CN109423518A - Detect the composition and method of the methylate DNA of Septin9 gene - Google Patents
Detect the composition and method of the methylate DNA of Septin9 gene Download PDFInfo
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- CN109423518A CN109423518A CN201710754697.5A CN201710754697A CN109423518A CN 109423518 A CN109423518 A CN 109423518A CN 201710754697 A CN201710754697 A CN 201710754697A CN 109423518 A CN109423518 A CN 109423518A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The composition and corresponding kit that the present invention provides a kind of for detecting the methylate DNA of SEPT9 gene；The composition includes at least one methylation sensitive restriction restriction endonuclease, and the nucleic acid at least one region for expanding SEPT9 gene and its segment.The present invention also provides a kind of methods of methylate DNA for detecting SEPT9 gene.Compared with prior art, the present invention has the advantage that: 1) compared with the methylate DNA detection method based on bisulfite conversion, method detection time provided by the invention is shorter；2) compared with the methylate DNA detection method based on bisulfite conversion, the reagent stability that the present invention uses is strong；3) operating procedure is simple；4) DNA loss is few, and detection sensitivity is higher.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of composition and its use in the methylate DNA of detection gene
On the way, more particularly it relates to it is a kind of for detecting the composition of the methylate DNA of Septin9 gene.
Background technique
(SEPT9) gene of source of people Septin 9 is located at human chromosomal 17q25.3, is septin gene family member, ginseng
With the regulation cell biological processes such as cytokinesis and cell cycle.Data shows that SEPT9 gene is likely to be a kind of suppression
Oncogene.Research discovery to kinds cancer cell SEPT9 genomic DNA: SEPT9 gene occurs in kinds cancer cell
The phenomenon that DNA methylation.
DNA methylation is a kind of regulatory mechanism of epigenetics.Pass through the CpG base to genome sequence specific region
Cytimidine in combination carries out methylation modification, produces 5-methylcytosine, and then realize the expression of regulation corresponding gene.DNA
The expression of methylation suppressor, and the expression of DNA demethylation activated gene.
This phenomenon of DNA methylation, therefore the methyl in SEPT9 gene occur in kinds cancer cell for SEPT9 gene
Changing DNA becomes a kind of cancer marker, has extensive and important clinical value, such as: passing through SEPT9 in detection peripheral blood
The methylate DNA of gene realizes the Non-invasive detection of early-stage cancer；Pass through SEPT9 gene methylation DNA content in detection peripheral blood
Variation auxiliary monitoring to the therapeutic effect etc. of cancer.
It is often non-methylate DNA using prior art biological sample DNA obtained due to forming the diversity of cell
With the mixture of methylate DNA, such as: 1) the cancerous tissue DNA obtained, source include the cancer cell that DNA methylation occurs,
It also include the normal cell that DNA methylation does not occur in cancerous tissue；2) the peripheral blood dissociative DNA (circulating obtained
Cell-free DNA, cfDNA), main source is the normal white cell that DNA methylation does not occur, and with cancer cell
The content for the cancer cell DNA that apoptosis and decline enter in peripheral blood is generally less than the 0.1% of cfDNA.Therefore, for detecting
The method of SEPT9 gene methylation DNA is required to effectively distinguish the DNA of methylation and non-methylation, specifically to sample
This part to methylate in DNA is detected.
Since the 5-methylcytosine in methylate DNA is matched with the cytimidine base having the same in non-methylate DNA
To behavior, therefore the detection method based on base pairing can not be directly used, such as: DNA hybridization, PCR.Detection methylation at present
The technology of DNA with based on bisulfite conversion (bisulfite conversion) method based on, it may be assumed that bisulfites with
Special biochemical reaction occurs for the cytimidine of non-methylation, and by subsequent basic hydrolysis, the cytimidine of non-methylation is converted into
Uracil, and 5-methylcytosine does not change under the same conditions.It therefore, cannot by DNA hybridization in initial DNA sequence dna
The cytimidine and methylated cytosine of differentiation become uracil and methylated cytosine, thus energy after bisulfite conversion
It is enough to be detected by the Protocols in Molecular Biology based on DNA hybridization.
Although this method has been used widely in basic research and clinical detection, bisulfites is used
Conversion this technology of DNA itself comes with some shortcomings, and such as: 1) since bisulfites is unstable, the holding time is limited, thus base
It is typically only 6 months in the validity period of the kit of bisulfite conversion；2) time-consuming for bisulfite conversion process, tradition
Method needs overnight, even if after improving and optimizing, it is still necessary to a few hours；3) process of bisulfite conversion DNA leads to DNA's
Fracture and degradation cause the loss of DNA, therefore reduce the sensitivity of detection；4) DNA after bisulfite conversion needs to purify
It just can be carried out subsequent detecting step with recycling, and the purification and recovery of DNA will cause the loss of DNA, therefore further decrease
The sensitivity of detection.
Methylation sensitive restriction restriction endonuclease (methylation-sensitive restriction enzyme) is one
Class contains the sensitive restriction enzyme of methylated base to its recognition site, can effectively distinguish born of the same parents in its restriction enzyme site sequence
The methylation state of pyrimidine only carries out digestion to the restriction enzyme site sequence that cytosine methylation does not occur.But it is currently used this
Fermentoid detects the methylation of DNA, usually southern blotting technique method.The method in addition to need a large amount of high relative molecular mass DNA (>=
5ug) outside, it can only detect that copy number ratio is greater than the methylated alleles of a few percent, and be only capable of providing MSRE knowledge
The information of those island CpG methylation states in other sequence.
Therefore, currently the sensitive convenient and fast method for using methylation sensitive restriction restriction endonuclease detection DNA methylation is deposited
In demand.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of for detecting the combination of the methylate DNA of SEPT9 gene
Object and corresponding kit, the present invention also provides the method that detection is executed based on the kit, methods provided by the invention
The methylation of gene is detected for the selectivity of restriction enzyme site methylation state using methylation sensitive restriction restriction endonuclease
DNA, compared with traditional methylate DNA detection method based on bisulfite conversion, the present processes detection time is short,
Reagent stability is strong, operating procedure is simple, DNA loss less, detection sensitivity it is higher, be particularly suited for basis scientific research and
Clinical detection.
According to an aspect of the present invention, the present invention provides a kind of for detecting the first of SEPT9 gene in biological sample
The composition of base DNA, the composition include at least one methylation sensitive restriction restriction endonuclease, and for expanding SEPT9
The nucleic acid at least one of gene or its segment region.
Preferably, the nucleic acid includes to be equal to, be complementary to or hybridize under moderate stringency or stringent condition in selected from SEQ
The segment or its complementary series of at least 15 nucleotide of ID NO:1, and the nucleic acid includes at least one methyl-sensitive
The restriction enzyme site of restriction enzyme；
It is highly preferred that the nucleic acid include be equal to, be complementary to or under moderate stringency or stringent condition hybridization in being selected from
The segment or its complementary series of at least 15 nucleotide of SEQ ID NO:2, and the nucleotide fragments include at least one first
The restriction enzyme site of base sensitive restriction restriction endonuclease.
It is further preferred that nucleic acid primer selected from the following or probe sequence:
SEPT9 primers F:
SEQ ID NO:3:CCCACCAGCCATCATGT
SEPT9 primer R:
SEQ ID NO:4:CCACCTTCGAAGTCCGAAAT
SEPT9 probe P:
SEQ ID NO:5:CCATCCAGCTGCGCGTTGAC
Preferably, the methylation sensitive restriction restriction endonuclease is used to digest not methylate in biological sample DNA
SEPT9 gene DNA, the methylation sensitive restriction restriction endonuclease be selected from Hap II, Hha I, Not I, Nru I, Sac II or
At least one of Sma I；
It is highly preferred that the methylation sensitive restriction restriction endonuclease is Hha I and/or Sac II.
It was found by the inventors of the present invention that after using methylation sensitive restriction endonuclease digestion, digestion product can be without
Nucleic acid purification can be applied to following amplification reaction directly as PCR reaction template, to further simplify test procedure.
Further, it was found by the inventors of the present invention that the component of the enzyme cutting buffering liquid of methylation sensitive restriction restriction endonuclease allows for
It is compatible with pcr amplification reaction, however there are significant differences with pcr amplification reaction compatibility between different restriction endonucleases, specifically,
It was found by the inventors of the present invention that Hap II, Not I, the digestion products of Nru I and Sma I these types enzyme are different to subsequent PCR
The inhibiting effect of degree.Therefore, methylation sensitive restriction restriction endonuclease Hha I and/or Sac II are more preferably used.This two
It is anti-that the ion concentration of the most suitable enzyme cutting buffering liquid of kind methylation sensitive restriction restriction endonuclease is particularly suitable for PCR amplification of the invention
It answers.
According to the second aspect of the invention, the kit including the composition is additionally provided.
Preferably, the kit includes the container for accommodating the biological sample.Also, the kit
Explanation including using and explaining kit results.
According to the third aspect of the present invention, a kind of method of methylate DNA for detecting SEPT9 gene, institute are additionally provided
State method the following steps are included:
1) DNA of biological sample to be measured is separated；
2) the methylation sensitive restriction endonuclease digestion DNA is used, later to methylation sensitive restriction restriction endonuclease
It is inactivated；
3) using nucleic acid amplification step 2 provided by the invention) obtained product, thus obtain because DNA methylation occurs, and
The DNA fragmentation that do not sheared by the methylation sensitive restriction restriction endonuclease；
4) methylation state of SEPT9 gene is judged by the segment that step 3) obtains.
Preferably, in step 2), it is described digestion the following steps are included:
At 35-37 DEG C, the methylation sensitive restriction endonuclease digestion processing step 1 is used) obtained DNA 1-2
Hour
Preferably, in step 2), methylation sensitive restriction restriction endonuclease is inactivated using heat inactivation；More preferably
Ground, it is described inactivation the following steps are included:
It after being handled using methylation sensitive restriction endonuclease digestion, is heated to 80-99 DEG C and maintains 5-10 minutes, realize
Heat inactivation to methylation sensitive restriction restriction endonuclease.
Preferably, it in step 3), will be contacted through the processed DNA of step 2) with amplification enzyme, nucleic acid of the present invention,
So that the processed gene DNA is amplified and generates amplified production or be not amplified；
Preferably, in step 4), probe in detecting amplified production is used；It whether there is based on the amplified matter, judge
The methylation state of SEPT9 gene.
Preferably, the step 3) uses at least one method chosen from the followings: using hot resistant DNA polymerase as described in
Amplification enzyme, using lack the polymerase of 5 ' -3 ' 5 prime excision enzyme activities, using polymerase chain reaction (PCR), generate with detectable
The amplified production nucleic acid molecules of label.
Preferably, the biological sample is selected from cell line, Histological section, tissue biopsy/paraffin embedding tissue, body
Liquid, excrement, colonic effluent, urine, blood plasma, serum, whole blood, the haemocyte of separation, the cell that is separated from blood or its group
It closes.
Preferably, the sample is blood plasma.
According to certain preferred embodiments, the methylation state of the DNA sequence dna of the target gene is by polymerase chain
What the cycle threshold Ct value of reaction determined, wherein the target gene is selected from SEPT9 gene or its segment.
Finally, the detection for SEPT9 gene is conveniently realized by the method for the DNA in real-time PCR analysis sample,
And it can be according to the Ct value of real-time PCR come the methylation state of quickly and conveniently judgement sample SEPT9 gene.
Compared with prior art, the present invention has the advantage that:
1) compared with the methylate DNA detection method based on bisulfite conversion, when method provided by the invention detects
Between it is shorter；
2) compared with the methylate DNA detection method based on bisulfite conversion, reagent stability that the present invention uses
By force；
3) operating procedure is simple；
4) DNA loss is few, and detection sensitivity is higher, and see Table 2 for details.
Unless otherwise defined, term and those skilled in the art in relation to technology and scientific in this specification lead to
What is understood is equivalent in meaning.Although can be applied and the similar or identical method and material around here in experiment or practical application
Material, herein still hereinafter describes material and method.It include wherein fixed with this specification in conflicting situation
Subject to justice, in addition, material, method and example are only for explanation, and it is without limitation.
Other features and advantages of the invention will be explained in detail by following illustrating with claims.
Detailed description of the invention
Above and other feature of the invention will by with reference to the accompanying drawing and its detailed description be described further.It should
Understand, these attached drawings illustrate only several illustrative embodiments according to the present invention, therefore are not considered as pair
The limitation of the scope of the present invention.Unless stated otherwise, attached drawing is not necessarily to scale, and wherein similar label indicates class
As component.
Fig. 1 is the gene methylation DNA detection method based on methylation sensitive restriction endonuclease digestion and is based on sulfurous
The process CIMS of the gene methylation DNA detection method of sour hydrogen salt conversion.
Specific embodiment
This application provides a kind of compositions and method for detecting SEPT9 gene methylation DNA.Be based on bisulfites
The methylate DNA detection method of conversion is compared, and composition provided by the present application and method have the advantages that following several respects: 1) being examined
It is short to survey the time；2) reagent stability is strong；3) operating procedure is simple；4) DNA loss is few, and detection sensitivity is higher.(referring to Fig. 1)
Following embodiments for the composition of the application, kit, nucleic acid sequence and detection method.It is appreciated that examining
Consider general description presented above, it is possible to implement various other embodiments.
In embodiments, it discloses a kind of for detecting the composition of the methylate DNA of SEPT9 gene, the composition
Including at least one methylation sensitive restriction restriction endonuclease, and at least one region for expanding SEPT9 gene or its segment
Nucleic acid.
Specifically, the nucleic acid includes to be equal to, be complementary to or hybridize under moderate stringency or stringent condition in selected from SEQ
The piece of at least 15 nucleotide of ID NO:1 or and its complementary series, and the nucleic acid includes at least one methyl-sensitive
The restriction enzyme site of restriction enzyme；More specifically, the nucleic acid includes to be equal to, be complementary to or in moderate stringency or tight item
Hybridize under part in the segment or its complementary series of at least 15 nucleotide selected from SEQ ID NO:2, and the nucleotide fragments
Restriction enzyme site comprising at least one methylation sensitive restriction restriction endonuclease.
SEQ ID NO:1 sequence:
CTGGGCGCCCGCCCTCCTCGCCATGGCCCGGCCTCCACATCCGCCCACATCTGGCCGCAGCGGGGCGCC
CGGGGGGAGGGGCTGAGGCCGCGTCTCTCGCCGTCCCCTGGGCGCGGGCCAGGCGGGGAGGAGGGGGGCGCTCCGGT
CGTGTGCCCAGGACTGTCCCCCAGCGGCCACTCGGGCCCCAGCCCCCCAGGCCTGGCCTTGACAGGCGGGCGGAGCA
GCCAGTGCGAGACAGGGAGGCCGGTGCGGGTGCGGGAACCTGATCCGCCCGGGAGGCGGGGGCGGGGCGGGGGCGCA
GCGCGCGGGGAGGGGCCGGCGCCCGCCTTCCTCCCCCATTCATTCAGCTGAGCCAGGGGGCCTAGGGGCTCCTCCGG
CGGCTAGCTCTGCACTGCAGGAGCGCGGGCGCGGCGCCCCAGCCAGCGCGCAGGGCCCGGGCCCCGCCGGGGGCGCT
TCCTCGCCGCTGCCCTCCGCGCGACCCGCTGCCCACCAGCCATCATGTCGGACCCCGCGGTCAACGCGCAGCTGGAT
GGGATCATTTCGGACTTCGAAGGTGGGTGCTGGGCTGGCTGCTGCGGCCGCGGACGTGCTGGAGAGGACCCTGCGGG
TGGGCCTGGCGCGGGACGGGGGTGCGCTGAGGGGAGACGGGAGTGCGCTGAGGGGAGACGGGACCCCTAATCCAGGC
GCCCTCCCGCTGAGAGCGCCGCGCGCCCCCGGCCCCGTGCCCGCGCCGCCTACGTGGGGGACCCTGTTAGGGGCACC
CGCGTAGACCCTGCGCGCCCTCACAGGACCCTGTGCTCGTTCTGCGCACTGCCGCCTGGGTTTCCTTCCTTTTATTG
TTGTTTGTGTTTGCCAAGCGACAGCGACCTCCTCGAGGGCTCGCGAGGCTGCCTCGGAACTCTCCAGGACGCACAGT
TTCACTCTGGGAAATCCATCGGTCCCCTCCCTTTGGCTCTCCCCGGCGGCTCTCGGGCCCCGCTTGGACCCGGCAAC
GGGATAGG
SEQ ID NO:2 sequence:
CCCACCAGCCATCATGTCGGACCCCGCGGTCAACGCGCAGCTGGATGGGATCATTTCGGACTTCGAAGG
TGG
It is further preferred that nucleic acid primer selected from the following or probe sequence:
SEPT9 primers F:
SEQ ID NO:3:CCCACCAGCCATCATGT
SEPT9 primer R:
SEQ ID NO:4:CCACCTTCGAAGTCCGAAAT
SEPT9 probe P:
SEQ ID NO:5:CCATCCAGCTGCGCGTTGAC
Preferably, the methylation sensitive restriction restriction endonuclease is for digesting the SEPT9 not methylated in sample DNA
Gene DNA, the methylation sensitive restriction restriction endonuclease are selected from Hap II, Hha I, Not I, Nru I, Sac II or Sma I
At least one of；
It is highly preferred that the methylation sensitive restriction restriction endonuclease is Hha I and/or Sac II；
Specifically, in preferred embodiments, the described method comprises the following steps:
In the first step, the DNA of biological sample to be measured is separated.The source of the biological sample to be measured can be any suitable
Source, such as cell line, Histological section, biopsy, the tissue of paraffin embedding, body fluid, excrement, colonic effluent, urine, blood
Slurry, serum, whole blood, the haemocyte of separation, the cell from blood separation and its all possible combination.Preferably, the source
For excrement or body fluid, selected from colonic effluent, urine, blood plasma, serum, whole blood, separation haemocyte, separate the cell of autoblood.
Then it can be separated from sample separation genomic DNA by any standard approach in the prior art, including using can quotient
The kit of purchase.In short, the biological sample must be broken and by enzyme, change when target DNA is wrapped in cell membrane
It learns or mechanical means is cleaved.Albumen and other pollutants are for example then removed by the digestion of protein kinase K.Then from
Solution recycles genomic DNA.This can realize by various methods, including saltouts, organic extraction or DNA is integrated to solid phase
Support.The influence that will receive many factors to the selection of method, the amount including time, expense and required DNA.When the sample
When product DNA is not wrapped in cell membrane (such as Circulating DNA from blood sample), it can be used and separate in the prior art
And/or the standard method of purifying DNA.These methods include using protein degradation reagent, such as chaotropic salt, such as guanidine hydrochloride or urea；
Or detergent, such as dodecyl sodium sulfate (SDS), cyanogen bromide.Other methods include but is not limited to ethanol precipitation or propyl alcohol precipitating,
Pass through the vacuum concentration etc. of centrifugation.Those skilled in the art also can use device, such as the filter of such as ultrafiltration, silicon face or
Film, magnetic-particle, granules of polystyrene, polystyrene surface, positively charged surface and the film with positive charge, electrification
Film, powered surfaces, electrification conversion film charge transfer surface.
Once nucleic acid is extracted, just genome double-stranded DNA is used to analyze.In the second step of the method, institute is used
State at least one of methylation sensitive restriction restriction endonuclease to DNA sample 35-37 DEG C progress digestion process 1-2 hours, with
It is cut so that unmethylated SEPT9 gene or at least one region of its segment are methylated sensitive restriction inscribe enzyme selectivity
It cuts, and the corresponding region to methylate keeps its integrality.Then, above-mentioned methylation sensitive restriction endonuclease digestion system is added
Heat is to 80-99 DEG C and maintains 5-10 minutes, realizes and inactivates to the heat of above-mentioned methylation sensitive restriction restriction endonuclease, prevents from methylating
Influence of the sensitive restriction restriction endonuclease to subsequent step.
In the third step of the method, using prime nucleotide of the invention and the amplification processed DNA of enzymatic amplification
Segment.The amplification of several DNA fragmentations can be carried out simultaneously in the same reaction vessel.In general, the amplified reaction is using polymerization
Enzyme chain reaction (PCR) carries out.Preferably, the length of the amplified production is 60 to 140 base-pairs.For SEPT9 gene
And its detection of the methylation of segment, utilize the primer and probe for being directed to SEPT9 gene.Such as: it is designed according to SEQ ID NO:2
Primer and probe:
SEPT9 primers F:
SEQ ID NO:3:CCCACCAGCCATCATGT
SEPT9 primer R:
SEQ ID NO:4:CCACCTTCGAAGTCCGAAAT
SEPT9 probe P:
SEQ ID NO:5:CCATCCAGCTGCGCGTTGAC
The marker that can directly or indirectly detect can be carried by the segment that amplification obtains.Preferably, marker
For the form of fluorescent marker, radionuclide or the molecule fragment that can adhere to.
In the 4th step of the method, the amplified production obtained in the third step of the method is judged, to determine
In sample whether the methylate DNA containing SEPT9 gene.In the 4th step, the detection to amplified production is to pass through real-time detection
Probe carries out.
In the present invention, it can use standard operation according to prior art in various business real-time PCR instrument equipment
Carry out the detection of real-time PCR.It is enterprising in Life Technologies instrument (7500Fast) according to certain specific embodiments
The detection of the real-time PCR of row.PCR reaction mixture is by DNA profiling and 300- through methylation sensitive restriction endonuclease digestion
600nM primer, 150-300nM probe, 1UTaq polymerase, each dNTP of 50-400uM, 1 to 10mM MgCl2And 2XPCR
It is buffered to the volume of final 2 μ l to 100 μ l.Continue 10 minutes initial denaturations at 90-99 DEG C, and then carry out 35-55 circulation:
1) it carries out annealing for 10 to 30 seconds at 50 to 65 DEG C, extends 15 to 90 seconds at 60 to 75 DEG C, 5 to 90 are denaturalized at 90 to 99 DEG C
Second.Only Observable expands in the SEPT9 genetic fragment of methylation.Also, egg in some specific embodiments, is moved with β flesh
Digestion reference material and PCR reference material of the white gene (ACTB) as detection, by using the primer complementary with ACTB gene order
To create ACTB gene amplicon, restriction enzyme site of the digestion reference material comprising above-mentioned methylation sensitive restriction restriction endonuclease, PCR ginseng
The restriction enzyme site that product do not include above-mentioned methylation sensitive restriction restriction endonuclease is examined, and is expanded with specific probe in detecting ACTB gene
Increase son.
In conclusion the application by compositions as described above, methylation sensitive restriction restriction endonuclease, nucleic acid sequence,
Kit and application thereof and above-mentioned detection method realize the detection of SEPT9 gene methylation DNA, and the combination
Object, kit and application thereof are in many-sided detection method for being better than the methylate DNA based on bisulfite conversion.
It will be described specific embodiment below.
The method of the invention of embodiment 1 is compared with traditional method
Through the identification of the detection methods such as sequencing: the SEPT9 gene of Jurkat cell system genome is non-methylation, and Hela
The SEPT9 gene of cell line genome is methylation.
The step of the present embodiment, is as follows:
1) DNA of both cell lines is extracted respectively, then with the ratio of 1000:1 by Jurkat and Hela cell line
Genomic DNA is added in TE buffer, configures positive DNA solution.The final concentration of positive DNA solution is 35ng/ μ l
The Hela genomic DNA of Jurkat genomic DNA and 35pg/ μ l；It is molten that Jurkat cell system genomic DNA is separately added to TE
Negative DNA solution is configured in liquid, the final concentration of negative DNA solution is the Jurkat genomic DNA of 35ng/ μ l.
2) molten to negative DNA solution and positive DNA respectively using methylation sensitive restriction restriction endonuclease Hha I or Sac II
Liquid carries out endonuclease reaction, and reaction system is 20 μ l, comprising: 3.5 μ l of negative or positive DNA solution, 2 μ l of methyl-sensitive restriction endonuclease
(10 units/μ l), 2 μ l of 10x enzyme cutting buffering liquid；Reaction condition is 37 DEG C and is incubated for 1 hour.After endonuclease reaction, digestion is heated
Reaction solution is incubated for 10 minutes to 85 DEG C, carries out hot inactivation to methyl-sensitive restriction endonuclease.
3) SEPT9 gene primer, probe combinations and reference gene ACTB then, is added into above-mentioned endonuclease reaction liquid respectively
Gene primer, probe combinations detect the methylation state of SEPT9 gene by PCR.
Wherein, the PCR amplification condition taken in this experimental example are as follows: enterprising in Life Technologies instrument (7500)
The real-time PCR of row.PCR reaction mixture by through methylation sensitive restriction endonuclease digestion DNA profiling and 450nM primer,
225nM probe, 1UTaq polymerase, 200um each dNTP, 4.5mM MgCl2It forms with 2XPCR buffer to final
The volume of 50ul.Continue 10 minutes initial denaturations at 94 DEG C, and then carry out 45 circulations: 1) carrying out annealing for 15 seconds at 60 DEG C,
Extend 15 seconds at 72 DEG C, is denaturalized 15 seconds at 94 DEG C.Finally, it is real-time for SEPT9 gene to measure configured DNA solution sample
The Ct value of PCR detection.
In addition, using the SEPT9 gene methyl based on bisulfite conversion of Bo Er really (Beijing) Science and Technology Ltd.
Change detection kit and the detection of SEPT9 gene methylation is carried out to equivalent DNA solution sample.
Testing result is as shown in table 1: negative and positive reference product digestion internal references are no amplified productions, illustrate that digestion is anti-
Should completely, therefore the non-methylate DNA of SEPT9 gene is by complete degestion；Negative and positive reference product PCR internal references show amplification
Product, therefore PCR reaction is normal；The Ct value of positive reference product shows amplified production, and the Ct value of negative reference product shows do not have
There is amplified production.Therefore it may determine that: the SEPT9 gene methylation DNA inspection based on methylation sensitive restriction endonuclease digestion
Survey method can be used for detecting SEPT9 gene methylation DNA.
Table 1 is that the methylate DNA detection method using of the invention based on methylation sensitive restriction endonuclease digestion is examined
Survey SEPT9 gene methylation sample (Hela cell line) and the non-methylation sample of SEPT9 gene (Jurkat cell system).
On the other hand, compared with the SEPT9 gene methylation detection method based on bisulf iotate-treated, it is based on methyl
The Ct value significant (p < 0.05) for changing the SEPT9 gene methylation DNA detection method of sensitive restriction endonuclease digestion is relatively low, therefore
The sensitivity of SEPT9 gene methylation DNA detection method based on methylation sensitive restriction endonuclease digestion it is significant (p <
0.05) it improves, see Table 2 for details:
Table 2 be the methylate DNA detection method more of the invention based on methylation sensitive restriction endonuclease digestion and
Methylate DNA detection method based on bisulfite conversion detects SEPT9 gene methylation sample
It is experimentally confirmed, can be realized using the method based on methylation sensitive restriction restriction endonuclease sensitive, special
The detection of SEPT9 gene methylation DNA, and can be improved the effect phase of kit, simplify testing process, reduce detection time and
Cost improves detection sensitivity.
Embodiment 2 detects the methylate DNA of SEPT9 gene using different restriction enzymes
It was found by the inventors of the present invention that the digestion products of Hap II, Not I, Nru I and Sma I these types enzyme are to subsequent
PCR has different degrees of inhibiting effect.The most suitable enzyme of both methylation sensitive restriction restriction endonucleases of Hha I and/or Sac II
The ion concentration for cutting buffer is particularly suitable for pcr amplification reaction of the invention.
The step of the present embodiment, is as follows:
1) DNA of both cell lines is extracted respectively, then with the ratio of 1000:1 by Jurkat and Hela cell line
Genomic DNA is added in TE buffer, configures positive DNA solution.
2) using methylation sensitive restriction restriction endonuclease Hap II, Not I, Nru I, Sma I, Hha I or Sac II points
Other to carry out endonuclease reaction to positive DNA solution, reaction system is 20 μ l, comprising: 3.5 μ l of negative or positive DNA solution, methylation
Sensitive 2 μ l of restriction endonuclease (10 units/μ l), 2 μ l of 10x enzyme cutting buffering liquid；Reaction condition is 37 DEG C and is incubated for 1 hour.Endonuclease reaction knot
Shu Hou, heats endonuclease reaction liquid to 85 DEG C, is incubated for 10 minutes, carries out hot inactivation to methyl-sensitive restriction endonuclease.
3) SEPT9 gene primer, probe combinations and reference gene ACTB then, is added into above-mentioned endonuclease reaction liquid respectively
Gene primer, probe combinations detect the methylation state of SEPT9 gene by PCR.
Wherein, the PCR amplification condition taken in this experimental example are as follows: enterprising in Life Technologies instrument (7500)
The real-time PCR of row.PCR reaction mixture by through methylation sensitive restriction endonuclease digestion DNA profiling and 450nM primer,
225nM probe, 1UTaq polymerase, 200 μm each dNTP, 4.5mM MgCl2It forms with 2XPCR buffer to final
The volume of 50ul.Continue 10 minutes initial denaturations at 94 DEG C, and then carry out 45 circulations: 1) carrying out annealing for 15 seconds at 60 DEG C,
Extend 15 seconds at 72 DEG C, is denaturalized 15 seconds at 94 DEG C.Finally, it is real-time for SEPT9 gene to measure configured DNA solution sample
The Ct value of PCR detection.
Testing result is as shown in table 3: methylation sensitive restriction restriction endonuclease Hap II, Not I, Nru I and Sma I are to sun
Property reference material detection Ct value (including SEPT9 and ACTB) be apparently higher than both methyl-sensitives of Hha I and Sac II limitation
Property restriction endonuclease.Since the DNA detection ACTB digestion control of all methylation sensitive restriction inscribe enzymatic treatments is negative,
Endonuclease reaction is normal and complete.Therefore, the detection Ct value increase of Hap II, Not I, Nru I and Sma I are PCR reactions
The amplification efficiency of system is lower than Hha I or Sac II.It is purified after being cut due to DNA enzymatic to carry out PCR amplification directly as template,
Therefore enzyme cutting buffering liquid can have an impact PCR amplification efficiency.Example two is as the result is shown: Hap II, Not I, Nru I and
The digestion products of Sma I these types enzyme have different degrees of inhibiting effect to subsequent PCR.Both first of Hha I and/or Sac II
The ion concentration of the most suitable enzyme cutting buffering liquid of base sensitive restriction restriction endonuclease is particularly suitable for pcr amplification reaction (table of the invention
3)。
Table 3 is the methylate DNA that more different restriction enzymes detects SEPT9 gene
Although various aspects of the invention and embodiment are disclosed, other aspect and embodiment are for this field skill
It is also obvious for art personnel.Various aspects and embodiment disclosed herein are for illustration purposes only, rather than limit mesh
's.Protection scope of the present invention and purport are only determined by appended claims.
Sequence table
<110>Bo Er really (Beijing) Science and Technology Ltd.
<120>composition and method of the methylate DNA of Septin9 gene are detected
<130> DIC16110088
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1001
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> allele
<222> ()..()
<223>partial sequence of SEPT9 gene
<400> 1
ctgggcgccc gccctcctcg ccatggcccg gcctccacat ccgcccacat ctggccgcag 60
cggggcgccc ggggggaggg gctgaggccg cgtctctcgc cgtcccctgg gcgcgggcca 120
ggcggggagg aggggggcgc tccggtcgtg tgcccaggac tgtcccccag cggccactcg 180
ggccccagcc ccccaggcct ggccttgaca ggcgggcgga gcagccagtg cgagacaggg 240
aggccggtgc gggtgcggga acctgatccg cccgggaggc gggggcgggg cgggggcgca 300
gcgcgcgggg aggggccggc gcccgccttc ctcccccatt cattcagctg agccaggggg 360
cctaggggct cctccggcgg ctagctctgc actgcaggag cgcgggcgcg gcgccccagc 420
cagcgcgcag ggcccgggcc ccgccggggg cgcttcctcg ccgctgccct ccgcgcgacc 480
cgctgcccac cagccatcat gtcggacccc gcggtcaacg cgcagctgga tgggatcatt 540
tcggacttcg aaggtgggtg ctgggctggc tgctgcggcc gcggacgtgc tggagaggac 600
cctgcgggtg ggcctggcgc gggacggggg tgcgctgagg ggagacggga gtgcgctgag 660
gggagacggg acccctaatc caggcgccct cccgctgaga gcgccgcgcg cccccggccc 720
cgtgcccgcg ccgcctacgt gggggaccct gttaggggca cccgcgtaga ccctgcgcgc 780
cctcacagga ccctgtgctc gttctgcgca ctgccgcctg ggtttccttc cttttattgt 840
tgtttgtgtt tgccaagcga cagcgacctc ctcgagggct cgcgaggctg cctcggaact 900
ctccaggacg cacagtttca ctctgggaaa tccatcggtc ccctcccttt ggctctcccc 960
ggcggctctc gggccccgct tggacccggc aacgggatag g 1001
<210> 2
<211> 72
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> allele
<222> ()..()
<223>partial sequence of SEPT9 gene
<400> 2
cccaccagcc atcatgtcgg accccgcggt caacgcgcag ctggatggga tcatttcgga 60
cttcgaaggt gg 72
<210> 3
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> allele
<222> ()..()
<223>SEPT9 primers F
<400> 3
cccaccagcc atcatgt 17
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> allele
<222> ()..()
<223>SEPT9 primer R
<400> 4
ccaccttcga agtccgaaat 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> allele
<222> ()..()
<223>SEPT9 probe P
<400> 5
ccatccagct gcgcgttgac 20
Claims (10)
1. a kind of for detecting the composition of the methylate DNA of SEPT9 gene in biological sample, the composition includes at least one
Kind methylation sensitive restriction restriction endonuclease, and the nucleic acid at least one region for expanding SEPT9 gene or its segment.
2. composition according to claim 1, which is characterized in that the nucleic acid includes to be equal to, be complementary to or medium
Hybridize under tight or stringent condition in the segment or its complementary series of at least 15 nucleotide selected from SEQ ID NO:1, and
The nucleic acid includes the restriction enzyme site of at least one methylation sensitive restriction restriction endonuclease；
Preferably, the nucleic acid includes to be equal to, be complementary to or hybridize under moderate stringency or stringent condition in selected from SEQ ID
The segment or its complementary series of at least 15 nucleotide of NO:2, and the nucleotide fragments include that at least one methylation is quick
Feel the restriction enzyme site of restriction enzyme；
It is highly preferred that nucleic acid primer selected from the following or probe sequence:
SEPT9 primers F:
SEQ ID NO:3:CCCACCAGCCATCATGT
SEPT9 primer R:
SEQ ID NO:4:CCACCTTCGAAGTCCGAAAT
SEPT9 probe P:
SEQ ID NO:5:CCATCCAGCTGCGCGTTGAC.
3. composition according to claim 1 or 2, which is characterized in that the methylation sensitive restriction restriction endonuclease is used for
The SEPT9 gene DNA not methylated in the biological sample DNA of detection is digested, and in the methylation sensitive restriction
Enzyme cutting is selected from least one of Hap II, Hha I, Not I, Nru I, Sac II or Sma I；
It is highly preferred that the methylation sensitive restriction restriction endonuclease is Hha I and/or Sac II.
4. a kind of for detecting the kit of the methylate DNA of SEPT9 gene in biological sample, the kit includes that right is wanted
Seek composition described in any one of 1-3；
Preferably, the kit includes the container for accommodating the biological sample；
It is highly preferred that the kit also includes the explanation for using and explaining the result of kit.
5. a kind of method for the methylate DNA for detecting SEPT9 gene, the described method comprises the following steps:
1) DNA of biological sample to be measured is separated；
2) the methylation sensitive restriction endonuclease digestion DNA in the composition of any of claims 1-3 is used, it
The methylation sensitive restriction restriction endonuclease is inactivated afterwards；
3) using the nucleic acid amplification step 2 in the composition of any of claims 1-3) obtained product, to obtain
The DNA fragmentation for obtaining because DNA methylation occurs, and not sheared by the methylation sensitive restriction restriction endonuclease；
4) methylation state of SEPT9 gene is judged by the segment that step 3) obtains.
6. according to the method described in claim 5, it is characterized in that, in step 2), the digestion the following steps are included:
At 35-37 DEG C, the methylation sensitive restriction restriction endonuclease in the composition of any of claims 1-3 is used
Obtain DNA 1-2 hours of digestion process step 1).
7. according to the method described in claim 5, it is characterized in that, being inactivated using heat in claim 1-3 in step 2)
Methylation sensitive restriction restriction endonuclease in described in any item compositions is inactivated；Preferably, the inactivation includes following
Step:
It after being handled using the methylation sensitive restriction endonuclease digestion, is heated to 80-99 DEG C and maintains 5-10 minutes, realize
Heat inactivation to the methylation sensitive restriction restriction endonuclease.
8. according to the method described in claim 5, it is characterized in that, through the processed DNA of step 2) and will expand in step 3)
Nucleic acid contact in increasing enzyme, composition of any of claims 1-3, so that the processed gene DNA is expanded
Increase and generates amplified production or be not amplified；
Preferably, at least one method chosen from the followings is used in step 3): using hot resistant DNA polymerase as the amplification
Enzyme has detectable label using the polymerase of 5 ' -3 ' 5 prime excision enzyme activities of shortage, using polymerase chain reaction (PCR), generation
Amplified production nucleic acid molecules.
9. according to the method described in claim 5, it is characterized in that, using any one of claim 1-3 institute in step 4)
Probe in detecting amplified production in the composition stated；It whether there is based on the amplified matter, judge the methylation shape of SEPT9 gene
State.
10. composition according to any one of claim 1-3, kit according to claim 4, or according to power
Benefit requires method described in any one of 5-9, which is characterized in that the biological sample is selected from cell line, Histological section, tissue
Biopsy/paraffin embedding tissue, body fluid, excrement, colonic effluent, urine, blood plasma, serum, whole blood, the haemocyte of separation, from blood
The cell separated in liquid, or combinations thereof；
Preferably, the sample is blood plasma.
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