferably select usually different between condition and/or source sample, but be not only to the mark of single conditional diagnosis ideally.In this mode, the differential methylation pattern across outpost produces the state of diagnosis instead of some condition specific gene seat.Following table shows this principle.

Therefore, should understand in method of the present invention, preferably selected locus subgroup is made up of locus different between condition and/or source sample, but is not the diagnosis only to single condition and/or source sample.

In supposed situation, have 6 kinds to invite sharp (A-F) and 8 locus (1-8), wherein response is different invites sharp methylation state scoring for methylating (+) or do not methylate (-).Following table 1 (a) shows a kind of possible result.

Table 1-a invites sharp type (A-F)

In contained 8 locus, locus 6 and 7 is eliminated as potential outpost, because it to invite in sharp training group not change (i.e. locus 6 all methylate and locus 7 does not all methylate).

Locus 3 and 4 is change not, and is respectively a kind ofly invite sharp diagnosis (being respectively A and F).These may be thought of as outpost's material standed for, but are not desirable, because it seems instruction, one invites sharp/locus, and unlikely inviting in the overview of sharp (if having) at a lot of other plays an important role.

Not to be that any one is specific invite sharp diagnosis to remaining locus, but jointly to separate in (separation) training group all invites sharp (see the following form 1 (b)).

Table 1-b invites sharp type (A-F)

If introduce other subsequently to invite sharp (G, arrow), it can mark with selected outpost and distinguish mutually, not with eliminate those distinguishes, although this invites sharp not for selection course.

Therefore, desirable outpost is marked at roughly inviting in sharp/sample source of half and occurs, and in complementary fashion invite swash between " distinctiveness exist (segregate) ".

DNA methylation assay step completes by using any available method, include but not limited to: methyl-sensitive amplified fragments analyzes (MSAP), the variant of AFLP (AFLP), wherein, compare and use with splitting voluminous thing general introduction that Restriction Enzyme generates (see Xiong etc. 1999); With sodium hydrogensulfite process DNA, then cycle sequencing (see Frommer etc. 1992), Manganic pyrophosphate complex initiation (see Uhlmann etc. 2002), high flux single-molecule sequencing (as Zeschnigk etc. 2009), or qPCR (see Munson etc. 2007), HPLC (as Sandhu etc. 2009), mass spectrum (as lgarashi etc. 2009) or directly by high-resolution melting curve analysis (WO2009/010776).In one embodiment, methylation state uses (namely occur or disappear) with strict qualitative fashion.In another embodiment, methylation state is measured by the methylated quantitative data of locus, such as, by arranging decision threshold or using the difference analysis system (such as principal component analysis (PCO)) based on quantitative data.

It is standard technique that PCO analyzes, and wherein measures the variable across multidimensional, and extracts new axle, thus the first axle (being called the first main component) catches variable quantity maximum in one dimension.Second axle is captured in disalignment the second largest variable quantity crossing over several dimension, and is called the second main component.Therefore, described first and second compositions do not refer to any particular variables, and this is standard analytical protocol.

" interested cell sample " comprises the cell derived from body outer cell line, and derived from or form that biomaterial is such as organized, the cell of organ or all biological.

Growth of Cells in the outer or body of occlusion body of " cultivating interested cell sample " under condition.Such as, cell sample can in for the Suitable assays room environment division of Growth of Cells growth in vitro.Or cell sample can " in body " growth, i.e. interested individual biological (as plant, animal or human).

" contrast growth conditions " comprises the active implication controlling (i.e. specificity and adjustment) growth conditions or good qualification growth conditions.How contrast growth conditions is grown by cell sample and measures, and active control may more suitable growth in vitro condition and the collating condition of good qualification may be more suitable for growth conditions in vivo.But such was the case with for this.

' atypical growth condition ' refers to the growth conditions different from the condition of equivalence in contrast growth conditions, such as, compares the temperature that contrast growth conditions is higher.

Described ' atypical growth condition ' can including (but not limited to) the individual condition changed, such as temperature, pH, light expose, but also can comprise physiological status in all tissue or biological level, morbid state, biology be exposed to chemistry, medicine or biological reagent, etc.

' phenotype and/or physiological change ' comprises the measured change of any cell, tissue or full biological level.Such as, be exposed to high temperature can have and compare full biological level in the different observable effects of cellular level.

Described change can be qualitative (as physiological reaction) or quantitative (increase of existing phenotype or physiological property or minimizing).

Of the present invention second and the third aspect an embodiment in, step (ii) in multiple samples are 1-10, invite sharp condition for 000, preferred 10-500 invites sharp condition, and more preferably 30-100 invite sharp condition.

Preference as step (viii) in the cell sample distinguishing the sample of experience atypical growth condition and control sample or select for it of locus as described in qualification;

Either side in the present invention first, second or the third aspect, after step (vii), (viii) and optional step (ix), can other steps be carried out:

X. locus subgroup and its methylation state and cell sample or the phenotype obtained in the tissue of described cell sample, organ or biology and/or physiological reaction are associated.

In one embodiment, the method for the either side in first, second or the third aspect of the present invention can comprise optional step:

Xi. DNA isolation from cell sample, the growth conditions of wherein said cell is unknown;

Xii. the methylation state of step (vii) institute identified gene seat subgroup in the DNA be separated in authentication step (xi);

Xiii. the locus methylation state of qualification in (xii) is compared with the result of step (viii), thus whether the cell sample of determining step (xi) once went through atypical growth condition.

In another embodiment, in first, second or the third aspect of the present invention, the method for either side can comprise (after optional step (x) above) following optional step:

Xi. from cell sample DNA isolation, wherein when being in one or more atypical growth conditions, the phenotype of described cell and/or physiological reaction (or the tissue/biology in its source) the unknown.

Xii. the methylation state of step (vii) institute identified gene seat subgroup in the DNA be separated in authentication step (xi).

Xiii. the locus methylation state of qualification in step (vi) is compared with the result of step (x), thus determine whether described cell (or the tissue/biology in its source) answers known or unknown atypical growth condition and phenotype and/or physiological reaction occur.

Preferably, step (xii) goes back the type of the atypical growth condition that identification of cell was once gone through.

Preferably, step (xii) identifies the atypical growth condition of not tested in described cell experience step (ii).

Preferably, the such as method of step (viii) also identifies the physiological reaction type to the atypical growth condition that cell/tissue/organ/individuality exposes.

Preferably, step (xiii) identifies after once going through one or more atypical growth conditions different with (ii) cell correlated response from (i), and described cell (or its tissue of originating or biology) experienced by phenotype and/or physiological reaction.

Preferably, described contrast growth conditions is identical with the biology normal range (NR) of cells of interest growth conditions in vivo.Technical staff can understand cell type or the biology that normal growing conditions depends on research.The example of normal growing conditions is configured for the cultivation of designated cell system or tissue, or the open scheme of the species or genus be even used to specify or the more biology survival of high-class group.

For some in a lot of specific cells system used in the present invention, the example of normal growing conditions includes but not limited to:

Prostate cancer DU145, Alimirah F, waits (2006). " DU-145and PC-3humanprostate cancer cell lines express andogren receptor:implications for the androgenreceptor functions and regulations(DU-145 and PC-3 PC-3 express androgen receptor: the function of instruction androgen receptor and adjustment) " .FEBSLett.580 (9): 2294-300

Breast cancer MCF-7Dickson RB etc. (1986) " in Characterisation of estrogenresponsive transforming activity in human breast cancer cell lines(MCF-7, estrogen responds the qualification of activity of conversion) " .CancerRes.46:1707-13

Leukaemia THP-1Dickson RB etc. (1986) " in Characterisation of estrogenresponsive transforming activity in human breast cancer cell lines(MCF-7, estrogen responds the qualification of activity of conversion) " .CancerRes.46:1707-13

(1973) Morphology and growth such as neuroblastoma SHSY5Y Biedler JL, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuousculture (in Continuous Cultivation the growth and morphology of human neuroblastoma cells, oncogenicity and cytogenetics) .Cancer Res.33 (11): 2643-52

Osteocarcinoma Saos-2BiedlerJL etc. (1973) Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture (in Continuous Cultivation the growth and morphology of human neuroblastoma cells, oncogenicity and cytogenetics) .Cancer Res.33 (11): 2643-52

Rat SP12Greene LA and Tischler AS (1976). " Establishment of anoradrenergic clonal line of rat adrenal pheochromocytoma cells with respond tonerve growth factor(responds the foundation of the norepinephrine energy cloned cell line of the PC12 cells of nerve growth factor) " .Proc.Natl.Acad.Sci.U.S.A.73 (7): 2428-8

(2002) .Principles of Bone Biology(" bone biology principle " such as mouse MC3T3Bilezikian). Santiago: academic press (Academic Press) .1506

(1992) .Tobacco BY-2cell line as the " Hela " cell in the cell biology ofhigher plants(tobacco BY-2 cells systems such as tobacco plant BY-2Nagata t are as " Hela " cell in higher plant cell biology) .International Review ofCytology.132:1-30

The DNA locus number forming the subgroup comprising outpost's mark is preferably 1-5000 DNA methylation site in the genome of research, but preferably 10-1000 comprises the targeting regions of DNA methylation, and most preferably 20-100 comprise the targeting regions of DNA methylation.

Expediently, the different atypical growth conditions in the step (ii) of second aspect are non-convergency difference.Non-convergency difference refers to that atypical growth condition is not same or analogous type or kind.Such as, temperature and nutrient availability can regard as non-convergency difference condition; The different strains being exposed to identical Pathogen category then can not.

Described atypical growth condition can be selected from: temperature, physics invites the sharp (atmospheric pressure comprising increase or reduce, invite sharp to the direction of object, stir, gravity, physical stress), physical damnification, nutrient availability, be exposed to chemical toxicant, hormone or signaling molecule, be exposed to other chemical reagent, visible ray is invited sharp (spectral quality or intensity), be exposed to black light radiation (the such as X-ray on background level, gamma-rays, infrared light or ultraviolet light, microwave etc.), be exposed to pathogen or parasite, be exposed to other biological or other members mutually of the same race, be exposed to other biological, individual exudate, food grass (herbivory) or another biology take target organisms as food, water is invited sharp (too much or very little), be exposed to the signal or the condition that stimulate described object to enter rest stage, oxygen is invited and is swashed or be exposed to excessive or secondary good free radical, etc..

Preferably, the gene-correlation of the locus subgroup selected of step (vi) and cellular stress path component.Described locus can be gene itself or the regulatory region with one or more gene-correlation.

This locus can from the DNA extron relevant to the functional expression of conservative cellular stress path or regulatory region.The example of plant is including (but not limited to) some gene, and described gene directly and indirectly participates in jasmonic path and (such as sees Dempsey etc. 1999; Wasternack, 2007; Fan etc. 2007; Zhao etc. 2007; Reinbothe etc. 2009), NO pathway in cultured (such as seeing Wilson etc. 2008), salicylic acid path (Fan etc. 2007; Zabala etc. 2009), ethene path (such as seeing Zhao etc. 2007), abscisic acid path (Zabala etc. 2009; Fan etc. 2009), and participate in the gene (such as seeing Saltveit and Choi, 2007) of phenolic compound control.

The example of people and mammalian biological is including (but not limited to) the main transcriptional pathway of p53 (being such as shown in Millau etc. 2009); P13K path (is such as shown in Assinder etc. 2009; Maira etc. 2009), PTEN-AKT3 signal cascade reaction (such as seeing Madhunapantula and Robertson2009), endoplasmic reticulum stress response gene hire1p and its bond (Tirasophon etc. 1998) and RET transmembrane receptor and the cascade reaction gene (Myers and Mulligan2004) of being correlated with.

Expediently, the gene of described gene locus at least one component in each stress reaction path of cells of interest or regulatory region.

When described biology/tissue/cell type is in different atypical growth condition (typical case invite sharp), according to methylating, the clarity changed selects DNA locus (outpost) subgroup with uniformity and the methylation patterns in difference methylated outpost.DNA locus can common ' later ' diagnosis causing the overview that methylates change described in invite sharp.May identify that multiple induction invites sharp, because outpost's mark shows invite sharp complementation (inconsistent) reaction (or not reacting) to dissimilar.Described method makes multiple site as described biology or organ or tissue or cell type to the multiple outpost inviting the general physiological of sharp derivant to react.

Expediently, DNA locus is chosen to ' pervasive (generalists) ', and therefore can for the identification of widely uncorrelated invite sharp.Invite that to swash diagnosis be based on the aggregated model across all selected outpost marks, have such as (but being not limited to) multi-variables analysis or a decision tree analysis qualification single invite sharp.Crucially, this scheme also can diagnose that display is normal, the sample of contrast overview, and invites and swash display atypia but be not previously accredited as those of non-convergency differential profile, and prompting is exposed to the unknown and invites sharp.

There is the required characteristic of these outpost several, described characteristic can increase the known or the unknown of its diagnosis and invite sharp ability, invite swash for desired by genome Stochastic choice, invite sharp relevant targeting regions or even whole genome (wherein jam-to-signal may hinder resolution to invite sharp difference) with specific.

The preferred feature of sharp outpost is invited to be listed below:

1. with mode identical with regard to homologue's type of same biology respond same invite swash and can repeat change methylation state.

2. the methylated state between repeat samples that same time takes from same individual homologue that should be presented at ideally does not change or preferably <20% change.

3. produce changing from different qualitative the methylating of inviting sharp state to be associated of easily detecting.

4. response some invite and swash and do not respond other and invite and swash to change methylation state (preferably invite sharp change to 15-85%, more preferably 25-75% invites and to swash and most preferably 40-60% invites sharp).

5. comprise preferably represent most of or preferably whole stress reaction paths based on methylated locus.

6. combine respectively according to each tissue/organ type of biology.

7. constant between tissue/stage of development or preferred <10% change ideally, unless be only intended to for specifically being organized, organ or stage of development.

Preferably, one or more these characteristics are had for DNA locus of the present invention.

Preferably, the selection of the locus subgroup of step (vi) is completed automatically by computer program.

Computer program how Select gene seat generic instance Water demand survey multiple locus of methylation state.A possible embodiment needs gene subgroup, and collects and invite sharp gene expression dose and display methylation state for multiple.Then the coding of the computer program of scheme assembling is below adopted.

Data

Described gene expression data changes into three state, and by 4 times in (coding) gene expression or larger change are expressed as " 1 ", and 4 times of reductions are changed into "-1 ", and do not change in gene " 0 ".

Anyly invite that swash can not be identified and shift out the expression of any gene is not mutagenic.There is inviting of identical expression response to swash and can not identify separately, and therefore shift out all (or except for one all).

Algorithm (A)

Measure and need qualification given number to invite sharp minimum number gene.Described algorithm is that Stochastic sum is incomplete, and therefore can not ensure to generate optimum answer, although always generate and best very close answer.Its also abundant one show allow experiment between statistical comparison.

Term

N, the intended gene number of use;

Eval, orderly group that gets N number of gene, and return unique qualification invite sharp number.

Climbing method (Hill climber) (is such as shown in Goldfeld, SM, Quandt RE and Trotter HF (1966) Econometrica34541; Prigel-Bennett A (2004) Theoretical Computer Science320,135-153)

1. generate the random groups of N number of gene, wherein N is the intended gene number used.

2. replace one of N number of gene in described group with the gene do not had in current group.

3. use the genome of step 2, use function Eval, what find unique qualification invites sharp number.

If step 2 and 3 solution produce than current retained better solution, with one of step 2/3 replacement current reservation solution.

If all invite swash identified by uniqueness, report solution and stop other, get back to step 2.

Climbing method (hill climber) is 10, and 000 reruns, and isolated operation 1000 times.If can not find that unique qualification is all to invite sharp optimum solution, return uniqueness and identify and invite at most sharp solution.

In a fourth aspect of the present invention, providing the method with regard to identifying one or more groups DNA locus of qualification with regard to one or more atypical growth conditions, comprising step:

A () adopts one or more methods of first, second or the third aspect of the present invention to be associated with one or more atypical growth conditions to make one or more groups DNA locus simultaneously; Or

B () to combine in first, second or the third aspect of the present invention one or more step (viii) and (ix) result, thus identify one or more groups DNA locus by one or more atypical growth conditions.

In a fifth aspect of the present invention, provide the DNA locus group of one or more atypical growth conditions of qualification, wherein DNA locus group methods described herein define.

In a sixth aspect of the present invention, the method for the atypical growth condition of one or more cells of interest of qualification experience is provided, comprises step:

I. DNA isolation from cells of interest, the growth conditions of wherein said cell is unknown.

Ii identifies the methylation state of the methylated locus group of energy in the DNA be separated in step (i).

Iii. by the methylation state of the locus of qualification in (ii) compared with the known methylation state group of homologous genes seat group, wherein said known methylation state group is that one or more invite sharp characteristic.

Iv. identify whether cells of interest once went through atypical growth condition.

Preferably, step (iv) goes back the type of the atypical growth condition that identification of cell was once gone through.

In a seventh aspect of the present invention, the application in the atypical growth condition providing DNA locus group as defined above to experience at identification of cell.

The application of the 6th and the 7th aspect or method, be wherein still in described atypical growth condition.

The application of the 6th and the 7th aspect or method, wherein, described in be exposed to atypical growth condition be in the past (namely before test sometime) experienced.

DNA locus and relevant diagnosis methylation patterns can be used for regard to extensive use qualification atypical growth conditioned disjunction cell (or the tissue/biology in its source).Described qualification can comprise now and exposure in the past, comprises in sample the atypical growth condition not having to occur, includes but not limited to:

Diagnostic symptom or silent disese (further details see under).

Acquired Infection neurological susceptibility.

Irritated.

Old and feeble

Be exposed to medical medicine or poison.(do not need before exposure to occur active component.

Be exposed to performance enhancement medicine.Embodiment can include but not limited to steroids, nandrolone, erythropoietin(EPO), nikethamidum, cathidine, clenbuterol, remove first androsterone (Norandrosterone), stanozolol and hydrodiuril.

Biology is made to be exposed to agricultural chemicals.

Check the state of commercially available clone.

Test is used for the clone physiological status of biomedical object.Example includes but not limited to: assess the vigor of synthesis or separate stem cells, usefulness and/or totipotency; Evaluate vigor and the ability of the Skin Cell system for transplanting; Guarantee the homogeneous physiological status of standard cell lines system culture relative to reference standard.

Clinical front Shi Yan – test cell system or organ or biology be not to prevention, the needing or the reaction of required atypia of medical treatment or toxic biological chemical reagent.

The diagnosis of disease comprises asymptomatic, that symptom front or other occur Fuzzy symptoms.Much possible example comprises diagnosis multi-form cancer, metabolic disease, is exposed to virus or bacteriological infection, comprises latent infection (as pulmonary tuberculosis), invites and swash relevant disease/disorder, parasitic infection or invasion and attack.The disease example of outpost's diagnosis is used to include but not limited to:

Not there is or occur the bacterial disease of Fuzzy symptom or delay symptom, such as tuberculosis (symptom and symptomless infection), meningitis, septicemia, Chlamydia.

Comprise all cancer types of asymptomatic stage and remission stage.

Not there is or occur the virus disease of Fuzzy symptom or delay symptom.Possible example includes but not limited to: HIV, bleb, influenza and Ebola virus (ebola).

Having been noted that methylates changes and the disease of this system energy assisted diagnosis in itself, comprises leukaemia, head and neck cancer, Hodgkin's disease, cancer of the stomach, prostate cancer, kidney, carcinoma of urinary bladder, breast cancer, Burkitt lymphoma, wilms' tumor, handkerchief-Wei Er Shi, angelman syndrome, ICF syndrome, histiocytoma, hypertension, paediatrics Neurobiology.

In a eighth aspect of the present invention, provide the computer program of the locus subgroup of selective discrimination methylation state with qualification and distinguish atypical growth condition.

In the of the present invention 8 9th, the DNA locus group of one or more atypical growth conditions of qualification arabidopsis cell sample experience is provided, wherein different methylation state and/or different gene locus of expressing in following one or more (as all) arabidopsis gene inner or its upstream/downstream as many as 5kb place (see Lister etc. (2008).

AT2G15800

AT2G38240

AT5G54720

AT1G76650

AT3G31540

AT3G28820

AT5G02580

AT1G07120

AT1G61800

AT1G48110

AT5G28235

AT5G28430

AT5G28760

AT3G53300

AT5G46200

AT3G57260

AT4G02330

AT3G05400

AT5G25110

AT3G28770

AT5G62750

In another aspect of this invention, provide the method for arbitrary aspect above, wherein said locus group as the 9th aspect define, and described locus is for the identification of from the atypical growth conditioned disjunction physiology/phenotypic response in the cells of interest of arabidopsis.Or described locus is for the identification of the atypical growth conditioned disjunction physiology/phenotypic response in the cells of interest from non-arabidopsis thaliana.

In another aspect of this invention, the DNA locus group of one or more atypical growth conditions providing surveyor's cell sample to experience, wherein different methylation state and/or different gene locus of expressing in following one or more (as all) people's gene chromosome position is inner or its upstream/downstream 5kb place of as many as (see Eckhardt etc. (2006).

ZNRF3

SLC7A4

Myosin-18B (MYO18B)

Glycoprotein ibalpha (blood platelet), beta polypeptides

RP1-47A17.8

Oncostatin M (OSM)

CTA-299D3.6

CTA-941F9.6

CAP-4

PIB5PA

SUSD2

chr22:35,256,796-35,277,053,NCBI36

RP3-438O4.2

RP4-756G23.1

The somatostatin receptor 3 type (SSTR3)

Bcl-2 interacts and kills and wounds albumen (BIK)

GAS2L1

RP3-355C18.2

γ-cellule albumen

CARMA3

TMPRSS6

PDGF-B chain precursor (PDGFB)

CELSR1 cadherin

T-box transcription factor (TBX1)

Q6ZRW2_HUMAN

NKG2DL4(RAET1E)

DAAM2

RP1-47M23.1

RP11-397G17.1

Nesprin-1(Syne-1)

Myogenicity suppresses sub-I-mf (MDFI)

RP11-174C7.4

CMAH

PKHD1

Glutathione peroxidase 5

GRIK2

SLC22A1

RP11-235G24.1

TBX18

TGM3

RIN2

SLC24A3

Q9ULE8_HUMAN

C20orf117

Breast cancer extension increasing sequence 4 (BCAS4)

Nuclear factor of activated T cells (NFATC2)

SCUBE1

JAG1

According to a further aspect in the invention, the exposure providing the compilation of the target DNA sequence dna group being positioned at biological genome DNA and using method to experience with diagnosis and identification of organism sample and/or the kind relating to tissue and/or cell or source, described method comprises the DNA sequence dna selecting to have methylation state, and described methylation state is different per sample and change still can not diagnose separately only single condition (exposure that biological sample has experienced) or source (kind of tissue and/or cell or source).

According to a further aspect in the invention, there is provided the compilation of the target DNA sequence dna group being positioned at biological genome DNA and using method with the kind of diagnosis and appraisement organization and/or cell or source, described method comprises the DNA sequence dna selecting to have methylation state, and described methylation state is different per sample and change still can not diagnose separately only single source (kind of tissue and/or cell or source).

According to a further aspect in the invention, the exposure that the compilation of the target DNA sequence dna group being positioned at biological genome DNA and using method have experienced with diagnosis and identification of organism sample is provided, described method comprises the DNA sequence dna selecting to have methylation state, and described methylation state is different per sample and change still can not diagnose separately only single condition (exposure that biological sample has experienced).

According to a further aspect in the invention, provide the compilation of the target DNA sequence dna group being positioned at biological genome DNA and using method with the kind of diagnosis and appraisement organization and/or cell or source, described method comprises

I () be DNA isolation from the cell sample in two or more known variety classeses or source;

(ii) methylation state of qualification methylated locus of energy from the DNA that each sample is separated;

(iii) methylation state of the DNA from each sample separation is compared; With

(iv) select to have the locus subgroup of following characteristics, the feature of described subgroup is: to have in some sample different with sample but not only make a definite diagnosis the locus methylation state in single kind or source.

In another aspect of this invention, provide the method that identification of dna locus (inviting sharp outpost) is organized with regard to qualification atypical growth condition (inviting sharp), comprise step:

I () cultivates interested cell sample under contrast growth conditions;

(ii) in the environment that there is at least one atypical growth condition, identical cells of interest sample described with step (i) is cultivated;

(iii) from step (i) and DNA isolation each cell sample of (ii);

(iv) methylation state of the methylated locus of energy in the DNA be separated in authentication step (iii);

V () compares from the methylation state of step (i) with (ii) the middle DNA be separated;

(vi) the locus subgroup with following characteristics is selected, the feature of wherein said subgroup is: in the DNA be separated in step (ii), the methylation state of these locus is different from the DNA be separated in step (i), and in described subgroup, each locus responds some atypical growth condition and do not respond other conditions and change methylation state.

Alternatively, described method comprises step:

(vii) by the methylation state of selected genes seat subgroup with notbe associated for the atypical growth condition of Select gene seat and/or tissue/cell type and/or its combination.

In another aspect of this invention, provide the method for qualification many groups DNA locus (inviting sharp outpost) with regard to qualification atypical growth condition (inviting sharp), comprise step:

I () cultivates various kinds of cell sample under contrast growth conditions, wherein interested various kinds of cell has different genotype;

(ii) exist in the environment of at least one atypical growth condition, multiple cells of interest sample described in incubation step (i);

(iii) from step (i) and DNA isolation each cell sample of (ii);

(v) comparison step (i) and (ii) or (i) methylation state with the middle DNA be separated of (iii) or (ii) and (iii).

(vi) select the locus subgroup with following characteristics, the feature of wherein said subgroup is: in the DNA be separated in step (ii), the methylation state of these locus is different from the DNA be separated in step (i) and/or (iii); And/or methylation state difference compared with (i) of these locus in the DNA be separated in step (iii); And each locus responds some atypical growth condition and does not respond other conditions and change methylation state in described subgroup.

Alternatively, also in steps:

(viii) by the methylation state of selected genes seat subgroup with notbe associated for the atypical growth condition of Select gene seat and/or tissue/cell type and/or its combination.

In another aspect of this invention, providing the method with regard to identifying identification of dna locus group with regard to multiple atypical growth condition, comprising step:

I () cultivates interested cell sample under contrast growth conditions;

(ii) in the environment that there is at least one atypical growth condition, cultivate the multiple cells of interest sample identical with step (i), wherein each sample grows respectively under different atypical growth conditions;

(iii) from step (i) and DNA isolation each cell sample of (ii);

(vi) the locus subgroup with following characteristics is selected, the feature of wherein said subgroup is: in the DNA be separated in each sample step (ii), the methylation state of these locus is different from the DNA be separated in step (i), and in described subgroup, each locus responds some atypical growth condition and do not respond other conditions and change methylation state.

Alternatively, other steps are had:

Preferably, the methylation state of selected genes seat subgroup is diagnosed jointly notfor the atypical growth condition of Select gene seat and/or tissue/cell type and/or its combination.

Preferably, the methylation state of selected genes seat subgroup is diagnosed jointly for the atypical growth condition of Select gene seat and/or tissue/cell type and/or its combination.

Preferably, the inventive method comprise use at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more plant different sharp, organization type or the cell type invited with Select gene seat subgroup.

Such as, in some embodiments, method of the present invention comprises and uses about 1-about 10,000, preferably about 10-about 500, and more preferably from about 30-about 100 kinds of differences invite sharp, organization type or cell type with Select gene seat subgroup.

As described herein, in a preferred embodiment, each locus in locus subgroup does not only diagnose condition separately.

Preferably, each locus in locus subgroup has one or more, such as two or more, three or more, four or multiple, five or multiple, six or multiple, seven or eight following properties,

(i) to identical sharp repeatability ground of inviting in the same manner or to change methylation state with the mode that identical source organism homologue type is associated;

(ii) between the repeat samples taking from same individual homologue at same time, methylated state does not change or is preferably less than about 20% change;

(iii) produce that easily detect and different qualitative methylating of inviting sharp state or cell and/or tissue types or source to be associated to change;

(iv) produce that easily detect and different invite sharp state or cell and/or tissue types or source to be associated significantly quantitatively methylate and change;

(v) respond some invite swash and do not respond other invite swash and change methylation state (preferably to 15-85% invite swash change, more preferably 25-75% invite swash and most preferably 40-60% invite sharp);

(vi) comprise preferably represent major part or preferably in whole (a) biology stress reaction path or (b) cell and/or organization type based on methylated locus;

(vii) combine respectively according to each tissue/organ type of biology;

(viii) constant between different tissues/stage of development or be preferably less than about 10% change, unless be only intended to for specifically organizing, organ or stage of development.

Preferably, the locus in locus subgroup is not diagnosed separately and is arbitraryly invited sharp, source material or physiological status.

Preferably, locus subgroup can use one to invite sharp, source material or physiological status to identify, but when be applied to be exposed to difference invite swash or the sample of source material or physiological status time, still retain diagnosis attribute.

Preferably, each locus in described locus subgroup complementally distinctiveness is present in the inviting in sharp or sample source for Select gene seat of roughly half.

The present invention also provides one or more groups target DNA sequence dna or locus subgroup of being identified by methods described herein.Also be provided in the application such as herein described in other places described herein and use one or more target DNA sequence dnas described or locus subgroup, following in one or more:

Diagnostic symptom or silent disese (further details see under).

Acquired Infection neurological susceptibility.

Irritated.

Old and feeble

Be exposed to performance enhancement medicine.Embodiment can include but not limited to steroids, nandrolone, erythropoietin(EPO), nikethamidum, cathidine, clenbuterol, remove first androsterone, stanozolol and hydrodiuril.

Biology is made to be exposed to agrochemicals thing.

Check the state of commercially available clone.

Test is used for the physiological status of the clone of biomedical object.Example includes but not limited to: assess the vigor of synthesis or separate stem cells, usefulness and/or totipotency; Evaluate vigor and the ability of the Skin Cell system for transplanting; Guarantee to demonstrate,prove the homogeneous physiological status of standard cell lines system culture relative to reference standard.

By drawings and Examples, the present invention is described now.That should understand embodiment tells about the restriction formed never in any form scope of the present invention.

Fig. 1 principal component analysis (PCO)

According to the figure of methyl-sensitive AFLP (MSAP) data analysis inviting sharp arabidopsis from difference.The first two composition is only responsible for the overall changeability (non-training data) of 23.2%.(S=salt is invited sharp, and N=temperature is invited sharp, and H=eats grass (herbivory) and C=contrast).

The PCO of the polymorphism MSAP mark of Fig. 2 only between salinity and temperature and control sample.The first two composition is responsible for the changeability (in the upper training of C, N+S) of 24.8%.

(S=salt is invited sharp, and N=temperature is invited sharp, and H=eats grass (herbivory) and C=contrast).

The all PCO inviting the polymorphism MSAP between sharp (outpost's group) to mark of Fig. 3.The first two composition is responsible for the changeability of 32.7%.

Fig. 4 analyzes data by adding up paired comparisons between four kinds of process.Compare from each 6,100 amplicons the most remarkable of totally 381 amplicons.The entire change that the first two composition is contained is 15.2%.(S=salt is invited sharp, and N=temperature is invited sharp, and H=eats grass (herbivory) and C=contrast).

Fig. 5. analyze data by adding up paired comparisons between four kinds of process.Compare from each 6,50 amplicons the most remarkable of totally 217 amplicons.The entire change that the first two composition is contained is 17.9%.(S=salt is invited sharp, and N=temperature is invited sharp, and H=eats grass (herbivory) and C=contrast).

Fig. 6 analyzes data by adding up paired comparisons between four kinds of process.Compare from each 6,25 amplicons the most remarkable of totally 116 amplicons.The entire change that the first two composition is contained is 20.5%.(S=salt is invited sharp, and N=temperature is invited sharp, and H=eats grass (herbivory) and C=contrast).

Fig. 7 analyzes data by adding up paired comparisons between four kinds of process.Compare from each 6,10 amplicons the most remarkable of totally 52 amplicons.The entire change that the first two composition is contained is 25.4%.(S=salt is invited sharp, and N=temperature is invited sharp, and H=eats grass (herbivory) and C=contrast).

Fig. 8 analyzes data by the difference between four kinds of process.Compare at each 6, in 380 amplicons, show 100 amplicons of maximum difference totally.The entire change that the first two composition is contained is 23.5%.(S=salt is invited sharp, and N=temperature is invited sharp, and H=eats grass (herbivory) and C=contrast).

Fig. 9 analyzes data by the difference between four kinds of process.Compare at each 6, in 206 amplicons, show 50 amplicons of maximum difference totally.The entire change that the first two composition is contained is 28.3%.(S=salt is invited sharp, and N=temperature is invited sharp, and H=eats grass (herbivory) and C=contrast).

Figure 10 analyzes data by the difference between four kinds of process.Compare at each 6, in 112 amplicons, show 25 amplicons of maximum difference totally.The entire change that the first two composition is contained is 33.3%.(S=salt is invited sharp, and N=temperature is invited sharp, and H=eats grass (herbivory) and C=contrast).

Figure 11 analyzes data by the difference between four kinds of process.Compare at each 6, in 53 amplicons, show 10 amplicons of maximum difference totally.The entire change that the first two composition is contained is 39.2%.(S=salt is invited sharp, and N=temperature is invited sharp, and H=eats grass (herbivory) and C=contrast).

Figure 12 is according to master (Principle) the constituent analysis figure of overall training analysis of AFLP data inviting sharp mouse RAW cell from difference.The first two composition represents entire change (■) IL4 of 15.8%, (◆) LPS, and (▲) nutrition invites sharp, and (●) contrasts.

Figure 13 is according to the principal component analysis figure inviting the contrast of the AFLP data of sharp mouse RAW cell, IL4 and nutrition to invite sharp training analysis from difference.The first two composition represents the entire change of 20.7%.(■) IL4, (◆) LPS, (▲) nutrition invites sharp, and (●) contrasts

Figure 14 is according to the contrast of AFLP data, the principal component analysis figure of IL4 and LPS training analysis that invite sharp mouse RAW cell from difference.The first two composition represents the entire change of 19.8%.(■) IL4, (◆) LPS, (▲) nutrition invites sharp, and (●) contrasts

Figure 15 is according to the principal component analysis figure inviting the contrast of the AFLP data of sharp mouse RAW cell, LPS and nutrition to invite sharp training analysis from difference.The first two composition represents the entire change of 19.9%.(■) IL4, (◆) LPS, (▲) nutrition invites sharp, and (●) contrasts

Figure 16 Genevestigator output display 82 kinds invites the effect swashed finding to be marked by the arabidopsis (Athaliana) of Regulation by Methylation Stochastic choice expression.(Lister etc. (2008)).Row represent 82 kinds and invite sharp.Row represents the selection of genetic marker.The gray tone of each box represents when arabidopsis experience correspondence invites sharp (row), the expression of corresponding gene (OK).Black instruction is expressed and is not changed, and light gray is lower mediation Dark grey is raise.

9 stages of development of Figure 17 Genevestigator output display are to finding the effect (Lister etc. (2008)) being marked Stochastic choice expression by the arabidopsis of Regulation by Methylation.Row represent 9 stages of development.Row represents the selection of genetic marker.The gray tone of each box represents the expression from the arabidopsis material corresponding gene (OK) being in each stage of development (being shown by list).

Figure 18 Genevestigator output display 40 organs are to finding the effect being marked Stochastic choice expression by the arabidopsis (Athaliana) of Regulation by Methylation.(Lister etc. (2008)).Row represent 40 organs.Row represents the selection of genetic marker.The gray tone of each box represents the expression of the corresponding gene (OK) from each arabidopsis organ (row).

Figure 19 uses three kinds of outpost to separate 27 kinds to invite sharp, state (the continuous connecting line of grey that each expression three is possible; Raise or hyper-methylation outpost; Grey intermittent attachment lines; Lower or hypomethylation outpost; Black connecting line: expression or the methylation level of outpost do not change).

Figure 20 is about inviting the ternary tree swashing and regulate and change, and the display of two genes causes 8 kinds potentially to invite sharp S1-S8."+" represents rise, "-" represent and lower.As fruit gene 1 lowers (-) and gene 2 raises (+), this corresponding S6 invites sharp overview.

Figure 21 PCoA schemes (the first principal coordinate is to the second principal coordinate) and shows separately (with the standardized expression overview of dual format) that experience 78 kinds of differences invite 15 sharp locus outpost overviews.Describedly sharp manual sort is invited to become following unofficial group: biological (black bars), chemistry (hollow square), hormone (open triangles), light (black triangles), nutrients (empty circles), PCO (black circles), cold (hollow parallel quadrangle), dry (black parallelogram), heat (gray parallelograms), anoxic (asterisk), infiltration (grey is square), salt (cross) and damage (gray circles).

Figure 22 PCoA schemes (the first principal coordinate is to the second principal coordinate) and shows separately (with the standardized expression overview of dual format) that experience 78 kinds of differences invite 36 sharp locus outpost overviews.Describedly sharp manual sort is invited to become following unofficial group: biological (black bars), chemistry (hollow square), hormone (open triangles), light (black triangles), nutrients (empty circles), PCO (black circles), cold (hollow parallel quadrangle), dry (black parallelogram), heat (gray parallelograms), anoxic (asterisk), infiltration (grey is square), salt (cross) and damage (gray circles).

Figure 23 PCoA schemes (the first principal coordinate is to the 3rd principal coordinate) and shows separately (with the standardized expression overview of dual format) that experience 78 kinds of differences invite 36 sharp locus outpost overviews.Describedly sharp manual sort is invited to become following unofficial group: biological (black bars), chemistry (hollow square), hormone (open triangles), light (black triangles), nutrients (empty circles), PCO (black circles), cold (hollow parallel quadrangle), dry (black parallelogram), heat (gray parallelograms), anoxic (asterisk), infiltration (grey is square), salt (cross) and damage (gray circles).

Figure 24 PCoA schemes (the first principal coordinate is to Second principal component) and shows separately (with the standardized expression overview of dual format) that experience 78 kinds of differences invite the sentry post overview of 59 sharp locus.Describedly sharp manual sort is invited to become following unofficial group: biological (black bars), chemistry (hollow square), hormone (open triangles), light (black triangles), nutrients (empty circles), PCO (black circles), cold (hollow parallel quadrangle), dry (black parallelogram), heat (gray parallelograms), anoxic (asterisk), infiltration (grey is square), salt (cross) and damage (gray circles).

Figure 25 Genevestigator output display 506 kinds invites the effect (Eckhardt etc. (2006)) swashed the methylated 49 kinds of marker expressions that find differences in people.Coloud coding instruction black does not change for expressing, and light gray is lower mediation Dark grey is raise.

Figure 26 Genevestigator output display 8 age groups are to the effect (Eckhardt etc. (2006)) of the methylated 49 kinds of marker expressions that find differences in people.

Figure 27 Genevestigator output display 40 organs are to the effect (Eckhardt etc. (2006)) of the methylated 49 kinds of marker expressions that find differences in people.

Embodiment

The molecular biology method therefor applied in the examples below that is that those skilled in the art are known.The general teaching material describing conventional molecular biological well known by persons skilled in the art, microbiology and recombinant DNA technology comprises; such as: Sambrook etc.; Molecular Cloning:ALaboratory Manual(" molecular cloning: laboratory manual "); the second edition, the CSH Press (ColdSpring Harbor Laboratory Press) (2001) at cold spring port, New York; Glover compiles, DNA Cloning:PracticalApproach(" DNA clone: hands-on approach "), I and II rolls up, the MRL Press LLC (MRL Press, Ltd) (1985) of England Oxford; And Ausubel, F., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.GT., Smith J.A., Struhl, K.Current protocols inmolecular biology(" fine works molecular biology experiment guide "). the Green in New York publishes affiliated company and Wei Li publishing company (Green Publishing Associates/Wiley Intersciences).

Embodiment 1 – invites sharp for the identification of inviting sharp methyl-sensitive AFLP in arabidopsis

Candidate outpost's DNA methylation be marked at arabidopsis (environmental: Colombia) group experience outsides that two kinds is non-convergency difference invite swash after collection be incorporated in MSAP (methylation-sensitive amplified polymorphism) overview of gained and screen not isolabeling.Invite the overview that methylates of sharp detected object to compare mutually, and make comparisons with reference (contrast) group grown under standard laboratory conditions.Sharp group is invited not identify the ability (example of method) of inviting and swashing lower plant growth to set up outpost's mark diagnostic by referring to the 3rd.Therefore, four groups of candidate outpost (be selected from three and invite any two and the outpost that tests on a third in sharp group) can be gathered and detect.

Plant growth

In 24 single lattice plates, cultivate plant, in the mono-lattice of each 4cm x4cm, have 2 strain plants.Remove single lattice and make bottom water injection.Therefore, each process has 46 strain plants.Before transferring to the experiment condition in 4 controlled growth cabinets, seed is inoculated at 4 ° of C, and grows 1 week under glass.Described plant is at 8 hours of photoperiod (about 70 μm of ol/m ²/ s) under grow to suppress to bloom.

In four cabinets, the temperature of three is set as 22 ° of C; The following setting in detail of temperature (in order to test as inviting sharp temperature) of the 4th cabinet.The some growth that plant separates in two at somewhat different conditions.

Table 2 – plant growing condition

DNA extracts

Use the kit of Qiagen to carry out all DNA according to manufacturer's description to extract. plant Mini Kit is used for extracting DNA from the arabidopsis sample of all 46 strain plants of each process.This kit all taken from by reagent discussed below.

Scissors is used to destroy about 100mg plant tissue in liquid nitrogen in 1.5ml microcentrifugal tube.At once, do not make tissue melt, often pipe adds the 400 μ l lysis buffer AP1 and 4 μ l RNaseAs that are preheating to 65 ° of C.By upset contents were mixed, and hatch 10 minutes at 65 ° of C, every 2-3 minute biased sample once in a while.

Then, 130 μ l AP2 buffer solutions are joined in each sample, and described pipe incubated on ice 5 minutes with protein precipitation and polysaccharide.Then 13,000rpm centrifuge tube 5 minutes is to precipitate thickness lysate and other solids.

Then QIAshredder transferred to by supernatant ^tMpost (having silica matrix) and centrifugal 2 minutes of 13,000rpm to remove precipitation and cell fragment.Collect post efflux and transfer in fresh tube, then mixing with 0.5 volumes of wash buffer and 1 volume ethanol.This mixture transfers to the little centrifugal column of the 2nd DNeasy and at 8,000rpm centrifugal 1 minute.Because DNA molecular is retained in post, abandon described efflux.By making 500 μ l lavation buffer solution AW flow through post, centrifugal 1 minute of 8,000rpm, the DNA that washing combines twice.

Then, the 100 μ l buffer A E and incubated at room after 5 minutes that are preheating to 65 ° of C are added, by centrifugal 13,000rpm centrifugal 1 minute dry described films.

Ago-Gel is quantitative

Aliquot DNA (1-5 μ l) carries out 1% (w/v) agarose gel electrophoresis to measure the DNA quality and quantity that exist.By dissolve in the 1x TAE buffer solution (40mM tris-acetic acid esters, 1mMEDTA) of suitable volumes appropriate amount agarose and subsequently in micro-wave oven heating until agarose fusing, prepare described gel.Described gel solution is cooled to about 50 ° of C, then adds 10mg/ml Ethidum Eremide solution to final concentration 0.35 μ g/ml, and pours into subsequently in cast sheet, place suitable comb to produce well.

When provided, described gel forwards horizontal strip electrophoresis equipment to, and gel comb is in cathode end.Remove described gel comb, add enough 1x TAE buffer solutions to electrode chamber and be about 1mm to cover gel.Then the DNA sample (the blue loading dye [0.23% (w/v) bromophenol blue, 60mMEDTA, 40% (w/v) sucrose] of 5 μ l DNA:1 μ l) of preparation is joined in gel pore.Large for HyperLadderII (than Ao Li company (Bioline), BIO-33040) tick marks is joined side swimming lane.Described gel constant voltage 3-5V/cm carries out electrophoresis 15-60 minute.Described DNA UV ultraviolet transillumination platform (320nm wavelength) is observed.

Methyl-sensitive AFLP

Methyl-sensitive AFLP (AFLP) carries out in random selected 8 DNA sample of each process, and based on the AFLP scheme that (1995) such as Vos describe, but use the isoschizomers of target same identification motif.

The basis of described technology is the restriction fragment being detected genomic DNA by polymerase chain reaction (PCR) amplification.This can use limited universal primer group to create fingerprint from the DNA with any source or complexity, and does not need the priori of sequence.Use and make the method be suitable for detection to the Restriction Enzyme of methyl-sensitive to methylate.

With 2 Restriction Enzyme restricted dnas, one rare and one be frequent cutter to cytosine methylation sensitivity.Use and 2 kinds of different restrictions are carried out to the isoschizomers of the frequent cutter of dissimilar cytosine methylation sensitivity.All enzymes are available from Canadian Fu Meitaisi company (Fermentas).

MspI enzyme: cut between two cytimidines of sequence 5 ' CCGG3 ', and prevent it from acting on by methylating on first C instead of second C.

HpaII enzyme: cut between two cytimidines of sequence 5 ' CCGG3 ', and prevent it from acting on by methylating on second C instead of first C.

The restricted reaction of table 3 –

The adapter special to restriction site is connected to use universal primer amplified fragments on DNA, and does not need first to obtain sequence information.All enzymes are from Fu Meitaisi company, and adapter is from Ji Nuosi Co., Ltd of Sigma (Sigma-Genosys).

Table 4-adapter structure

Adapter mixture is generated by combination 1nM EcoRI adapter and 10nM MspI/HpaII adapter.

Table 5 – coupled reaction

An Oligonucleolide primers of corresponding EcoRI end and an Oligonucleolide primers of corresponding MspI/HpaII end is used to complete amplification wheel.By adding a unnecessary base at primer 3 ' end, first round amplification reduces possible segment number, and by adding one or two other bases at primer 3 ' end, second takes turns the amplification segment number that reduction is possible further.Second takes turns EcoRI primer 6-Fam(carboxyl fluorescin) mark to make the described product of observation.

The pre-amplimer of table 6-

Table 7 – increases PCR mixture in advance

Table 8 – increases PCR program in advance

Table 9 – selective amplification primer

Table 10-selective amplification PCR mixture

DNA (1/15) before amplification	5μl
		Instant PCR mixture	10μl
EcoRI primer+AX (10 μMs)	0.8μl
		HpaII/MspI primer+AXX (1 μM)	1μl
Water	3.2μl

Table 11 – is used for the touchdown PCR program of selective amplification

The product of restrictive amplification step delivers to mark genome company (Macrogen) of Korea S, and the genetic analyzer of Applied Biosystems, Inc. (Applied Biosystems) is separated by Capillary Electrophoresis.Described result uses GeneMapper analysis software to observe and explains, and outputs to Microsoft Excel and analyze further.Use the MVSP (http://www.kovcomp.co.u/mvsp/) described in Kovach, W. (1998); MVSP-multivariate statistics software kit, Windows3.0 version, British Wales Pentraeth:KCS company (Kovach Computing Services)) or Minitab15 (http://www.minitab.com/en-GB/default.apsx WT.srch=1 & WT.mc_id=SE004815) carry out multi-variables analysis (principal coordinate analysis).

Each band (amplicon) in AFLP scheme thinks the single allele of individual gene seat.For each process, the allele kind of each locus first with simple qualitative fashion 1 (appearance) or 0 (disappearance) to the individual assignment of each repetition.If (such as 11111111 compare 11111000 can regard as difference to the individual allele overview difference of the locus of three or more individuality, 11111111 compare 00111111 then can not), think locus invite swash process between or contrast and inviting swash between process different.The possible candidate outpost that all locus meeting these standards are considered to follow-up multi-variables analysis marks (see below).

4 are had to take turns training to assemble outpost's DNA methylation mark.For this reason, in four groups (comprising contrast) of three kinds of process, (table 11) is made comparisons between process.Then merge the solidification group that the data organized from these four provide outpost, be then applied to all samples.

Table 12 – is for training the group of outpost

Principal coordinate analysis is for analyzing the result of self-training outpost.

Result

All marks (non-selectivity training)

When training the polymorphic locus being limited to qualification leap all conditions (combined training), 959 locus of marking, polymorphism between all individualities all showing certain level.When data for collect multi-variables analysis time, invite sharp group to have reasonably separately, experience identical invite sharp plant to invite to swash trend towards flock together (PCO, Fig. 1).But, experience is different invite swash individuality between process particularly temperature invite swash with eat grass (herbivory) or contrast between some is overlapping.The first two composition is responsible for the entire change of appropriateness 23.2%.

When only use meet two invite swash in outpost's choice criteria those mark add contrast time, although use number of labels decline, training invite swash between separately improve.Such as, when invite with regard to control sample and salt and temperature swash select outpost time, the number of labels of use drops to 525 from 959.But, separately improve (not having overlap between training group) between these groups, and be increased to 24.8% a little by the change ratio of the first two principal component accounts.

Importantly, experience do not train invite the individuality of sharp (food grass (herbivory)) also show clear many separately, only have a fuzzy prototype (H8) (Fig. 2).

When training package containing all three invite sharp time, locus number drops to 137, but invite swash between separately significantly improve, there is no fuzzy individuality (Fig. 3).In addition, significantly increased (to 32.7%) by the entire change ratio of the first two component accounts.

For the statistical method that outpost selects

Use the software kit available from R website (www.r-project.org) under R environment (R environment), complete the calculating of data in a organized way.Relatively process is to processing the most relevant to differentiation four kinds statistically to measure which characteristic (band).Use three kinds of diverse ways selectivity characteristics, random forest, Welch t-statistic and TG-AUC (AUC), and produce from the result combination of all three the characteristic group (table 13) being most suitable for distinguishing different disposal.

The subgroup of selected characteristic uses principal coordinate analysis to observe to measure maximum optimum number (figure 4 – 7) of separating between four kinds of process.

Differentiating method

In a organized way data for calculate with regard in four kinds of process with regard to each at the potential digital 8 outer band total numbers occurred.Computing between difference, and select these process between the maximum characteristic of difference as outpost (table 14).

Table 13 – Using statistics bag R is by front 100 bands that selected by paired comparisons, often pair is compared.

Often pair of front 100 bands compared that table 14 – just has maximum difference and selects.

The subgroup of selected characteristic uses principal coordinate analysis to observe to measure maximum optimum number (Fig. 8-11) of separating between four kinds of process., know difference between reservation group herein, because the number of outpost's mark declines, but population proportion is increased to from 16.1% (Fig. 8) when using front 100 marks and only uses front 10 marks as 27% during sentry post (Figure 11).

Embodiment 2 – invites sharp methyl-sensitive AFLP to invite sharp for the identification of in mouse (Mus musculus) clone.

Cell culture condition

The impact swashed mouse monokaryon granulomacrophage RAW264.7 (No. ECACC 91062702) apparent gene group is invited in order to study outside.Cultured cell in 32 hole microwell plates, with 2.5x10 ⁵cell/ml inoculation, and at 37 ° of C5%CO before collecting ₂hatch 20 hours.Except as otherwise noted, the culture medium of use is the DMEM being supplemented with 2mML glutamine and 10% hyclone.

These cell masses are used for three kinds and invite sharp:

1. (I) 20mg/ml IL-4 (IL4)

2. (L) 1mg/ml lipopolysaccharides (LPS)

3. there is no L glutamine in (S) culture medium.

DNA extracts

Use the kit of Qiagen to carry out all DNA according to manufacturer's description to extract. blood and Tissue kit are used for extracting DNA from macrophage sample.The all reagent related to below from kit.

By upset from culture hole removing culture medium, with 200 μ l PBS, 20 μ l protease and 200 μ l buffer A L (without ethanol) replace.Content by vortex mixed, and hatches 10 minutes at 56 ° of C.Then, 200 μ l ethanol (95-100%) are joined in each sample, and described plate passes through vortex mixed.Then described mixture from each hole is transferred to little centrifugal column at 8,000rpm centrifugal 1 minute.

Because DNA molecular is retained in post, abandon described efflux.By making 500ul lavation buffer solution AW1 flow through post, centrifugal 1 minute of 8,000rpm, washs the DNA once combined, and then makes 500 μ l lavation buffer solution AW2 flow through post, centrifugal 1 minute of 8,000rpm.Subsequently, by 13,000rpm centrifugal 1 minute dry described film, then add 100 μ l buffer A E and incubated at room 1 minute, then 8,000rpm carry out eluted dna in centrifugal 1 minute.

The quality of DNA and quantity are as tested as described in embodiment 1 above.

AFLP

AFLP (AFLP) is the scheme according to (1995) such as Vos, and as embodiment 1 above describe in detail and carry out.

Select outpost

Mouse outpost selects in the mode identical with arabidopsis in above-described embodiment 1.

Result

All marks (non-selectivity training)

When training the polymorphic locus being limited to qualification leap all conditions (combined training), 734 locus of marking, polymorphism between all individualities all showing certain level.When data for collect multi-variables analysis time, invite sharp group have good separately, experience and identically invite sharp clone to trend towards flock together (PCO, lower Figure 12).In this situation, be only 15.8% by the change of the first two principal component accounts.

Select display two to invite to swash the mark of the most consistent difference between control sample to maintain (or slightly increasing) and invite difference between sharp group, but be the increase in the change ratio that the first and second main components contain, with regard to three figure inviting two in swashing training, it drops on (Figure 13-15) in the entire change close limit of 19.8-20.7%.

Therefore, select to emphasize two invite swash difference between process polymorphic marker subgroup still equally can to use in all polymorphic marker PCO that carry out separately (separate) the third invite sharp.

In addition, in the training outpost group that the first two main component being responsible for group differentiation occupies, entire change is more than the markd figure of use, and therefore comprises less ' noise ' change.Notice that outpost's group of training comprises several all different locus in process and between processing, and if therefore only for inviting sharp diagnosis can not be useful.

Embodiment 3-generates the sample method of outpost and its application in arabidopsis

Target gene is selected to be used as outpost by cross reference two complementary data storehouses.

Described first database (the arabidopsis apparent gene picture group spectrum of high integration list base discrimination rate, http://neomorph.salk.edu/epigenome/epigenome.html) comprise the regulatory gene list that methylates that recent research produces, described recent research compare arabidopsis wild type with maintains (met1-3) at DNA methylation and set up defect in (drm1-2drm2-2cmt3-11) mutant transcript profile with to methylate group (Lister etc. 2008).Because after the DNA methylation that mutant compares wild type totally reduces, the expression of these genes increases, and can infer that these genes are regulated by DNA methylation, and major part is by the downward that methylates.Therefore, invite the expression swashed between lower or any stage of development or tissue to change in any test of comparing contrast, its overview change that methylates can be pointed out to be induced by the condition changed; Make its suitable alternatively outpost.

Genevestigator (https: //www.genevestigator.com) provides and invites sharp, organ and information (Fig. 16 – 18) that microarray analysis that under the stage of development, 484 arabidopsis genes are expressed is derivative certain limit is specific.

If invite sharp Explicit Expression to compare contrast increase by 4 times when experience is invited sharp, gene is classified as swash lower rise given inviting.Downward is defined as expression and compares contrast decline 4 times.Increase to 1 and decline 4 times or change into-1 more greatly by the expression of 4 times or more of encoding, express and do not have change to be encoded to 0, output data transformations is three state.Therefore, in analysis subsequently, use cDNA abundance as the thick correlative of methylation state of DNA, and because herein is provided the potential indicant of worst case with regard to the outpost that methylates of these locus.The direct measurement of DNA methylation also can reduce shown without information change, because mRNA is more prone to change (Stavreva etc. 2009 unexpected and unstable in abundance; The .2007 such as Kageyama), and also because each independent locus respectively comprises several potential methylation sites that can be used as outpost with self performance separately.

Invite sharp 4 and 75 in any gene, not cause expression to change, and therefore can not identify and thus remove from analysis.Invite sharp S51, S52 and S57 to have identical expression response, therefore can not identify separately, and thus remove S51 and S52 (retaining S57 in analysis).Invite sharp according to initial 82 kinds, therefore use 78 kinds of overviews in this analysis.

Checked 39,688 genes altogether invites sharp to combination; Each gene 82 kinds is had to invite 484 sharp genes.These centerings, 1806 (4.56%) show >4 expression doubly changes, and invites under swashing and has 931 (2.35%) upper mediations 875 (2.21%) to lower.

Computer program

Software development and programming

The same with all Bayesian analyses, the algorithm of this analysis is random with incomplete; Similarly, therefore program is not guaranteed to generate absolute answer still often like this, and is always to provide the value enough close with the absolute value for comparing object in addition.

Task definition and algorithm design

Program object is defined as the given minimum number gene invited needed for sharp number of Testing and appraisal.In this example, because select all gene expressions of alternatively outpost to change (see on) methylating in mutant, 4 times of methylation states of expressing to change for inferring these locus change.

Algorithm (A)

N, the intended gene number of use

The sharp number of inviting that the gene of expression three different phases (-1 lowers, and 0 does not have to change and 1 rise) can be used to separate is three.Therefore, identify from one group of gene expression profile invite sharp theoretical amount be in described group gene cube.In addition, according to combinatorial formula, three a collection of (three by three) repeatedly do not get any group of N number of element and can generate N ³possible combination (example is see Figure 19).

Compile written as computer programs and invite sharp minimal genome to identify can classify 81 kinds from 488 genes.Ternary tree is for representing solution, and the corresponding gene of the joint on tree and branch indicate or raise, lower or regulate the response (Figure 20) not having to change.Two genes of display produce 8 kinds and potentially invite sharp S1-S8."+" instruction is raised, "-" instruction is lowered.As fruit gene 1 lowers (-) and gene 2 raises (+), this corresponding S6 invites sharp overview.

Stochastic hill-climbing invites sharp number for the identification of the optimum can identified from given number gene.It is possible that complete solution swashs minority gene and/or invite, because computation requirement or can invite sharp number to increase and exponential form increases along with gene, and therefore this solution provides the most useful solution to described problem.

Climbing method

10,000 iteration of running climbing method repeats and isolated operation 1,000 time.If can not find that unique qualification is all to invite sharp optimum solution, invite to swash and return uniqueness and identify and invite at most sharp solution.

Result

Use above-mentioned data run algorithm A.Minimum of a value N is initial is asserted 20, and declines in units of 1 until can not find that qualification is all to invite sharp solution.Using this scheme, identifying that 15 for identifying that all 78 kinds are invited the minimal amount needed for swashing.Selected genes and its application order are shown in table 15.Table 16 show how to use the gene expression list in table 15 to diagnose each invite sharp.Table 17 summarises often kind and invites sharp how to raise, to lower or not have the mode changed to affect these genes.

Can identify that 78 kinds are invited sharp redundant gene group, rerun process above to available gene pool, remove the gene in table 15 to set up.Invite sharp 80 by means of only an identified for genes, described gene is used for first and analyzes and no longer because of it can identify and remove subsequently.Find that N originates in 15, and increase until can solution be found in units of 1.Can whole 77 kinds of qualification sharp N minimum of a value be invited to be 21.Table 18 shows qualification 77 kinds and invites 21 redundant genes needed for swashing and its order.

Table 15. uniqueness qualification 78 kinds invites and swashs 15 required genes and its order.

Position	Gene
		1	AT3G42090
2	AT5G62210
		3	AT3G12580
4	AT2G36750
		5	AT5G39870
6	AT1G33850
		7	AT5G26620
8	AT5G28910
		9	AT5G35715
10	AT3G01250
		11	AT1G61340
12	AT4G10260
		13	AT1G35183
14	AT5G28810
		15	AT3G45360

Table 16. invites sharp key (key) from table 15 15 identified for genes 78 kinds used.

Invite sharp	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
																S1	-1	1	0	0	-1	-1	-1	-1	-1	-1	-1	1	1	0	0
S2	-1	-1	1	1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1
																S3	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1
S5	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1
																S6	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	1
S7	1	1	-1	-1	1	-1	-1	0	-1	0	-1	1	1	-1	-1
																S8	-1	0	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S9	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1

S10	-1	-1	-1	-1	-1	-1	-1	1	-1	0	-1	1	-1	-1	-1
																S11	-1	-1	1	1	-1	1	-1	-1	-1	-1	1	-1	1	-1	-1
S12	-1	-1	-1	1	-1	-1	-1	-1	-1	0	1	-1	0	-1	-1
																S13	-1	0	-1	-1	1	0	-1	-1	-1	-1	-1	-1	1	-1	0
S14	-1	-1	-1	-1	-1	-1	1	1	-1	-1	1	-1	0	0	-1
																S15	-1	-1	-1	-1	-1	0	-1	1	-1	0	-1	-1	-1	-1	0
S16	-1	-1	-1	-1	0	-1	-1	1	-1	-1	-1	1	-1	-1	0
																S17	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1
S18	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
																S19	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S20	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	0	0	0
																S21	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	0
S22	-1	-1	1	1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	0
																S23	-1	-1	1	1	1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
S24	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	0
																S25	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1
S26	-1	-1	-1	-1	0	0	-1	1	-1	-1	-1	-1	0	0	-1
																S27	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	0	1	0
S28	0	-1	-1	0	0	-1	-1	1	-1	0	-1	-1	-1	0	-1
																S29	-1	-1	1	1	-1	0	-1	0	-1	-1	1	-1	-1	0	-1
S30	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	0
																S31	-1	-1	-1	0	-1	0	-1	1	-1	0	-1	-1	-1	-1	0
S32	-1	-1	-1	-1	-1	-1	-1	1	-1	0	-1	-1	0	-1	0
																S33	-1	-1	-1	0	-1	-1	-1	1	-1	-1	-1	-1	0	0	0
S34	0	-1	-1	-1	-1	0	-1	1	-1	-1	-1	-1	-1	0	-1
																S35	0	-1	1	1	-1	-1	0	0	1	-1	-1	-1	-1	-1	1
S36	-1	-1	1	1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	0
																S37	-1	-1	-1	-1	-1	1	-1	1	-1	0	-1	-1	-1	-1	-1
S38	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	0	-1	0
																S39	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0
S40	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	0	-1
																S41	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	0	-1
S42	-1	-1	1	-1	-1	1	-1	-1	-1	0	1	-1	-1	-1	-1
																S43	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
S44	0	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	0	-1
																S45	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	1	-1	0	-1
S46	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	0
																S47	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0
S48	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1
																S49	0	0	-1	0	-1	-1	-1	1	-1	0	-1	-1	0	0	-1
S50	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1
																S53	-1	1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S54	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	0
																S55	-1	-1	1	-1	-1	-1	-1	-1	1	-1	-1	-1	1	-1	-1
S56	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																S57	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1
S58	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1
																S59	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1
S60	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	0	-1	-1	-1	1
																S61	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S62	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1
																S63	0	1	1	-1	-1	-1	1	-1	-1	-1	1	-1	-1	1	-1

S64	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	0
																S65	-1	-1	-1	1	-1	1	-1	-1	-1	0	-1	-1	-1	-1	0
S66	-1	-1	1	-1	1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
																S67	-1	-1	-1	1	1	-1	-1	-1	-1	-1	-1	-1	-1	1	1
S68	-1	-1	-1	-1	1	-1	1	-1	-1	-1	-1	-1	0	-1	0
																S69	0	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S70	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	1
																S71	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
S72	-1	1	0	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1
																S73	-1	-1	-1	1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1
S74	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1
																S76	-1	-1	1	-1	-1	-1	-1	-1	-1	1	1	-1	-1	-1	-1
S77	0	-1	1	-1	-1	-1	-1	-1	-1	-1	1	0	0	-1	-1
																S78	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	1	-1	-1	-1
S79	-1	-1	1	1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
																S80	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0
S81	-1	-1	-1	1	-1	-1	1	-1	-1	-1	1	-1	-1	-1	-1
																S82	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1

Table 17. changes the number gene of expression under sharp condition inviting, broken by upper mediation downward.

Invite sharp	The number that gene expression changes	Raise	Lower
				S1	81	36	45
S2	19	18	1
				S3	12	7	5
S5	10	1	9
				S6	12	5	7
S7	36	21	15
				S8	7	5	2
S9	14	12	2
				S10	12	7	5
S11	44	23	21
				S12	15	9	6
S13	44	14	30
				S14	36	21	15
S15	18	4	14
				S16	18	5	13
S17	20	8	12
				S18	16	14	2
S19	11	3	8

S20	57	33	24
				S21	23	6	17
S22	13	7	6
				S23	17	17	0
S24	18	7	11
				S25	37	16	21
S26	22	5	17
				S27	29	6	23
S28	32	7	25
				S29	28	22	6
S30	20	5	15
				S31	26	10	16
S32	22	8	14
				S33	16	7	9
S34	18	7	11
				S35	69	28	41
S36	27	12	15
				S37	20	5	15
S38	6	3	3
				S39	22	18	4
S40	3	2	1
				S41	5	3	2
S42	58	29	29
				S43	5	2	3
S44	4	0	4
				S45	7	2	5
S46	13	7	6
				S47	5	0	5
S48	6	4	2
				S49	39	17	22
S50	2	1	1
				S53	5	5	0
S54	4	2	2

S55	4	4	0
				S56	2	2	0
S57	3	2	1
				S58	12	6	6
S59	21	6	15
				S60	21	6	15
S61	3	2	1
				S62	3	1	2
S63	34	30	4
				S64	16	13	3
S65	26	13	13
				S66	18	17	1
S67	53	40	13
				S68	10	6	4
S69	21	17	4
				S70	63	22	41
S71	21	17	4
				S72	26	13	13
S73	15	9	6
				S74	2	1	1
S76	8	8	0
				S77	64	34	30
S78	30	15	15
				S79	32	23	9
S80	1	0	1
				S81	29	27	2
S82	3	2	1

Use GeneAlex software (Peakall and Slouse2005) according to above-mentioned expression overview, analyze (PCoA) by principal coordinate euclidean and measure the different similitude (Gower, 1966) between swashing of inviting.This is analyzed inviting of some level of display and swashs grouping (Figure 21).

Second group of 21 gene is joined above in 15 genes, thus improve separately described and grouping (table 18).

Table 18: unique qualification 77 kinds invites and swashs 21 required redundant genes and its order.

Position	Gene
		1	AT5G28235
2	AT2G15800
		3	AT5G28430
4	AT2G38240
		5	AT5G28760
6	AT5G54720
		7	AT3G53300
8	AT1G76650
		9	AT5G46200
10	AT3G31540
		11	AT3G57260
12	AT3G28820
		13	AT4G02330
14	AT5G02580
		15	AT3G05400
16	AT1G07120
		17	AT5G25110
18	AT1G61800
		19	AT3G28770
20	AT1G48110
		21	AT5G62750

This generates matrix shown in table 19, and wherein the gene outpost 1-21 of responsive genes S1-82 does not change (-1), lowers (0) or raise (1).

Table 19. invites sharp key (key) from table 18 21 identified for genes 77 kinds used.

Invite sharp
																						S1	0	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	1	-1	0	-1	0	0	1	-1	-1
S2	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	1	-1	-1	1	-1	-1	-1
																						S3	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1
S5	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1
																						S6	-1	-1	-1	-1	-1	0	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S7	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S8	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	1	-1	-1	-1
S9	0	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	1	-1	-1	-1	-1	-1	-1

S10	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1
																						S11	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	0	-1	-1	1	-1	-1	-1	-1	-1	1	1
S12	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0
																						S13	-1	-1	1	-1	-1	0	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	0	-1	-1	-1
S14	0	-1	-1	-1	0	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S15	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
S16	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S17	-1	-1	-1	-1	0	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S18	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S19	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	0
S20	0	-1	1	-1	-1	1	-1	1	-1	-1	1	-1	1	-1	-1	-1	-1	1	0	-1	-1
																						S21	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
S22	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0
																						S23	-1	-1	-1	-1	-1	1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1
S24	-1	-1	0	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
																						S25	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	0
S26	0	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	0	-1	-1	-1	-1	-1
																						S27	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S28	0	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
																						S29	0	-1	-1	-1	-1	1	-1	1	-1	-1	1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1
S30	0	-1	-1	-1	0	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	1
																						S31	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S32	-1	-1	-1	-1	0	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1
																						S33	0	-1	-1	-1	0	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1
S34	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
																						S35	-1	-1	-1	-1	0	-1	1	-1	-1	-1	-1	-1	0	-1	0	-1	-1	1	-1	-1	-1
S36	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
																						S37	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	1
S38	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S39	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	1	1	0
S40	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
																						S41	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	1	-1	-1
S42	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	0	0	1	1	-1	-1	-1	-1	-1
																						S43	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0
S44	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S45	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0
S46	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1
																						S47	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S48	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S49	0	-1	-1	-1	-1	1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1
S50	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S53	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1
S54	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	1	-1
																						S55	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1
S56	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S57	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	1	-1
S58	0	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
																						S59	-1	1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S60	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
																						S61	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S62	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1
																						S63	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	1	-1
S64	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	1	-1	-1	1	-1	-1	-1
																						S65	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	0	1	-1	-1	1	-1	0	-1	-1
S66	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1
																						S67	1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	0	1	-1	1	-1	1	-1	-1	1
S68	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S69	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1
S70	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	0	1	-1	-1	-1	1	-1	-1	-1
																						S71	-1	-1	1	-1	-1	1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	1	1	-1	1	-1
S72	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	1	-1	-1	0
																						S73	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	1	-1	-1	0	1	1	-1	1	-1
S74	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																						S76	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S77	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	0	1	0	-1	-1	-1	0	0	-1	-1	-1
																						S78	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	0	-1	-1	0	-1	0	1	-1	-1
S79	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	1	-1	1	-1	-1	-1
																						S81	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	1	-1	1	-1	-1	-1
S82	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1

Again, difference invites the similitude between swashing to analyze (PCoA) mensuration by above-mentioned principal coordinate euclidean.Second group of outpost's gene display is according to inviting the higher cluster level (Figure 22 and 23) swashing grouping.

Therefore, these data show to invite the outpost swashing qualification to organize to distinguish experience according to 39 kinds these are invited sharp and experience and do not invite sharp for the identification of the overwhelming majority of described outpost.In addition, sharp cluster is invited to provide some instruction to show that optimization detected the unknown of can extensively classifying to a certain extent invites sharp.

3rd group of 23 genes are joined above in 36 genes, thus improve invite sharp separately and strengthen cluster.Table 20.

Table 20. uniqueness qualification 77 kinds invites and swashs 23 required redundant genes and its order.

Position	Gene
		1	AT5G17860
2	AT2G38230
		3	AT4G08430
4	AT2G23510
		5	AT5G44415
6	AT4G08430
		7	AT5G06510
8	AT5G59310
		9	AT1G49920
10	AT3G28810
		11	AT2G06095
12	AT5G52760
		13	AT3G49580
14	AT1G73800
		15	AT5G44890
16	AT2G14560
		17	AT1G68875
18	AT3G23460
		19	AT1G48100
20	AT3G28270
		21	AT4G25040
22	AT1G04670
		23	AT4G36450

Again, difference invites the similitude between swashing to analyze (PCoA) mensuration by above-mentioned principal coordinate euclidean.This second group of gene display invites the higher cluster level (Figure 24) of swashing in grouping.

This generates matrix shown in table 21, and wherein the gene outpost 1-21 of responsive genes S1-82 does not change (-1), lowers (0) or raise (1).

Table 21. invites sharp key (key) from table 18 21 identified for genes 77 kinds used.

S1	0	-1	-1	-1	-1	0	0	0	-1	1	1	-1	0	-1	1	-1	-1	1	-1	0	-1	-1	-1
																								S2	1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S3	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	1
																								S5	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0
S6	-1	-1	-1	-1	0	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1
																								S7	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	1
S8	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S9	-1	-1	-1	-1	-1	0	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S10	-1	-1	1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1
																								S11	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	0	-1	-1	-1	1	1	0	-1	0
S12	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1
																								S13	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	0	-1	-1	-1	-1	0	-1	-1	0	-1	-1
S14	1	-1	1	-1	-1	0	-1	-1	-1	-1	0	1	1	-1	-1	-1	-1	0	-1	-1	-1	-1	0
																								S15	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	1	-1	-1
S16	-1	-1	1	0	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S17	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1
S18	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	1	-1	-1	-1	0	-1	-1	-1	-1	-1
																								S19	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S20	-1	-1	1	-1	-1	0	-1	-1	-1	-1	-1	1	-1	1	-1	-1	-1	-1	-1	0	-1	0	-1
																								S21	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	1	-1	-1
S22	-1	-1	-1	-1	-1	-1	0	0	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S23	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S24	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	0
																								S25	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	0	0	0	-1	0	-1	-1	-1	-1	-1	-1	-1
S26	-1	-1	1	-1	-1	0	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1
																								S27	0	-1	-1	-1	-1	1	-1	0	0	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	0	-1	-1	-1
S28	-1	-1	1	0	-1	0	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1
																								S29	1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	1	-1	1	-1	1	-1	0	-1	-1	-1	-1	-1
S30	-1	-1	1	0	-1	0	-1	-1	-1	-1	0	-1	-1	-1	0	-1	0	-1	-1	-1	-1	-1	-1
																								S31	-1	-1	1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1
S32	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1
																								S33	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1
S34	-1	-1	1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S35	-1	-1	1	-1	-1	-1	0	0	-1	-1	-1	1	0	1	-1	-1	-1	-1	-1	0	-1	-1	-1
S36	1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	1	-1	-1
																								S37	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
S38	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S39	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
S40	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S41	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1
S42	-1	0	-1	-1	-1	-1	1	0	-1	-1	-1	0	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	1
																								S43	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1
S44	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1
																								S45	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
S46	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1
																								S47	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S48	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1
																								S49	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	1	-1	1	-1	1	0	0	-1	-1	-1	-1	-1
S50	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S53	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
S54	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
																								S55	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
S56	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S57	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S58	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1
																								S59	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	0	-1	0	-1	-1	-1	-1	-1	-1	-1
S60	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	0	-1	0	-1	-1	-1	-1	-1	-1	-1
																								S61	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S62	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1
																								S63	-1	-1	-1	-1	-1	1	-1	1	-1	-1	-1	1	1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1
S64	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S65	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1
S66	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S67	-1	-1	-1	-1	-1	1	1	-1	-1	-1	-1	1	1	-1	-1	1	-1	1	-1	0	-1	-1	-1
S68	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S69	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1

S70	-1	1	-1	-1	-1	-1	-1	-1	0	-1	-1	0	-1	-1	-1	-1	-1	1	-1	-1	-1	0	-1
																								S71	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	1	-1	0	-1	-1	1	-1	-1	-1	-1
S72	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																								S73	-1	-1	-1	-1	-1	-1	-1	1	-1	1	-1	-1	-1	-1	-1	-1	0	-1	1	0	-1	-1	-1
S74	-1	-1	-1	-1	-1	-1	-1	1	-1	1	-1	-1	-1	-1	-1	-1	0	-1	1	0	-1	-1	-1
																								S76	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S77	0	-1	-1	-1	-1	-1	-1	-1	-1	1	1	-1	-1	-1	-1	1	0	-1	-1	1	-1	-1	-1
																								S78	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1
S79	1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1
																								S81	-1	-1	-1	-1	-1	-1	-1	1	-1	-1	-1	1	1	1	-1	-1	-1	-1	-1	-1	-1	-1	-1
S82	-1	-1	-1	-1	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1

The two-dimensional space that following figure shows as a PcoA gained information part represents.In fact, generating a certain space, wherein inviting sharp group separately to invite sharp by forming the volume of as many as 6 dimension.This can not illustrate, but information can be used for simulating (in silico).

These data show three the arabidopsis outpost genes training group being continuously developed to the program generation enumerating outpost's principle and comprising 15,21 and 23 genes respectively.Each outpost organizes and the experience 78 kinds studied can be diagnosed to invite sharp, shows as the expression different from control sample/methylate.

Therefore, can assemble and have a series of selection marquee subgroups of appropriate properties to express and/or the change of methylation state of DNA with showing as during these mark to detect the different of " normally " physiological status relevant with standard control growth conditions.The independence of these groups means that in all outpost's groups of instruction, the uniformity different from contrast state provides the confidence level increased in diagnosis accuracy.Outpost's group continue to increase accumulation and it is that scatter diagram becomes structuring more (Figure 21 and 24), because outpost's number of labels also increases according to inviting the cluster of sharp type to increase for another characteristic of multi-variables analysis jointly.

When being applied to often kind and inviting sharp repeat samples (as in embodiment 1 and 2), increase number of labels by this way and make the identical noise changing generation between sharp sample of inviting of experience minimum.Under this mode, described cluster analysis or other decision making algorithms inviting of more previously not tested may be swashed or stress reaction is correctly categorized into suitable grouping.

Checking

In order to verify the predictive ability of described method, generating 1000 data groups, repeatedly not inviting sharp half described in grab sample.Described sampling invites sharp half to be marked as training group, unsampled invite swash be called checking group.

Each described training group algorithm A analyzes 4 times, and setting N is 10,20,30 and 40.

Twice test (table 22) is carried out to described result.Test A takes out successively and eachly invites sharp from described checking group, and finds that it whether can unique qualification from training group.Test b found the number organized and when combination is all invite sharp time only there is the group number of a member.If invite to swash and do not show any change with regard to the expression of selected genes, it is called that sky is invited sharp.

Table 22

Test the result that A verifies research, display uniqueness can be identified and be not used in the number gene in 39 genes of the described method of training.

N	Unique number	Non-unique number	Empty number
				10	22.28±3.43	10.89±3.09	5.83±2.49
20	28.5±3.38	5.42±2.4	5.08±2.41
				30	31.46±2.87	3.52±1.93	4.02±2.16
40	33.39±2.48	2.37±1.57	3.23±1.9

Test b

N	Group number	Tool a kind invites sharp group number	Empty number
				10	59.03±3.34	50.32±4.73	6.34±2.47
20	66.15±3.45	61.18±4.66	5.24±2.46
				30	69.52±2.88	66.11±3.9	4.15±2.17
40	71.66±2.55	69.17±3.46	3.35±1.92

Embodiment 4 – invites sharp with regard to biological sample qualification

Can to swash and not for the identification of its stage of development to distinguish to invite by exploitation described sentry post locus.

The partial methylation group covering human chromosome 6,20 and 22 is delivered by (2006) such as Eckhardt.Use this information, select 2345 people's marks as possible outpost, wherein 40 identified for genes are have methylation states (table 22) different between organ.These 40 genes express with comprising different tissues application Genevestigator software cross reference (Zimmerman etc. 2004 and Zimmerman etc. 2005) Network Based changed, and also find differential expression (Figure 27) in these and other different tissues not including the research that methylates in, demonstrating its expression is methyl-sensitive.

In addition, when when multiple stage of development detects the expression of these genes and when cell/individuality stimulate widely, drug-treated, disease or genetic modification time, the described gene activating given organism and observe broad range difference express overview.These overviews can with regard to the unique overview of maximum (if not all) test condition set.

Use Genevestigator, can be measured these and mark 506 kinds of stimulation in corresponding people (comprise experience chemicals, organ transplant, disease and environment invite sharp) (Figure 16 and 25).Also the possible outpost's expression in end user in 8 age groups (Figure 17 and 26) and final 227 kinds of people's cell types (Figure 18 and 27) carries out this and analyzes.

If the expression changed often (ideally always) be associated with the change that methylates in these outpost's locus, the overview that methylates of so crossing over these locus changes disease, invites the medical diagnosis of sharp, physiological age and/or state or even identify there is a lot of application to biological material source.

In use research laboratory, hospital or medical diagnostic laboratory can the distinct methods of prior art this outpost's group can be used for be applied to sample.The possible technology of described outpost's methylation state of marking includes, but is not limited to the sulphite process using genomic DNA; then outpost described in the Auele Specific Primer pcr amplification using each outpost's locus is 1 afterwards) high-resolution melting curve (HRM) analyzes (Wojdacz and Dobrovic2007; White etc. 2007), 2) sulphite specificity order-checking (Frommer etc. 1992) or 3) sulphite specificity Manganic pyrophosphate complex initiation (Yang etc. 2004).Should understand each outpost's locus specific primers of design is conventional to those skilled in the art.

All three schemes have merits and demerits, but can high throughput analysis sample, can automation, and generate numerical data with easy analytical form.

The design of final scheme can depend on the scheme of selection.Once obtain the result of all outpost, it can introduce activity data storehouse, and described database comprises invites software (algorithm) described in the sharp preceding embodiment be associated by result with known.If do not find that positive overview is mated, then can carry out PCoA (principal coordinate analysis) (as in embodiment 3) with by as described in sample epigenetics overview with on show and invite sharp group to be associated.

In the embodiment that table 22 provides, provide methylation state with quantitative terms.Simple threshold values can be given (as value >50%=1 to these values; Value <50%=0) for the PCoA in such as embodiment 3.Or described data can be directly used in quantified system analysis such as principal component analysis and invite sharp Clustering to measure.

Table 23-is found to be the gene example of differential methylation.

In genome annotation to the mapping of (P<0.001) amplicons of 470 differential methylation can identify analyze the gene of differential methylation in tissue/cell type.48 genes of differential methylation are shown in described table display, have average methyl value respectively to each tissue.Color coding represents black=do not have, light gray=heterogeneous body and white=hyper-methylation.From Eckhardt etc., 2006 improvement.

Should be appreciated that those skilled in the art know that can to as herein described preferred embodiment at present

Carry out various change and improvement.Can do not depart from spirit and scope of the invention and do not reduce the present invention with

When advantage, carry out these and change and improvement.Therefore be intended to appended claims and comprise these changes

And improvement.

The content of all references cited herein is included in herein by reference of text.

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Claims

1. the method in the exposure experienced with diagnosis and identification of organism sample of the compilation target DNA sequence dna group that is positioned at biological genome DNA and/or tissue and/or cell category or source, described method comprises

(i) from two or more known but experience different expose and/or variety classes or source cell sample DNA isolation;

(ii) qualification is from the methylation state of the methylated locus of energy in the DNA of each sample separation;

(iii) methylation state of the DNA be separated from each sample is compared; With

(iv) select to have the locus subgroup of following characteristics, the feature of described subgroup is: different but can not make a definite diagnosis separately the methylation state of each locus in single exposure and/or kind or source with sample in some sample;

Wherein, described method is not used in the medical diagnosis on disease of humans and animals.

2. the method for claim 1, it is characterized in that, the methylation state of described target DNA sequence dna group or selected locus subgroup diagnoses (i) jointly not for the atypical growth condition of Select gene seat and/or tissue/cell type and/or its combination; (ii) the atypical growth condition that Select gene seat is used and/or tissue/cell type and/or its combination.

3. the method for claim 1, it is characterized in that, described method comprise use at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more plant different sharp, organization type or the cell type invited to select target DNA sequence dna group or locus subgroup.

4. the method for claim 1, is characterized in that, each locus in described locus subgroup or each selected DNA sequence dna have one or more following properties,

(ii), between the repeat samples taking from same individual homologue at same time, methylated state does not change or is less than the change of about 20%;

(v) respond some invite swash and do not respond other invite swash and change methylation state;

(vi) comprise represent major part or all (a) biological stress reaction path or (b) cell and/or organization type based on methylated locus;

(vii) combine respectively according to each tissue/organ type of biology;

(viii) constant between different tissues/stage of development or be less than about 10% change, unless be only intended to for specifically organizing, organ or stage of development.

5. the method for claim 1, is characterized in that, each locus in described locus subgroup or each selected DNA sequence dna complementally distinctiveness are present in the inviting in sharp or sample source for Select gene seat of roughly half.

6. identification of dna locus group (inviting sharp outpost) is to differentiate a method for atypical growth condition (inviting sharp), and described method comprises step:

I () cultivates interested cell sample under contrast growth conditions;

(ii) exist in the environment of at least one atypical growth condition, cultivate at least one identical cells of interest sample described with step (i);

(iii) from step (i) and DNA isolation each cell sample of (ii);

(vi) the locus subgroup with following characteristics is selected, the feature of described subgroup is: in the DNA be separated in step (ii), the methylation state of these locus is different from the DNA be separated in step (i), and in described subgroup, each locus responds some atypical growth condition and do not respond other conditions and change methylation state;

7. method as claimed in claim 6, it is characterized in that, the methylation state of described target DNA sequence dna group or selected locus subgroup diagnoses (i) jointly not for the atypical growth condition of Select gene seat and/or tissue/cell type and/or its combination; (ii) the atypical growth condition that Select gene seat is used and/or tissue/cell type and/or its combination.

8. method as claimed in claim 6, it is characterized in that, described method comprise use at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more plant different sharp, organization type or the cell type invited to select target DNA sequence dna group or locus subgroup.

9. method as claimed in claim 6, it is characterized in that, each locus in described locus subgroup or each selected DNA sequence dna have one or more following properties,

(vii) combine respectively according to each tissue/organ type of biology;

10. method as claimed in claim 6, is characterized in that, each locus in described locus subgroup or each selected DNA sequence dna complementally distinctiveness are present in the inviting in sharp or sample source for Select gene seat of roughly half.

11. 1 kinds of identification of dna locus groups are to differentiate the method for one or more atypical growth conditions, and described method comprises step:

I () cultivates interested cell sample under contrast growth conditions;

(iii) from step (i) and DNA isolation each cell sample of (ii);

(vi) the locus subgroup with following characteristics is selected, the feature of described subgroup is: the methylation state of these locus in the DNA be separated in step (ii) is different from the DNA be separated in step (i), and in described subgroup, each locus responds some atypical growth condition and do not respond other conditions and change methylation state;

(vii) the locus subgroup selected in authentication step (vi);

(viii) methylation state of selected subgroup locus is associated with atypical growth condition and/or cells of interest sample;

12. methods as claimed in claim 11, it is characterized in that, described method also comprises following optional step:

(ix) by the methylation state of selected genes seat subgroup be not used for the atypical growth condition of Select gene seat and/or tissue/cell type and/or its and combine and be associated.

13. methods as claimed in claim 11, it is characterized in that, described method also comprises following optional step:

(ix) locus subgroup and its methylation state and cell sample or tissue, organ or the phenotype obtained in the biology of described cell sample and/or physiological reaction are associated.

14. methods as claimed in claim 11, it is characterized in that, in described step (ii), multiple sample is 1-10, invites sharp for 000 kind, comprise 10-500 kind invite swash and/or 30-100 kind invite sharp.

15. methods as claimed in claim 11, it is characterized in that, described method also comprises following optional step:

(xi) DNA isolation from cell sample, the growth conditions of wherein said cell is unknown;

(xii) methylation state of the locus subgroup that step (vii) is identified in the DNA be separated in authentication step (xi);

(xiii) methylation state of the locus of qualification in (xii) is compared with the result of step (viii), thus determine whether the cell sample of (xi) once went through atypical growth condition;

Described method is not used in the medical diagnosis on disease of humans and animals.

16. methods as claimed in claim 11, it is characterized in that, described method also comprises following optional step:

(xi) DNA isolation from cell sample, wherein, when being in one or more atypical growth conditions the phenotype of described cell (or its derived tissues/biology) and/or physiological reaction unknown;

(xii) methylation state of step (vii) institute identified gene seat subgroup in the DNA be separated in authentication step (xi);

(xiii) by the locus methylation state of qualification in (vi) with the result of step (x) compared with, thus determine whether described cell (or its derived tissues/biology) answers known or unknown atypical growth condition and phenotype and/or physiological reaction occur;

17. methods as described in claim 15 or 16, it is characterized in that, described step (xiii) goes back the type of the atypical growth condition that identification of cell was once gone through.

18. methods as described in claim 15 or 16, is characterized in that, described step (xiii) identifies cell and once went through the atypical growth condition that step (ii) do not tested.

19. methods as claimed in claim 11, is characterized in that, described DNA locus subgroup comprises 1 –, 5000 locus, comprise 10-1000 locus and/or 20-100 locus.

20. methods as claimed in claim 11, it is characterized in that, described contrast growth conditions is identical with the normal growth conditions in vivo of the biology of cells of interest.

21. methods as claimed in claim 11, is characterized in that, the different atypical growth conditions in described step (ii) are non-convergency difference.

22. methods as claimed in claim 11, it is characterized in that, described atypical growth condition is selected from: temperature, physics invites the sharp (atmospheric pressure comprising increase or reduce, invite sharp to the direction of object, stir, gravity, physical stress), physical damnification, nutrient availability, be exposed to chemical toxicant, hormone or signaling molecule, be exposed to other chemical reagent, visible ray is invited sharp, the black light radiation be exposed on background level (comprises X-ray, gamma-rays, infrared light or ultraviolet light, microwave etc.), be exposed to pathogen or parasite, be exposed to other biological or other members mutually of the same race, be exposed to other biological, individual exudate, food grass or another biology take target organisms as food, water invites excitation to swash (comprise too much or very little), be exposed to the signal or the condition that stimulate described object to enter rest stage, oxygen is invited and is swashed or be exposed to excessive or secondary good free radical.

23. methods as claimed in claim 11, is characterized in that, the locus subgroup of selection be associated with the gene of cellular stress pathway component or its control band in described step (vii).

24. methods as claimed in claim 23, is characterized in that, described locus is present in the gene of at least one composition in each stress reaction path in cells of interest.

25. methods as claimed in claim 11, is characterized in that, in described step (vi), the selection of locus subgroup is carried out automatically by computer program.

26. 1 kinds of qualification many groups DNA locus are to differentiate the method for atypical growth condition, and described method comprises step:

(ii) in the environment that there is at least one atypical growth condition, multiple cells of interest sample described in incubation step (i);

(iii) from step (i) and DNA isolation each cell sample of (ii);

(iv) can the methylation state of methylated locus in the DNA that is separated of authentication step (iii);

(v) comparison step (i) and (ii) or (i) methylation state with DNA isolation in (iii) or (ii) and (iii);

(vi) select the locus subgroup with following characteristics, the feature of described subgroup is: in the DNA be separated in step (ii), the methylation state of these locus is different from the DNA be separated in step (i) and/or (iii); And/or step (iii) in be separated DNA in locus methylation state and difference compared with (i), in described subgroup, each locus responds some atypical growth condition and does not respond other conditions and change methylation state;

(vii) the locus subgroup selected in authentication step (vii);

(viii) locus subgroup and its methylation state are associated with the atypical growth condition and/or cells of interest sample being used for Select gene seat;

27. methods as claimed in claim 26, it is characterized in that, described method also comprises following optional step:

28. methods as claimed in claim 26, it is characterized in that, described method also comprises following optional step:

29. methods as claimed in claim 26, it is characterized in that, in described step (ii), multiple sample is 1-10, invites sharp for 000 kind, comprise 10-500 kind invite swash and/or 30-100 kind invite sharp.

30. methods as claimed in claim 26, it is characterized in that, described method also comprises following optional step:

31. methods as claimed in claim 26, it is characterized in that, described method also comprises following optional step:

32. methods as described in claim 30 or 31, it is characterized in that, described step (xiii) goes back the type of the atypical growth condition that identification of cell was once gone through.

33. methods as described in claim 30 or 31, is characterized in that, described step (xiii) identifies cell and once went through the atypical growth condition that step (ii) do not tested.

34. methods as claimed in claim 26, is characterized in that, described DNA locus subgroup comprises 1 –, 5000 locus, comprise 10-1000 locus and/or 20-100 locus.

35. methods as claimed in claim 26, it is characterized in that, described contrast growth conditions is identical with the normal growth conditions in vivo of the biology of cells of interest.

36. methods as claimed in claim 26, is characterized in that, the different atypical growth conditions in described step (ii) are non-convergency difference.

37. methods as claimed in claim 26, it is characterized in that, described atypical growth condition is selected from: temperature, physics invites the sharp (atmospheric pressure comprising increase or reduce, invite sharp to the direction of object, stir, gravity, physical stress), physical damnification, nutrient availability, be exposed to chemical toxicant, hormone or signaling molecule, be exposed to other chemical reagent, visible ray is invited sharp, the black light radiation be exposed on background level (comprises X-ray, gamma-rays, infrared light or ultraviolet light, microwave etc.), be exposed to pathogen or parasite, be exposed to other biological or other members mutually of the same race, be exposed to other biological, individual exudate, food grass or another biology take target organisms as food, water invites excitation to swash (comprise too much or very little), be exposed to the signal or the condition that stimulate described object to enter rest stage, oxygen is invited and is swashed or be exposed to excessive or secondary good free radical.

38. methods as claimed in claim 26, is characterized in that, the locus subgroup of selection be associated with the gene of cellular stress pathway component or its control band in described step (vii).

39. methods as claimed in claim 38, is characterized in that, described locus is present in the gene of at least one composition in each stress reaction path in cells of interest.

40. methods as claimed in claim 26, is characterized in that, in described step (vi), the selection of locus subgroup is carried out automatically by computer program.

41. 1 kinds of identification of dna locus groups are to differentiate the method for multiple atypical growth condition, and described method comprises step:

I () cultivates interested cell sample under contrast growth conditions;

(iii) from step (i) and DNA isolation each cell sample of (ii);

(vii) the locus subgroup selected in authentication step (vi);

(viii) different from (ii) with its methylation state for locus subgroup atypical growth condition is associated;

42. methods as claimed in claim 41, it is characterized in that, described method also comprises following optional step:

43. methods as claimed in claim 41, it is characterized in that, described method also comprises following optional step:

44. methods as claimed in claim 41, it is characterized in that, in described step (ii), multiple sample is 1-10, invites sharp for 000 kind, comprise 10-500 kind invite swash and/or 30-100 kind invite sharp.

45. methods as claimed in claim 41, it is characterized in that, described method also comprises following optional step:

46. methods as claimed in claim 41, it is characterized in that, described method also comprises following optional step:

47. methods as described in claim 45 or 46, it is characterized in that, described step (xiii) goes back the type of the atypical growth condition that identification of cell was once gone through.

48. methods as described in claim 45 or 46, is characterized in that, described step (xiii) identifies cell and once went through the atypical growth condition that step (ii) do not tested.

49. methods as claimed in claim 41, is characterized in that, described DNA locus subgroup comprises 1 –, 5000 locus, comprise 10-1000 locus and/or 20-100 locus.

50. methods as claimed in claim 41, it is characterized in that, described contrast growth conditions is identical with the normal growth conditions in vivo of the biology of cells of interest.

51. methods as claimed in claim 41, is characterized in that, the different atypical growth conditions in described step (ii) are non-convergency difference.

52. methods as claimed in claim 41, it is characterized in that, described atypical growth condition is selected from: temperature, physics invites the sharp (atmospheric pressure comprising increase or reduce, invite sharp to the direction of object, stir, gravity, physical stress), physical damnification, nutrient availability, be exposed to chemical toxicant, hormone or signaling molecule, be exposed to other chemical reagent, visible ray is invited sharp, the black light radiation be exposed on background level (comprises X-ray, gamma-rays, infrared light or ultraviolet light, microwave etc.), be exposed to pathogen or parasite, be exposed to other biological or other members mutually of the same race, be exposed to other biological, individual exudate, food grass or another biology take target organisms as food, water invites excitation to swash (comprise too much or very little), be exposed to the signal or the condition that stimulate described object to enter rest stage, oxygen is invited and is swashed or be exposed to excessive or secondary good free radical.

53. methods as claimed in claim 41, is characterized in that, the locus subgroup of selection be associated with the gene of cellular stress pathway component or its control band in described step (vii).

54. methods as claimed in claim 53, is characterized in that, described locus is present in the gene of at least one composition in each stress reaction path in cells of interest.

55. methods as claimed in claim 41, is characterized in that, in described step (vi), the selection of locus subgroup is carried out automatically by computer program.

56. 1 kinds of methods identifying one or more atypical growth conditions that cells of interest was once gone through, described method comprises step:

I () be DNA isolation from cells of interest, the growth conditions of wherein said cell is unknown;

(ii) can the methylation state of methylated locus group in DNA isolation in authentication step (i);

(iii) locus methylation state step (ii) identified is compared with the known methylation state group of homologous genes seat group, and wherein, described known methylation state group characterizes one or more and invites sharp;

(iv) determine whether cells of interest once went through atypical growth condition;

57. methods as claimed in claim 56, it is characterized in that, described step (iv) goes back the type of the atypical growth condition that identification of cell was once gone through.