CN106319059A - Methylation sequencing method of single-chromosome levels - Google Patents
Methylation sequencing method of single-chromosome levels Download PDFInfo
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- CN106319059A CN106319059A CN201610800002.8A CN201610800002A CN106319059A CN 106319059 A CN106319059 A CN 106319059A CN 201610800002 A CN201610800002 A CN 201610800002A CN 106319059 A CN106319059 A CN 106319059A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention relates to a methylation sequencing method of single-chromosome levels. The methylation sequencing method includes the steps of 1), culturing and collecting lymphocyte in to-be-detected samples; 2), selecting single cells with micro-injection and isolating chromosome to obtain monoploids; 3), releasing the monoploids and processing the same with Bisulfite, converting C base not subjected to methylation in genome into U which is changed into T after performing PCR (polymerase chain reaction) amplification, distinguishing from the C base originally having methylation modification, and utilizing the high-throughput sequencing technology to perform methylation sequencing detection. The methylation sequencing method has the advantages of low cost, simpleness in testing method, short time-consuming cycle, mature equipment and the like and can be widely used in clinical research of early diagnosis and differential diagnosis of cancers; the method has the advantages of methylation sequencing detection method of whole-genome and single cell levels, genetic origin of methylation information can be determined, and the method can be widely applied in research of epigenetic inheritance.
Description
Technical field
The present invention relates to area of medical genetics, particularly relate to the sequence measurement that methylates of a kind of monosome level.
Background technology
Methylation analysis is mainly based upon the analysis method of full-length genome and individual cell level at present.
1. full-length genome methylates order-checking.DNA methylation order-checking can in full-length genome level to greatest extent, complete
Obtain methylation state information and the multirelation of gene expression regulation, can efficiently be precisely accomplished full-length genome methylate order-checking and
High-resolution DNA methylation spectrum formula is drawn.But, due to full-length genome methylated detection means analysis is the overall of cell mass
Average response, thus the average result of this cell mass can flood the reaction of individual cells, such as, certain gene of cancerous tissue
Promoter region methyl rate is higher than a little higher than cancer beside organism, and the methylated detection means of full-length genome cannot judge this methyl
The faint rising of rate is to have a faint rising due to cell methylation level various in whole cancerous tissue, or due to
The methylation level of a certain cell drastically raises and causes；It the most also can obscure us to brain, blood system, immunity
Understanding heterogeneous between the cell of system and these systems of composition thereof.
The most unicellular DNA methylation detects.Different single celled DNA methylation assay contribute to recognizing the difference between cell,
And from whole system, select multiple different unicellular study, then can reconstruct whole system, this process of reconstruction
More more valuable information can be provided.But, the detection of unicellular DNA methylation is provided that single celled methylation information,
But monosomic methylation information cannot be accurate to.Methylating of DNA can be hereditary, such as, and the table entrained by sperm
See hereditary information and be passed to filial generation, and regulate and control the gene expression of early embryonic development.Or the journey that methylates of imprinted gene
Degree difference, this depends on that it is from father or mother.The detection of unicellular DNA methylation possibly cannot determine this methylated
Information is that heredity is in male parent or female parent.
In a word, above two kinds of conventional methods cannot analysis of methylation information in monosome level accurately.
Summary of the invention
In view of problems of the prior art, the invention provides the order-checking side that methylates of a kind of monosome level
Method, the method that the present invention provides has that low cost, test method cycle simple, time-consuming be short and the advantage such as equipment is ripe, can be wide
In the clinical research of the general early diagnosis being applied to cancer and Differential Diagnosis, it possesses full-length genome and individual cell level methylates
The advantage of detection method also can determine that the genetic origin of methylation information, thus can be widely applied to the research of epigenetic.
For reaching this purpose, the present invention by the following technical solutions:
The invention provides the sequence measurement that methylates of a kind of monosome level, said method comprising the steps of:
(1) cultivate and the lymphocyte of collecting in sample to be tested；
(2) microinjection picking is unicellular and separates chromosome to obtain monoploid；
(3) monoploid is diluted, with Bisulfite process, genome will not occur methylated C base transition
Become U, after carrying out PCR amplification, become T, distinguish with originally having the C base modified that methylates, utilize high throughput sequencing technologies to carry out
Methylate order-checking.
The present invention mainly provides a kind of use microinjection technique and separates monosomic method, utilizes the method real
Analysis of methylation information on Chromosome level now, and determine the source of hereditary information.
The method that the present invention provides has that low cost, test method cycle simple, time-consuming be short and the advantage such as equipment is ripe,
Can be widely applied in the early diagnosis of cancer and the clinical research of Differential Diagnosis and can determine that the genetic origin of methylation information.
According to the present invention, step (1) described cultivation and the lymphocyte collected in sample to be tested, including following operation:
A) gather the peripheric venous blood blood sample of human body, in cell culture fluid, drip 30-35 under aseptic condition bleed
Liquid sample；
B) in 37 DEG C after mixing, 5%CO2Under the conditions of, cultivate 69h；
C) add Colchicine under aseptic condition and cultivate 3-5h to final concentration of 0.2 μ g/mL, continuation；
D) collection is in the lymphocyte of mitosis metaphase, and is dispersed into cell suspending liquid, and 4 DEG C save backup.
In the present invention, the peripheric venous blood blood sample of the human body gathered can as little as 1-2mL, therefore, this detection method
Have the advantages that sample consumption is few.
In the present invention, it is preferred to use specification is that the syringe of 2mL drips blood sample in cell culture fluid, for example with
Specification is that the syringe of 2mL liquid sample drop of being bled by 30-35 is added in the Tissue Culture Flask containing cell culture medium；For cell
The number of culture bottle can be such as 2 bottles, 3 bottles etc., do not do particular determination at this.
According to the present invention, step (1) described cell culture fluid uses commercially available lymphocyte culture fluid, those skilled in the art
Can select according to actual needs, not do particular determination at this.
Exemplarily, monosome level of the present invention methylate in sequence measurement, step (1) specifically uses as follows
Operation:
Gather the peripheric venous blood blood sample 2mL of human body, it is provided that two Tissue Culture Flasks containing cell culture fluid, nothing
2mL syringe is used to add 30 blood samples respectively in cell culture fluid, in 37 DEG C, 5%CO after shaking up gently under the conditions of bacterium2
Hatching 69h, making its ultimate density after adding colchicine is 0.2 μ g/mL, continues to cultivate 3h；Receive after two bottles of culture fluid are merged
Collection lymphocyte, is dispersed into cell suspending liquid by the lymphocyte of collection, and 4 DEG C save backup.
According to the present invention, step (2) described microinjection picking is unicellular and separates chromosome to obtain monoploid, including
Below operation:
A) cell pyrolysis liquid of component it is formulated as follows:
B) under microscope from cell suspending liquid one medium cell of picking, be placed in the cell pyrolysis liquid of step a), aobvious
Under micro mirror after observation of cell cracking release chromosome, whole chromosomes are drawn in microinjection, and are discharged into being centrifuged containing pure water
Guan Zhong；
C) the pure water equimultiple containing chromosome of mixing diluted and divide equally to 8 centrifuge tubes containing pure water.
Preferably, step b) and the described centrifuge tube of step c) all there are 9 μ L pure water.
Exemplarily, monosome level of the present invention methylate in sequence measurement, step (2) specifically uses as follows
Operation:
Under microscope from cell suspending liquid one medium cell of picking, be placed in the most ready cell pyrolysis liquid,
After basis of microscopic observation cell cracking release chromosome, whole chromosomes are drawn in microinjection, and are discharged into and have 9 μ L pure water
In centrifuge tube, after soft piping and druming mixing, the 9 μ L of the mixing pure water equimultiple containing chromosome is diluted and divides equally to 8 centrifuge tubes,
Often pipe has the 9 μ L pure water containing chromosome.
Exemplarily, the sequence measurement that methylates of monosome level of the present invention, comprise the following steps:
(1) the peripheric venous blood blood sample 2mL of human body is gathered, it is provided that two cells containing cell culture fluid are cultivated
Bottle, under aseptic condition use 2mL syringe in cell culture fluid, add 30 blood samples respectively, after shaking up gently in 37 DEG C,
5%CO2Hatching 69h, making its ultimate density after adding colchicine is 0.2 μ g/mL, continues to cultivate 3h；Two bottles of culture fluid are closed
Collecting lymphocyte after and, the lymphocyte of collection is dispersed into cell suspending liquid, 4 DEG C save backup；
(2) cell pyrolysis liquid of component it is formulated as follows:
Under microscope from cell suspending liquid one medium cell of picking, be placed in the most ready cell pyrolysis liquid,
After basis of microscopic observation cell cracking release chromosome, whole chromosomes are drawn in microinjection, and are discharged into and have 9 μ L pure water
In centrifuge tube, after soft piping and druming mixing, the 9 μ L of the mixing pure water equimultiple containing chromosome is diluted and divides equally to 8 centrifuge tubes,
Often pipe has the 9 μ L pure water containing chromosome；
(3) monoploid is diluted, with Bisulfite process, genome will not occur methylated C base transition
Become U, after carrying out PCR amplification, become T, distinguish with originally having the C base modified that methylates, utilize high throughput sequencing technologies to carry out
Methylate order-checking；
Described method is not with treatment and is diagnosed as purpose.
In the present invention, step (3) described Bisulfite process is to use method well known in the art to carry out, and does not does at this
Particular determination.
In the present invention, step (3) described high throughput sequencing technologies is that those skilled in the art use according to actual needs
Known technology, does not do particular determination at this.
In the present invention, the cell of described sample to be tested is lymphocyte.
Compared with prior art, the present invention at least has the advantages that
(1) present invention is by carrying out the order-checking that methylates under monosome level, compares employing genome sequencing, permissible
Make result more accurately and reliable, and blood sample used can as little as 1-2mL, have the advantages that sample consumption is few；
(2) method that the present invention provides has that low cost, test method cycle simple, time-consuming be short and equipment is ripe etc. excellent
Point, can be widely applied in the early diagnosis of cancer and the clinical research of Differential Diagnosis, and it possesses full-length genome and unicellular water
The advantage of flat methylation detecting method also can determine that the genetic origin of methylation information, thus can be widely applied to epigenetic
Research.
Accompanying drawing explanation
Fig. 1 be microinjection separate one mid-term lymphocyte operation chart；
Fig. 2 is the schematic diagram of monosome stepwise dilution to 8 parts；
Fig. 3 is to build monosome to methylate the flow chart of sequencing library；
Fig. 4 is the sequencing data bioinformatic analysis figure that methylates.
The present invention is described in more detail below.But following example is only the simple example of the present invention, not generation
Table or restriction the scope of the present invention, protection scope of the present invention is as the criterion with claims.
Detailed description of the invention
Further illustrate technical scheme below in conjunction with the accompanying drawings and by detailed description of the invention.
For the present invention is better described, it is simple to understand technical scheme, the present invention's is typical but non-limiting
Embodiment is as follows:
The cultivation of embodiment 1 lymphocyte and collection
Gather the peripheric venous blood blood sample 2mL of human body, under aseptic condition, use 2mL syringe respectively to two bottles of lymphs
Cell culture fluid adds 35 blood samples, in 37 DEG C, 5%CO after shaking up gently2Hatch 69h, after adding colchicine and make
Its ultimate density is 0.2 μ g/mL, continues to cultivate 3h.Merge two bottles of culture fluid, collect lymphocyte, the lymphocyte that will collect
Being dispersed into cell suspending liquid, 4 DEG C save backup.
Cell suspending liquid formulation components is as follows:
The unicellular picking of embodiment 2 and monosome separate
It is formulated as follows the cell pyrolysis liquid of component:
10 μ L Lymphocyte suspension in extraction embodiment 1 are placed in culture dish, and basis of microscopic observation uses micro-note
System of penetrating draws a medium cell, and its concrete operations are as shown in Figure 1；It is placed on the most ready light and slow cell membrane lysis liquid
In, basis of microscopic observation to cell cracks, and after Chromosome spread, whole chromosomes are drawn in microinjection, and are discharged into and have 9 μ L
In the centrifuge tube of pure water, after soft piping and druming mixing, the 9 μ L of the mixing pure water equimultiple containing chromosome is diluted and divide equally to 8 from
In heart pipe, making often pipe have the 9 μ L pure water containing chromosome, Fig. 2 shows the process of monosome stepwise dilution to 8 parts.
Embodiment 3 chromosome amplification and library construction
There are 8 centrifuge tubes of the 9 μ L pure water containing chromosome, use the whole genome amplification method of WGA4 and WGA3 to dye
Colour solid expands.Chromosome after amplification is through Bisulfite process, and end adds primer and labelling, builds after PCR expands
Sequencing library, it builds the process of sequencing library as shown in Figure 3.
Embodiment 4 high-flux sequence and bioinformatic analysis
The monosome library built obtains initial data through high-flux sequence, in each centrifuge tube of bioinformatic analysis
The chromosome number contained and numbering, the sequencing data bioinformatic analysis process that methylates as shown in Figure 4, its detailed process
For: initial data is carried out pretreatment, by genome alignment, the BAM/SAM file obtained is carried out methylation analysis, respectively
Obtain the order-checking degree of depth and methylation, CpG/CHG/CHH site methylation differential and genome area methylation differential, obtain
Obtain the report of CpG island, thus obtain the methylation information of the monosome level of gene.The present invention uses a kind of visual annular
Figure shows the chromosome distribution situation at 8 centrifuge tubes.
By embodiment 1-4, present invention achieves and carry out the order-checking that methylates under monosome level, it is accurate that it has result
The feature that blood sample the most reliable, used is low, can be widely applied to the early diagnosis of cancer and the clinical research of Differential Diagnosis
In, it possesses full-length genome and the advantage of individual cell level methylation detecting method and can determine that the heredity of methylation information comes
Source, thus can be widely applied to the research of epigenetic.
Applicant states, the present invention illustrates the detailed construction feature of the present invention by above-described embodiment, but the present invention is also
It is not limited to above-mentioned detailed construction feature, does not i.e. mean that the present invention has to rely on above-mentioned detailed construction feature and could implement.Institute
Belonging to those skilled in the art it will be clearly understood that any improvement in the present invention, the equivalence to parts selected by the present invention is replaced
And the increase of accessory, concrete way choice etc., within the scope of all falling within protection scope of the present invention and disclosure.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited in above-mentioned embodiment
Detail, in the technology concept of the present invention, technical scheme can be carried out multiple simple variant, this
A little simple variant belong to protection scope of the present invention.
It is further to note that each the concrete technical characteristic described in above-mentioned detailed description of the invention, at not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to various can
The compound mode of energy illustrates the most separately.
Additionally, combination in any can also be carried out between the various different embodiment of the present invention, as long as it is without prejudice to this
The thought of invention, it should be considered as content disclosed in this invention equally.
Claims (7)
1. the sequence measurement that methylates of a monosome level, it is characterised in that said method comprising the steps of:
(1) cultivate and the lymphocyte of collecting in sample to be tested；
(2) microinjection picking is unicellular and separates chromosome to obtain monoploid；
(3) monoploid is diluted, with Bisulfite process, genome will not occur methylated C base transition become U,
Become T after carrying out PCR amplification, distinguish with originally having the C base modified that methylates, utilize high throughput sequencing technologies to carry out methyl
Change order-checking；
Described method is not with treatment and is diagnosed as purpose.
2. the method for claim 1, it is characterised in that step (1) described cultivation and the lymph collected in sample to be tested
Cell, including following operation:
A) gather the peripheric venous blood blood sample of human body, in cell culture fluid, drip 30-35 under aseptic condition bleed liquid sample
This；
B) in 37 DEG C after mixing, 5%CO2Under the conditions of, cultivate 69h；
C) add Colchicine under aseptic condition and cultivate 3-5h to final concentration of 0.2 μ g/mL, continuation；
D) collection is in the lymphocyte of mitosis metaphase, and is dispersed into cell suspending liquid, and 4 DEG C save backup.
3. method as claimed in claim 2, it is characterised in that in step a), use the syringe of 2mL specification under aseptic condition
Blood sample is dripped in cell culture fluid.
4. the method as described in one of claim 1-3, it is characterised in that step (2) described microinjection picking is unicellular also
Separation chromosome is to obtain monoploid, including following operation:
A) cell pyrolysis liquid of component it is formulated as follows:
B) under microscope from cell suspending liquid one medium cell of picking, be placed in the cell pyrolysis liquid of step a), microscope
After lower observation of cell cracking release chromosome, whole chromosomes are drawn in microinjection, and are discharged in the centrifuge tube containing pure water；
C) the pure water equimultiple containing chromosome of mixing diluted and divide equally to 8 centrifuge tubes containing pure water.
5. method as claimed in claim 4, it is characterised in that all have 9 μ L in step b) and the described centrifuge tube of step c) pure
Water.
6. the method as described in one of claim 1-5, it is characterised in that said method comprising the steps of:
(1) the peripheric venous blood blood sample 2mL of human body is gathered, it is provided that two Tissue Culture Flasks containing cell culture fluid, nothing
2mL syringe is used to add 30 blood samples respectively in cell culture fluid, in 37 DEG C, 5%CO after shaking up gently under the conditions of bacterium2
Hatching 69h, making its ultimate density after adding colchicine is 0.2 μ g/mL, continues to cultivate 3h；Receive after two bottles of culture fluid are merged
Collection lymphocyte, is dispersed into cell suspending liquid by the lymphocyte of collection, and 4 DEG C save backup；
(2) cell pyrolysis liquid of component it is formulated as follows:
Under microscope from cell suspending liquid one medium cell of picking, be placed in the most ready cell pyrolysis liquid, micro-
After Microscopic observation cell cracking release chromosome, whole chromosomes are drawn in microinjection, and are discharged into and have the centrifugal of 9 μ L pure water
Guan Zhong, after soft piping and druming mixing, dilutes the 9 μ L of the mixing pure water equimultiple containing chromosome and divides equally to 8 centrifuge tubes, often managing
There is the 9 μ L pure water containing chromosome；
(3) monoploid is diluted, with Bisulfite process, genome will not occur methylated C base transition become U,
Become T after carrying out PCR amplification, distinguish with originally having the C base modified that methylates, utilize high throughput sequencing technologies to carry out methyl
Change order-checking；
Described method is not with treatment and is diagnosed as purpose.
7. the method as described in one of claim 1-6, it is characterised in that the cell of described sample to be tested is lymphocyte.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610800002.8A CN106319059A (en) | 2016-08-31 | 2016-08-31 | Methylation sequencing method of single-chromosome levels |
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| CN201610800002.8A CN106319059A (en) | 2016-08-31 | 2016-08-31 | Methylation sequencing method of single-chromosome levels |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087484A (en) * | 2015-08-14 | 2015-11-25 | 南方科技大学 | Single chromosome segregation method, single chromosome high-throughput sequencing library construction method and application |
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2016
- 2016-08-31 CN CN201610800002.8A patent/CN106319059A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087484A (en) * | 2015-08-14 | 2015-11-25 | 南方科技大学 | Single chromosome segregation method, single chromosome high-throughput sequencing library construction method and application |
Non-Patent Citations (3)
| Title |
|---|
| 哈斯木其尔等: "DNA 甲基化及其在动物遗传育种上的研究进展", 《内蒙古民族大学学报（自然科学版）》 * |
| 李懿等: "DNA甲基化检测技术进展及经验总结", 《医学综述》 * |
| 赖伊杰等: "肿瘤细胞DNA甲基化检测技术与研究方法新进展", 《生命科学》 * |
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