CN110305940A - A kind of method and kit of the detection of 9 gene methylation of Septin - Google Patents
A kind of method and kit of the detection of 9 gene methylation of Septin Download PDFInfo
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Abstract
The present invention provides a kind of 9 gene methylation detection kits of Septin, it includes following three groups for at least one set in the specific primer and probe of Septin 9-v2 exon DNA methylation assay, SEQ ID NO.5-SEQ ID NO.7, SEQ ID NO.8-SEQ ID NO.10, SEQ ID NO.11-SEQ ID NO.13.The present invention also provides a kind of 9 gene methylation detection method of Septin and sample-pretreating methods comprising following steps: TET oxydasis；Pyridine borane reaction；Multiple fluorescence PCR reaction.Kit of the present invention and detection method have detection sensitivity high, specific good, detect the high advantage of flux.
Description
Technical field
The present invention relates to technical field of gene detection, in particular to a kind of method of human gene DNA methylation assay
And kit.
Background technique
Septin9 gene is one of Septin gene family member of the discoveries such as Hartwell, has guanosine triphosphatase
(GTPase) active.Septin9 gene is located at human chromosomal 17q25.3, contains 17 exons, is about 2.40 × 105Bp,
Encode 9 albumen of Septin.But since its transcription product includes multiple translation starting points and variation montage, Septin9 can make a variation generation
15 kinds of 18 kinds of variation spliceosomes, coding polypeptides.There is 75% nucleotide sequence similarity and 89% between each hypotype of Septin9
~96% amino acid identity.In addition, tissue specificity is distributed in these variation spliceosomes, such as the change of Septin9 gene
Different spliceosome Septin 9-v1 is distributed in the tissue in addition to brain and thymus gland, and make a variation spliceosome Septin9-v2, Septin9-
V3, Septin9-v4 are mostly expressed in fetal tissue, and variation spliceosome Septin 9-v5 expresses very high in fetal tissue.
Septin9 gene and its various physiology courses of expression product wide participation human body, such as polarization, Cell apoptosis and proliferation, thin
Outer matter transportation intracellular, cytoskeleton are adjusted etc..
Studies have shown that Septin9 gene plays tumor suppressor gene in colorectal cancer generation, methylation can inhibit its base
Because of normal expression, lose its cancer suppressing function, and eventually lead to cell division and canceration.The detection means of DNA methylation is various,
3 major class, respectively enzyme digestion, affine concentration method, bisulfite conversion method can be divided into according to sample preprocessing difference.At present
The Septin9 DNA methylation assay for being directed to colorectal cancer sample is mainly bisulfite conversion method.This method uses sulfurous acid
Hydrogen salt handles sample DNA, converts the cytimidine not methylated (C) to uracil (U), and the cytimidine to methylate
(such as 5mC and/or 5hmC) is remained unchanged；After PCR amplification, U is changed into thymidine (T), and the C of methylation is changed into C, from
And it realizes and infers the modification of each cytimidine in single base level.Septin 9 reported in the literature based on bisulfite conversion
Methylation detecting method includes methyl-sensitive high-resolution melting curve (MS-HRM), methylation status of PTEN promoter joint denaturation
High performance liquid chromatography (MSP-DHPLC), methylation fluorescence method (Methllight method) etc..
MS-HRM carries out PCR amplification after handling DNA fragmentation to be measured with bisulfite, methylates and does not methylate
There are sequence differences by DNA, so as to cause the variation of melting temperature and peak type, therefore, can pass through the variation of melting temperature and peak type
Distinguish exhaustive methylation, completely non-methylation or heterozygosis methylation.This method does not need fluorescence probe, directly analysis primer side
All methylation sites of wing sequence, but accuracy is to be improved.Before this method still lacks largely in clinical application at present
Look forward or upwards the support of Journal of Sex Research data.
MSP-DHPLC is a kind of deriving method of methylation status of PTEN promoter, i.e., is handling DNA to be measured with bisulfite
Methylation status of PTEN promoter is carried out after segment, amplified production is analyzed with DHPLC, and this method test program is easier to, susceptibility and
Specificity is higher.But current this method needs the support of a large amount of perspective study data also in conceptual phase.
Methllight method is anti-using specific real-time quantitative PCR i.e. after handling DNA fragmentation to be measured with bisulfite
The sequence to identify exhaustive methylation should be expanded, has the characteristics that susceptibility height, high specificity, template DNA dosage are few.Change
Good Methllight rule is to use hydrolysis probes instead on the original basis and increase blocking oligonucleotide to improve analysis susceptibility.
Methllight method is 9 methylation detecting method of Septin more mature at present, accordingly 9 methyl of Septin of method exploitation
Change the Epi that testing product has Epigenomics AG companyTest kit, Abbott Molecular
Real Time mS9 kit etc..Wherein, the Epi of Epigenomics AG companyTest kit is first
Obtain the production for 9 gene methylation of Septin in external qualitative detection human peripheral blood plasma of FDA, CE, CFDA tripartite certification
Product.But the kit carries out methylation state detection, and its applicable detection machine only for 9 gene of Septin, one region
Type popularity rate is lower, its promotion and application clinically is caused to be restricted.
Furthermore it is noted that although bisulfite conversion method is considered as " the gold mark of DNA methylation analysis
It is quasi- ", but its there are two major defects: 1) bisulfite conversion condition is excessively violent, and DNA may be degraded to reduce
The sensitivity of PCR and subsequent analysis technology；2) unmodified cytimidine is fully converted to thymidine by bisulfite conversion
All Cytosines are that thymidine is serious by (unmodified cytimidine account for total cytimidine in human genome about 95%)
Sequence complexity is reduced, causes to detect of poor quality, positioning rate is low, and genome covering is unevenly and a system such as testing cost increase
Column problem.Therefore, the inherent shortcoming of bisulfite conversion method, DNA methylation assay 9 genetic fragment Single-issue of Septin with
And Fit Models problem must be solved the problems, such as in current 9 gene methylation of Septin detection.
Summary of the invention
An object of the present invention is to provide a kind of 9 gene methylation detection kit of Septin, the kit have compared with
High detection flux, higher detection specificity and sensibility.
To achieve the goals above, technical scheme is as follows:
A kind of 9 gene methylation detection kit of Septin, including following three groups be directed to Septin 9-v2 exon first
At least one set in the specific primer and probe of baseization detection, the primer and probe are as follows: with SEQ ID 2 be the SEQ of target spot
ID NO.5-SEQ ID NO.7, using SEQ ID NO.3 as SEQ ID NO.8-SEQ ID NO.10 of target spot, with SEQ ID
NO.4 is the SEQ ID NO.11-SEQ ID NO.13 of target spot.
It further include TET enzyme and TET oxidation buffer liquid in wherein some embodiments.
It further include internal standard gene primer and probe in wherein some embodiments, base sequence group becomes SEQ ID
NO.14—SEQ ID NO.16。
It further include negative quality-control product and positive quality control product, the feminine gender quality-control product is by ox blood in wherein some embodiments
The human gene group DNA of albumin and the non-methylation of 9 gene of Septin composition；The positive quality control product by bovine serum albumin,
The human gene group DNA of 9 gene methylation of human gene group DNA and Septin of the non-methylation of 9 gene of Septin forms.
It further include pyridine borane in wherein some embodiments.
In wherein some embodiments, the TET oxidation buffer formula of liquid is as follows: 167 ± 10mM HEPES (4- ethoxy
Piperazine ethanesulfonic acid) pH of buffer=8.0,333 ± 10mM NaCl, 3.3 ± 0.2mM α-ketoglutaric acid disodium salt dihydrate,
6.67 ± 0.05mM L-AA, 4 ± 0.5mMATP, 8.33 ± 0.03mM dithiothreitol (DTT).
In wherein some embodiments, the fluorescent PCR pre-reaction liquid includes PCR buffer, MgCl2, dNTP, primer and
The water of probe, nuclease free.
In wherein some embodiments, the 5 ' of the specific probe and internal standard gene probe are terminal modified fluorescence report base
Group FAM, HEX, ROX or CY5,3 ' ends are connected with MGB modification group.
The present invention provides a kind of 9 gene methylation detection kit of Septin, which devises three groups of primers and spy
Needle, in SEQ ID NO.1Septin 9-v2 exons 1 (including promoter region and transcription initiation site) range
It designs to obtain, specificity with higher for target spot in three regions.The primer and probe designed with this, then cooperate and utilize TET
5mC and 5hmC complete oxidation is 5- carboxyl cytimidine (5caC) by enzyme, and 5caC is reduced to dihydro urine by subsequent pyridine borane completely
Pyrimidine (DHU)；The two is mutually coordinated, to realize the high sensitivity and the detection of high specific of 9 gene methylation of Septin.
Another aspect of the present invention, there is provided a kind of pre-treating methods of 9 gene methylation of Septin detection, should
Pre-treating method is suitble to the use of the detection kit of above-mentioned 9 gene methylation of Septin detection, DNA to be measured can be effectively reduced
The degradation rate of segment guarantees the quality of DNA fragmentation to be measured；Meanwhile the cytimidine modified in DNA sample to be measured can be converted completely
For thymidine, this guarantees the complexity of sequence, in conjunction with the above-mentioned primer and probe designed according to the method, to improve
The specificity entirely detected.
Realize that the technical solution of above-mentioned purpose is as follows.
A kind of sample-pretreating method of 9 gene methylation of Septin detection, comprising the following steps:
(1) TET oxydasis: DNA sample to be measured is aoxidized using TET enzyme；Then it is carried out after Proteinase K is handled
Purifying, obtains DNA sample after purification；
(2) pyridine borane reacts: the DNA sample that step (1) obtains carries out pyridine borane reaction, then carries out purification process i.e.
DNA sample after being converted.
In wherein some embodiments, in the above method, the reaction condition of TET oxydasis described in step (1) is 37 ± 2
DEG C be incubated for 80 ± 5min；And/or oxidation reaction system is as follows:
DNA sample to be measured,
TET oxidation buffer liquid, 15 μ l,
1.5mM Fe2+, 3.33 μ l,
TET enzyme, 4 μM, adding water to total volume is 50 μ l.
In wherein some embodiments, Proteinase K described in step (1) processing it is specific as follows: to DNA sample to be measured with
The Proteinase K juxtaposition that 1 ± 0.1 μ l concentration is 0.8U is added after the completion of the oxidation reaction of TET enzyme directly into oxidation reaction system
In 50 ± 2 DEG C of 1 ± 0.1h of incubation.
In wherein some embodiments, reaction system include: oxidation after DNA sample, the 3M sodium acetate solution of 10 ± 1 μ l, 5
The 10M pyridine borane of ± 0.5 μ l, 50 μ l of total volume.
In wherein some embodiments, pyridine borane reaction condition is to be incubated for 16 ± 2h in 37 ± 2 DEG C in step (2).
The technical solution that the above various optimizations obtain, can be finally complete by the cytimidine to methylate in DNA to be measured well
It is converted into thymidine entirely, and unmethylated cytimidine remains unchanged, to obtain DNA sample after the conversion of high quality.
Another aspect of the present invention is to provide a kind of 9 gene methylation detection method of Septin.
A kind of 9 gene methylation detection method of Septin, includes the following steps
According to the method described above, pre-treatment is carried out to sample to be tested, the DNA sample after being converted；
According to the primer and probe of above-mentioned 9 gene methylation detection kit of Septin, to DNA sample after the conversion
Carry out multiple fluorescence PCR reaction.
The multiple fluorescence PCR reaction condition is as follows in one of the embodiments:
1:94 DEG C of stage, 20min；1 circulation；
2:62 DEG C of stage, 5s；55.5 DEG C, 35s；93 DEG C, 30s；Above 45 circulations；
3:40 DEG C of stage, 5s；1 circulation.
5mC and 5hmC in the above pre-treating method, by TET oxydasis described in step (1), in DNA sample to be measured
Complete oxidation is 5caC；It is reacted again by pyridine borane described in step (2), 5caC is complete in the DNA sample obtained in step (1)
It is reduced to DHU entirely, thus DNA sample after being converted.Compared with bisulfite conversion method, TET enzyme oxygen described in step (1)
It is more mild to change the reaction condition reacted with pyridine borane described in step (2), can effectively reduce the degradation of DNA fragmentation to be measured
Rate guarantees the quality of DNA fragmentation to be measured；Meanwhile with bisulfite conversion method by cytimidine unmodified in DNA sample to be measured
Being fully converted to thymidine causes unlike the complexity of sequence seriously reduces, and this method is will to repair in DNA sample to be measured
The cytimidine of decorations is fully converted to thymidine, and this guarantees the complexity of sequence, to improve the specificity of subsequent detection.
In addition, the quadruple Fluorescence PCR used in this method can be realized to the trizonal methylation state of 9 gene of Septin
Detection, compared to the Epi of Epigenomics AG companyTest kit only detects 9 gene of Septin one
The methylation state of segment, 9 gene specific region methylation detecting method of Septin provided by the present invention have higher inspection
Survey flux, higher detection specificity and lower false positive ratio.Furthermore detection kit of the present invention respectively reacts examination
Agent component applies also for domestic more universal fluorescence quantitative PCR instrument, to be conducive to clinical expansion and popularize.
9 gene methylation detection method of Septin of the present invention and kit have the advantage that
(1) detection sensitivity is high, specificity is good: the present invention converts DNA to be measured using TET auxiliary pyridine borane and can effectively protect
The quality of DNA and the complexity of sequence after card conversion, to improve the sensitivity and specificity of subsequent detection；The present invention is directed to
Septin9-v2 exons 1 separately designs three groups of Methylation-specific primers and probe, specific primer can ensure that aim sequence
Accurately and effectively expand, so that the sensitivity of detection is improved, specific primer and the equal specific recognition methylation of specific probe
Region of DNA domain reduces false positive rate to improve detection specificity.
(2) detection flux is high: the present invention realizes disposable complete to the trizonal methylation state of 9 gene of Septin
Detection, to improve detection flux.
(3) testing result is accurate and reliable: present invention design has negative quality-control product and positive quality control product, can be better protected from vacation
The generation of positive findings and false negative result, so that it is guaranteed that testing result is accurate and reliable.
(4) it is suitable for the higher fluorescence quantitative PCR instrument of domestic popularity rate, is conducive to promote in clinical application.
(5) applicable DNA sample type multiplicity: it can be used for plasma dna sample, tissue DNA sample, faeces DNA sample etc..
Detailed description of the invention
Fig. 1 is the amplification curve diagram of methylation human genome DNA after detection 25pg/ml conversion in the embodiment of the present invention 2
Schematic diagram；
Fig. 2 is the amplification curve diagram of the negative quality-control product of the present invention；
Fig. 3 is the amplification curve diagram of positive quality control product of the present invention.
Specific embodiment
Unless otherwise specified, practice of the invention will use molecular biology, cell biology, immunology and recombinant DNA
Traditional technology, belong to art technology range.See, for example, Sambrook, Fritsch and Maniatis, molecular cloning
Experiment guide, the 3rd edition (2002).In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or
According to the normal condition proposed by manufacturer.Used various common chemical reagent, are commercial product in embodiment.
Unless otherwise defined, all technical and scientific terms used in the present invention and belong to technical field of the invention
The normally understood meaning of technical staff it is identical.Term used in the description of the invention is intended merely to describe specific reality
The purpose for applying example is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant listed
Any and all combinations of project.
Below in conjunction with embodiment, invention is further explained.It will be appreciated that following embodiment provides only
It is the purpose in order to play explanation, is not used to limit the scope of the present invention.Those skilled in the art is not carrying on the back
In the case where from spirit of the invention and spirit, the present invention can be carry out various modifications and be replaced.
Embodiment 1: a kind of method and kit of the detection of 9 gene methylation of Septin
A kind of method and kit of the detection of 9 gene methylation of Septin, the detection method being related to utilize TET enzyme will
5mC and 5hmC aoxidizes 5caC in DNA to be measured, and 5caC is reduced to dihydro DHU, reused for Septin9 by subsequent pyridine borane
The specific primer and probe of gene methylation design establish multiple fluorescence PCR system and carry out PCR amplification, in the process DHU
It is converted into T, 9 gene methylation state of Septin is judged according to the fluorescence signal that multiple fluorescence PCR reacts；The kit packet
Include TET oxidation buffer liquid, TET enzyme, fluorescent PCR pre-reaction liquid, polymerase, negative quality-control product, positive quality control product.9 base of Septin
Because methylation detection kit preparation the following steps are included:
(1) TET oxidation buffer formula of liquid according to the invention prepares TET oxidation buffer liquid, and packing saves backup.It is described
TET oxidation buffer formula of liquid is as follows: the hydration of 167mM HEPES pH=8.0,333mMNaCl, 3.3mM α-ketoglutaric acid disodium
Object, 6.67mM L-AA, 4mMATP, 8.33mM dithiothreitol (DTT).The packing of prepared TET oxidation buffer liquid and in-
80 DEG C of preservations；
(2) preparation of TET enzyme: TET enzyme is bought from Epigentek (article No. E12002-1), with original-pack storage, according to detection
Demand take in right amount be prepared into concentration be 4 μM/μ l working solution, packing be stored in -80 DEG C it is spare.
(3) design and preparation of 9 gene primer of Septin and probe: for the outer aobvious of 9 gene v2 transcript of Septin
(SEQ ID 2 to the specificity that SEQ ID 4) separately designs several pairs of DNA methylation assays draws in three regions of 1 (SEQ ID 1) of son
Each primer and probe is carried out preliminary experiment, compares the performances such as sensitivity, specificity by object and fluorescence probe, final preferably to go out to be used for
Three group-specific primers and probe of 9 gene methylation of Septin detection, it is specific such as SEQ ID NO.5 to SEQ ID NO.13.
Preferred specific primer and probe are stored with 100 μM of mother liquor, and the working solution for being prepared into 20 μM according to detection demand is spare.
The sequence of Septin 9-v2 exons 1 is as follows:
Wherein, first target area of Septin 9-v2 exons 1 sequence is as follows:
CAGGCGGGCGGAGCAGCCAGTGCGAGACAGGGAGGCCGGTGCGGGTGCGGGAACCTGATCCGCCCGGG
AGGCGGGGGCGGGGCGGGGGCGCAGCGCGCGGGGAGGGGCCGGCGCCCGCCTT(SEQ ID NO.2)
Second target area of Septin 9-v2 exons 1 sequence is as follows:
GCTCCTCCGGCGGCTAGCTCTGCACTGCAGGAGCGCGGGCGCGGCGCCCCAGCCAGCGCGCAG(SEQ
ID NO.3)
The third target area of Septin 9-v2 exons 1 sequence is as follows:
TCCGCGCGACCCGCTGCCCACCAGCCATCATGTCGGACCCCGCGGTCAACGCGCAGCTGGATGGGATC
ATTT(SEQ ID NO.4)
Shown in the primer and probe sequence table specific as follows of the kit:
(4) design and preparation of internal standard gene primer and probe, for ACTB gene design primer and probe, specifically such as
SEQ ID 14 to SEQ ID 16.The internal standard gene primer and probe is configured to 100 μM of mother liquor storage respectively, according to detection
The working solution that demand is prepared into 20 μM is spare.
(5) preparation of fluorescent PCR pre-reaction liquid: fluorescent PCR pre-reaction liquid preparation program according to the invention prepares fluorescence
PCR pre-reaction liquid saves.
The preparation program of fluorescent PCR pre-reaction liquid is as follows:
(6) preparation of negative quality-control product and positive quality control product: with bovine serum albumin and the non-methylation of 9 gene of Septin
Human gene group DNA is configured to negative quality-control product, with bovine serum albumin (4 μ L), the human genome of the non-methylation of 9 gene of Septin
The 20 μ L of human gene group DNA (1ng/ μ L) of DNA480 μ L (1ng/ μ L) and 9 gene methylation of Septin is configured to positive quality control
Product,.(setting of positive quality control product is weakly positive).
(7) packing and assembling of kit: kit specification is 24 person-portions/box, and packing and assembling scheme are as follows:
The detection process of the present embodiment the following steps are included:
(1) prepare the kit and relevant various reagents and material, water, Proteinase K including nuclease free,P-30 gel column, 1.8 × Ampure XP magnetic bead, Zymo-IC column (being purchased from Zymo Research company), PB are slow
Fliud flushing (be purchased from Qiagen company), ThermoMixer constant temperature blending instrument (Eppendorf), fluorescent PCR instrument be domestic popularity rate compared with
High type such as ABI 7500 and 1.5mM Fe2+, 3M sodium acetate solution, commercially purchase is former for 10M pyridine borane etc.
Material is voluntarily prepared according to the concentration of detection demand.Wherein, 1.5mM Fe2+It is prepared using frerrous chloride, ready-to-use (because
Fe2+Solution placement can be oxidized too long).
(2) TET oxydasis reaction system is prepared according to following table:
(3) after the TET oxydasis reaction system of DNA sample to be measured prepares, 37 DEG C of incubation 80min is placed in, 1 μ l is added
Proteinase K (0.8U) is placed in 50 DEG C of incubation 1h, then usesP-30 gel column (is purchased from Bole's life medical product
(Shanghai) Co., Ltd.) and 1.8 × Ampure XP magnetic bead (be purchased from Beckman Coulter Inc., the U.S.) press corresponding product
Specification carries out purification process, is finally eluted with the water of 35 μ l nuclease frees with DNA sample after being aoxidized.
(4) pyridine borane reaction system is prepared according to following table:
| Reagent name | Dosage |
| DNA sample after oxidation | 35μl |
| 3M sodium acetate solution (pH=4.3) | 10μl |
| 10M pyridine borane | 5μl |
| Total volume | 50μl |
(5) after pyridine borane reaction system prepares, it is placed in 37 DEG C of ThermoMixer constant temperature blending instrument (Eppendorf),
16h is incubated under 850rpm, then reaction product is carried out using Zymo-IC column and PB buffer according to corresponding product description pure
Change processing, DNA sample after finally being converted.
(6) Fluorescence PCR system is prepared according to following table:
| Reagent name | Dosage |
| Fluorescent PCR pre-reaction liquid | 40μl |
| Polymerase | 5μl |
| DNA sample after conversion | 5μl |
| Total volume | 50μl |
(7) after Fluorescence PCR system prepares, it is put into fluorescence quantitative PCR instrument, PCR reaction condition is arranged according to the form below, glimmering
The channel Fluorescent Quantitative PCR instrument fluorescence channel FAM, HEX, ROX and CY5, carries out amplification reaction.
(8) testing result interpretation: judged according to the Ct value of FAM, HEX, ROX and CY5 fluorescence signal detected.Fluorescence
Signal collection is the collection at 55.5 DEG C of the 2nd stage of Fluorescence PCR, each circulating collection first order fluorescence signal.Detection reaction
FAM, HEX, ROX and CY5 fluorescence intensity of system indicate DNA applied sample amount in allowed band when reaching the threshold value of setting with CY5
Interior, FAM, HEX, ROX signal results are credible；Reach required cycle-index Ct value when the threshold value of setting with FAM, HEX, ROX
As yin and yang attribute criterion, Ct value is 0 more than or equal to 45: negative；Ct value is less than 45: positive.9 gene of specific Septin
DNA methylation assay result judges that table is as follows:
Embodiment 2: sensitivity analysis experiment
(1) sample to be tested selects
Select methylation human genome DNA as sample to be tested in the present embodiment.In order to simulate dissociative DNA piece segment length
It spends, the above-mentioned methylation human genome DNA as sample to be tested of ultrasonication is used in this experiment, makes its fragmentation.
(2) conversion of DNA sample
The method and kit of a kind of detection of 9 gene methylation of Septin obtained in embodiment 1 are according to embodiment 1
In detecting step the methylation human genome DNA of fragmentation is carried out after TET auxiliary pyridine borane processing converted
DNA。
(3) multiple fluorescence PCR detects
According to 1 the method for embodiment obtain conversion after DNA concentration adjust to 10ng/ml, 1ng/ml, 100pg/ml,
50pg/ml, 25pg/ml, 10pg/ml, 1pg/ml, respectively as DNA profiling 9 gene methyl of Septin described in embodiment 1
For the method and kit for changing detection according to the detecting step progress multiple fluorescence PCR detection in embodiment 1, every kind of concentration sets up 2
A repetition, applied sample amount are 5 μ l, set up 1 blank control, analyze the sensitivity of kit of the present invention according to testing result.
(4) testing result and analysis
When testing result shows the methylation human genome DNA sample concentration after conversion down to 25pg/ml, CY5,
There are amplified signal, and all S-type amplification curve in tetra- channels FAM, HEX and ROX, and testing result is still that Septin 9 methylates
Positive (referring to Figure 1 and Fig. 3)；That is, the present invention can detecte the DNA profiling amount down to 2.5pg/ml, this is lower than
The Epi of similar product Epigenomics AG companyThe detection limit 4.7pg/ that test kit is reported
Ml, it was demonstrated that detection sensitivity of the invention is high, and the pattern detection result positive detection low to methylate DNA content can be improved
Rate.Concrete outcome see the table below:
Embodiment 3: specificity analysis experiment
(1) sample to be tested selects
Select that non-methylation human genome DNA, methylation human genome DNA, 8 from healthy person in the present embodiment
DNA sample, 8 be determined as the DNA sample of 9 methylation positive of Septin as sample to be tested through bisulfite PCR sequencing PCR.
Wherein, non-methylation human genome DNA, methylation human genome DNA are through ultrasonication fragmentation.
(2) conversion of DNA sample
The method and kit of the detection of 9 gene methylation of Septin described in embodiment 1 are according to the detection in embodiment 1
Step carries out DNA after TET auxiliary pyridine borane processing is converted to above-mentioned sample to be tested respectively.
(3) multiple fluorescence PCR detects
DNA is as a kind of DNA profiling 9 gene methylation of Septin obtained in embodiment 1 after the conversion of above-mentioned acquisition
The method and kit of detection carry out multiple fluorescence PCR detection according to the detecting step in embodiment 1, and applied sample amount is on 2ng/ μ l
5 μ l of sample, sets up 1 blank control, analyzes the specificity of kit of the present invention according to testing result.
(4) testing result and analysis
Testing result shows, 1 part of non-methylation human genome DNA and 8 DNA sample testing results from healthy person
It is all that Septin 9 methylates feminine gender, 1 part of methylation human genome DNA and 88 are determined as through bisulfite PCR sequencing PCR
The DNA sample testing result of 9 methylation positive of Septin is all 9 methylation positive of Septin, it was demonstrated that detection of the invention
Specific good, negative match-rate 100%, positive coincidence rate 100%.Concrete outcome see the table below that (N1-N9 respectively represents non-first
The DNA sample of base human genome DNA and healthy person, P1-P9 respectively represent methylation human genome DNA and Septin 9
The DNA sample of methylation positive):
Embodiment 4: similar product comparative experiments
(1) sample to be tested selects
It is apt to totally 100, plasma dna sample after making a definite diagnosis collected by medical test from Guangzhou benefit in the present embodiment, as to be measured
Sample.Wherein, 8 come from healthy person, and 21 come from Colorectal benign lesion, remaining 71 come from colorectal cancer patients.
(2) conversion of DNA sample
100 plasma dna samples of above-mentioned collection take in right amount respectively, 9 gene methylation of Septin described in embodiment 1
The method and kit of detection carry out TET auxiliary pyridine borane processing respectively according to the detecting step in embodiment 1 and are converted
DNA afterwards.
(3) multiple fluorescence PCR detects
DNA is as a kind of DNA profiling 9 gene methylation of Septin obtained in embodiment 1 after the conversion of above-mentioned acquisition
The method and kit of detection carry out multiple fluorescence PCR detection according to the detecting step in embodiment 1, set up 1 blank control, examine
Survey the accuracy of result and similar product testing result comparative analysis kit of the present invention.
(4) similar product detects
100 plasma dna samples of above-mentioned collection take it is appropriate respectively using bisulfite PCR sequencing PCR and
The Epi of EpigenomicsAG companyTest kit is detected.Bisulfite PCR sequencing PCR is to utilize weight
The cytosine deamination not methylated in DNA sample is changed into uracil by sulphite, and the cytimidine to methylate is protected
Hold constant, through PCR amplification, then uracil is converted into thymidine；Finally, PCR product is sequenced, with untreated sequence
Column compare, and judge whether that the site CpG methylates.The method is considered as " goldstandard " of DNA methylation assay.EpiTest kit is then a kind of blood testing for 9 gene methylation of Septin for obtaining FDA approval.
The lucky biology of bisulfite sequencing commission U.S. is completed, EpiTest is bought from Epigenomics AG company
Kit is carried out according to kit specification detection.
(5) testing result and analysis
Testing result shows detection method and kit test result and bisulfite sequencing assay result of the invention
Coincidence rate be 100%, with EpiThe coincidence rate 95% of test testing result, it was demonstrated that detection of the invention is quasi-
True property.In addition, EpiTest detects 5 positives altogether in healthy person and Colorectal benign lesion plasma dna, and
The present invention only detects 3 positives in Colorectal benign lesion plasma dna, illustrates compared to EpiTest, this hair
Bright detection method and kit is more specific, reduces false positive rate；EpiTest is in colorectal cancer blood
It starches and detects 55 positives in DNA, and the present invention detects 58 positives in plasma of colorectal cancer DNA, illustrates detection of the invention
Method and kit have more sensibility, reduce false negative rate.(feminine gender indicates that Septin 9 methylates yin to concrete outcome such as following table
Property, the positive indicates 9 methylation positive of Septin):
Embodiment 5: different DNA sample type detection Comparative result tests
(1) sample to be tested selects
It is apt to the plasma dna sample for 20 colorectal cancer patients made a definite diagnosis collected by medical test in the present embodiment from Guangzhou benefit
Sheet, tissue DNA sample, each portion of faeces DNA sample, as sample to be tested.
(2) conversion of DNA sample
The method and kit of the detection of 9 gene methylation of Septin described in embodiment 1 are according to the detection in embodiment 1
Step carries out TET auxiliary pyridine borane processing to above-mentioned plasma dna sample, tissue DNA sample, faeces DNA sample respectively and obtains
DNA after conversion.
(3) multiple fluorescence PCR detects
The side that DNA is detected as DNA profiling 9 gene methylation of Septin described in embodiment 1 after the conversion of above-mentioned acquisition
Method and kit are set up 1 blank control, are tied according to detection according to the detecting step progress multiple fluorescence PCR detection in embodiment 1
Fruit analyzes the applicable sample type of kit of the present invention.
(4) testing result and analysis
Testing result is shown, for providing 20 colorectal cancer patients of plasma dna, tissue DNA and faeces DNA simultaneously,
Three kinds of DNA sample testing results are identical, coincidence rate 100%, illustrate that detection method and kit of the invention are suitable for
A variety of DNA sample types, including plasma dna, tissue DNA and faeces DNA.Concrete outcome see the table below:
DNA and purifying DNA detection effect comparative test are not purified after embodiment 6:TET auxiliary pyridine borane processing
(1) sample to be tested selects
Non- methylation human genome DNA and methylation mankind base of the selection through ultrasonication fragmentation in the present embodiment
Because group DNA is as sample to be tested.
(2) conversion of DNA sample
The method and kit of the detection of 9 gene methylation of Septin described in embodiment 1 are according to the detection in embodiment 1
The first of the step non-methylation human genome DNA to the identical fragmentation of 6 parts of concentration and the identical fragmentation of 6 parts of concentration respectively
Base human genome DNA carries out TET auxiliary pyridine borane processing.Wherein, 3 parts of non-methylation human genome DNAs and 3 parts of first
Base human genome DNA its reaction product after completing pyridine borane reaction does not purify as without subsequent purification processing
DNA；Another 3 parts of non-methylation human genome DNAs and 3 parts of methylation human genome DNAs after completing pyridine borane reaction its
Reaction product carries out purification process according to corresponding product description using Zymo-IC column and PB buffer, obtains purifying DNA.
(3) multiple fluorescence PCR detects
Above-mentioned acquisition does not purify DNA and purifying DNA as 9 gene methyl of Septin described in DNA profiling embodiment 1
The kit for changing detection carries out multiple fluorescence PCR detection according to the detecting step in embodiment 1,1 blank control is set up, according to inspection
It surveys interpretation of result and does not purify DNA and purifying DNA detection effect.
(4) testing result and analysis
Testing result shows, unpurified non-methylation human genome DNA after 3 parts of TET auxiliary pyridine boranes processing
ACTB gene C t value is apparently higher than the non-methylation human genome DNA purified after 3 parts of TET auxiliary pyridine boranes processing, and 3 parts
TET assists the Septin 9 and ACTB gene (internal reference base of unpurified methylation human genome DNA after pyridine borane processing
Cause) the methylation human genome DNA that purifies after obviously higher than the 3 parts TET auxiliary pyridine boranes processing of Ct value, and wherein 1 part
TET assist pyridine borane processing after unpurified methylation human genome DNA's testing result for null result (reference gene
Value > 36 Ct), more preferably, testing result is accurate, and TET for the DNA detection effect through purifying after illustrating TET auxiliary pyridine borane processing
It is poor that DNA detection effect is not purified after auxiliary pyridine borane processing, thereby increases and it is possible to null result or false negative result occurs.It is possible that
It is because it includes 600mM sodium acetate that pyridine borane, which reacts final system, pH=4.3 and 1M pyridine borane may be to subsequent PCR
Have an impact, by the various investigations of inventor, final or confirmation is needed pyridine borane treated reaction product purifying.Cause
This, is in order to ensure the accuracy of testing result, the method and kit of the detection of 9 gene methylation of Septin described in embodiment 1
It also needs to carry out at purifying after carrying out TET auxiliary pyridine borane processing to DNA sample to be measured according to the detecting step in embodiment 1
Reason.Concrete outcome see the table below:
Embodiment 7:TET auxiliary pyridine borane processing converts methylated cytosine to the verification test of thymidine
(1) sample to be tested selects
Select the methylation human genome DNA through ultrasonication fragmentation as sample to be tested in the present embodiment.
(2) conversion of DNA sample
The method and kit of the detection of 9 gene methylation of Septin described in embodiment 1 are according to the detection in embodiment 1
Step carries out TET auxiliary pyridine borane processing to the methylation human genome DNA of the identical fragmentation of 3 parts of concentration and is converted
DNA afterwards.3 parts of methylation human genome DNA of the fragmentation of same concentrations are separately taken to handle without TET auxiliary pyridine borane,
As unconverted DNA.
(3) multiple fluorescence PCR detects
DNA and unconverted DNA is as DNA profiling 9 gene first of Septin described in embodiment 1 after the conversion of above-mentioned acquisition
The method and kit of baseization detection carry out multiple fluorescence PCR detection according to the detecting step in embodiment 1, set up 1 blank pair
According to DNA and unconverted DNA testing result can be by methylated cytosines to verify TET auxiliary pyridine borane processing after comparison conversion
It is converted into thymidine.
(4) sequencing detection
DNA and unconverted DNA is as template 9 gene first of Septin described in embodiment 1 after the conversion that step (2) obtains
Three group-specific primers (the SEQ for the detection of 9 gene methylation of Septin that the method and kit of baseization detection preferably go out
IDNO.5-6, SEQ ID NO.8-9 and SEQ ID NO.11-12) PCR amplification, the lucky biology of amplified production commission U.S. are carried out respectively
It is sequenced, TET auxiliary pyridine borane processing is further verified by sequencing result to convert thymus gland for methylated cytosine
Pyrimidine.
(5) testing result and analysis
Because multiple fluorescence PCR detection and sequencing detect related primer and probe both from implementation in the present embodiment
The method and kit of the detection of 9 gene methylation of Septin described in example 1, and these primers and probe are to assist pyridine according to TET
DNA sequence dna design after borine conversion, so, theoretically, unconverted DNA is that can not amplify using above-mentioned primer and probe
Septin9 gene methylation target fragment, DNA can amplify 9 base of Septin using above-mentioned primer and probe after only converting
Because of methylation target fragment.
Multiple fluorescence PCR testing result is shown in the present embodiment, and methylation human genome DNA detects knot after 3 parts of conversions
Fruit is 9 methylation positive of Septin, 3 parts of unconverted DNA without 9 gene methylation target fragment amplified signal of Septin,
This is consistent with theory, illustrates that TET auxiliary pyridine borane processing can convert thymidine for methylated cytosine.Sequencing result is aobvious
Show, the amplifiable 9 gene methylation target fragment of Septin out of the human genome DNA that methylates after 3 parts of conversions simultaneously obtains corresponding
Sequence information, and unconverted DNA fails to amplify 9 gene methylation target fragment of Septin, can not be sequenced；It will conversion
Methylation human genome DNA, which is sequenced, afterwards obtains 9 gene methylation target fragment sequence of Septin and 9 gene order of Septin
It is compared, methylated cytosine is converted into chest in the 9 gene methylation target fragment sequence of Septin that discovery sequencing obtains
Gland pyrimidine, and unmethylated cytimidine remains unchanged；Further demonstrating TET auxiliary pyridine borane processing can be by the born of the same parents that methylate
Pyrimidine is fully converted to thymidine.Concrete outcome is as shown in the table:
Table is unconverted DNA and conversion DNA multiple fluorescence PCR testing result
The human genome DNA that methylates after conversion using primer SEQ ID NO.5-6 carries out that detected sequence is sequenced
It is as follows:
CAGGTGGGTGGAGCAGCCAGTGTGAGACAGGGAGGCTGGTGTGGGTGTGGGAACCTGATCTGCCTGGGA
GGTGGGGGTGGGGTGGGGGTGCAGTGTGTGGGGAGGGGCTGGTGCCTGCCTT
The comparison knot for sequence and primer SEQ ID NO.5-6 detection the target area sequence SEQ ID NO.2 that above-mentioned sequencing obtains
Fruit is as follows:
The human genome DNA that methylates after conversion using primer SEQ ID NO.8-9 carries out that detected sequence is sequenced
It is as follows:
GCTCCTCTGGTGGCTAGCTCTGCACTGCAGGAGTGTGGGTGTGGTGCCCCAGCCAGTGTGCAG
The comparison knot for sequence and primer SEQ ID NO.8-9 detection the target area sequence SEQ ID NO.3 that above-mentioned sequencing obtains
Fruit is as follows:
The human genome DNA that methylates after conversion using primer SEQ ID NO.11-12 carries out that detected sequence is sequenced
It arranges as follows:
TCTGTGTGACCTGCTGCCCACCAGCCATCATGTTGGACCCTGTGGTCAATGTGCAGCTGGATGGGATCA
TTT
The comparison for sequence and primer SEQ ID NO.11-12 detection the target area sequence SEQ ID NO.4 that above-mentioned sequencing obtains
As a result as follows:
Embodiment 8: different method for transformation comparative tests
(1) sample to be tested selects
Non- methylation human genome DNA and methylation mankind base of the selection through ultrasonication fragmentation in the present embodiment
Because group DNA is as sample to be tested.
(2) conversion of DNA sample
The method and kit of the detection of 9 gene methylation of Septin described in embodiment 1 are according to the detection in embodiment 1
The first of the step non-methylation human genome DNA to the identical fragmentation of 3 parts of concentration and the identical fragmentation of 3 parts of concentration respectively
Base human genome DNA carries out DNA after TET auxiliary pyridine borane processing is converted.Separately take the fragmentation of same concentrations
It is phonetic to born of the same parents that each 3 parts of the methylation human genome DNA of non-methylation human genome and fragmentation uses current industry to generally acknowledge
Pyridine conversion ratio is more than the kit of 99% Qiagen company: EpiTect Fast Bisulfite Conversion Kits
(article No. 59824) carries out bisulfite conversion processing according to its specification and obtains DNA after bisulfite converts.
(3) multiple fluorescence PCR detects
The method that DNA 9 gene methylation of Septin described in embodiment 1 detects after above-mentioned TET auxiliary pyridine borane conversion
And kit carries out multiple fluorescence PCR detection according to the detecting step in embodiment 1.DNA makes after above-mentioned bisulfite conversion
With for bisulfite conversion after 9 gene order of Septin and ACTB gene order design with Septin described in embodiment 1
The method and kit primer and the identical primer of probe design position and probe of 9 gene methylations detection carry out multi-fluorescence
PCR detection.Compare the multiple fluorescence PCR detection knot of DNA after DNA and bisulfite convert after TET auxiliary pyridine borane converts
Fruit is to compare two kinds of method for transformation.
(4) testing result and analysis
Testing result shows, the non-methylation mankind of detection method of the invention and kit and the conversion of Qiagen kit
Genomic DNA testing result is all that Septin 9 methylates feminine gender, detection method of the invention and kit and Qiagen reagent
Methylation human genome DNA's testing result of box conversion is all 9 methylation positive of Septin, illustrates two kinds of method for transformation
Testing result is identical.But from the point of view of detecting Ct value, the Ct value of the DNA detection of detection method of the invention and kit conversion
It is slightly lower than Qiagen kit, illustrates the expanding effect of the DNA sample of detection method and kit conversion of the invention more
It is good；That is, detection method and kit changing effect of the invention are more preferably.In addition, detection method and reagent of the invention
Box conversion temperature is more mild, therefore more advantage.Concrete outcome is as follows, and (1-3 represents non-methylation human genome
DNA, 4-6 represent methylation human genome DNA):
Show that the present invention using 9 gene methylation of mankind Septin as test object, uses TET enzyme by above experiment
By in DNA sequence dna to be measured 5mC and 5hmC be oxidized to 5caC, 5caC is reduced to DHU by subsequent pyridine borane；It is directed to respectively again
9 gene methylation sequence design of Septin, three group-specific primers and probe are established specific multiple fluorescence PCR system and are carried out
Amplification, converts thymidine for DHU, to realize disposable complete to the trizonal methylation state of 9 gene of Septin
Detection.TET assists pyridine borane to handle reaction condition involved in the method for DNA to be measured more mild in the present invention, most
Eventually the result is that the cytimidine to methylate in DNA to be measured is fully converted to thymidine, and unmethylated cytimidine is kept not
Become, this both ensure that the complexity of DNA sequence dna after the guaranteed conversion of quality of DNA after conversion, to improve subsequent detection
Sensitivity and specificity；Therefore, compared with bisulfite conversion method, TET auxiliary pyridine borane handles the method for DNA to be measured more
Tool advantage.The multiple fluorescence PCR system established in the present invention is realized once based on domestic more universal fluorescence quantitative PCR instrument
Property detection to the trizonal methylation state of 9 gene of Septin, effectively increase detection flux, detection specificity, reduce
False positive rate；Therefore, with the Epi of Epigenomics AG companyTest kit is compared, in the present invention
Kit has more advantage.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Surexam Biotechnology Co., Ltd.
<120>a kind of method and kit of the detection of 9 gene methylation of Septin
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 363
<212> DNA
<213>Septin 9-v2 exons 1 (Artificial Sequence)
<400> 1
ccccaggcct ggccttgaca ggcgggcgga gcagccagtg cgagacaggg aggccggtgc 60
gggtgcggga acctgatccg cccgggaggc gggggcgggg cgggggcgca gcgcgcgggg 120
aggggccggc gcccgccttc ctcccccatt cattcagctg agccaggggg cctaggggct 180
cctccggcgg ctagctctgc actgcaggag cgcgggcgcg gcgccccagc cagcgcgcag 240
ggcccgggcc ccgccggggg cgcttcctcg ccgctgccct ccgcgcgacc cgctgcccac 300
cagccatcat gtcggacccc gcggtcaacg cgcagctgga tgggatcatt tcggacttcg 360
aag 363
<210> 2
<211> 121
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
caggcgggcg gagcagccag tgcgagacag ggaggccggt gcgggtgcgg gaacctgatc 60
cgcccgggag gcgggggcgg ggcgggggcg cagcgcgcgg ggaggggccg gcgcccgcct 120
t 121
<210> 3
<211> 63
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gctcctccgg cggctagctc tgcactgcag gagcgcgggc gcggcgcccc agccagcgcg 60
cag 63
<210> 4
<211> 72
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tccgcgcgac ccgctgccca ccagccatca tgtcggaccc cgcggtcaac gcgcagctgg 60
atgggatcat tt 72
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ctggccttga caggtgggtg 20
<210> 6
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ggaggaaggc aggcaccag 19
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
tggggtgggg gtgcagtgtg t 21
<210> 8
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cattcattca gctgagccag ggg 23
<210> 9
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gcagcaagga agcaccccca g 21
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gagtgtgggt gtggtgcccc 20
<210> 11
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tctgtgtgac ctgctgccca c 21
<210> 12
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gcacaccccc atcccacac 19
<210> 13
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
ttggaccctg tggtcaatgt g 21
<210> 14
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gttgttacag gaagtccctt gc 22
<210> 15
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
aaagcaatgc tatcacctcc cctg 24
<210> 16
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
acttctctct aaggagaatg gccc 24
Claims (10)
1. a kind of 9 gene methylation detection kit of Septin, which is characterized in that be directed to Septin 9-v2 including following three groups
At least one set in the specific primer and probe of exon DNA methylation assay, the primer and probe are as follows: be with SEQ ID 2
SEQ ID NO.5-SEQ ID NO.7 of target spot, using SEQ ID NO.3 as SEQ ID NO.8-SEQ ID NO.10 of target spot,
Using SEQ ID NO.4 as the SEQ ID NO.11-SEQ ID NO.13 of target spot.
2. 9 gene methylation detection kit of Septin according to claim 1, which is characterized in that further include TET enzyme
With TET oxidation buffer liquid.
3. 9 gene methylation detection kit of Septin according to claim 1, which is characterized in that further include internal standard base
Because of primer and probe, base sequence group becomes SEQ ID NO.14-SEQ ID NO.16.
4. 9 gene methylation detection kit of Septin according to claim 1, which is characterized in that further include negative matter
Control product and positive quality control product, it is described feminine gender quality-control product by bovine serum albumin and the non-methylation of 9 gene of Septin human genome
DNA composition；The positive quality control product is by bovine serum albumin, the human gene group DNA and Septin of the non-methylation of 9 gene of Septin
The human gene group DNA of 9 gene methylations forms.
5. 9 gene methylation detection kit of Septin according to claim 1-4, which is characterized in that also wrap
Include pyridine borane.
6. according to the described in any item 9 gene methylation detection kits of Septin of claim 2-4, which is characterized in that described
TET oxidation buffer liquid includes following component: 167 ± 10mM HEPES buffer solution pH=8.0,333 ± 10mM NaCl, 3.3 ±
0.2mM α-ketoglutaric acid disodium salt dihydrate, 6.67 ± 0.05mM L-AA, 4 ± 0.5mMATP, 8.33 ±
0.03mM dithiothreitol (DTT).
7. a kind of sample-pretreating method of 9 gene methylation of Septin detection, which comprises the following steps:
(1) TET oxydasis: DNA sample to be measured is aoxidized using TET enzyme, is then carried out after Proteinase K is handled pure
Change, obtains DNA sample after purification；
(2) pyridine borane reacts: the DNA sample that step (1) obtains carries out pyridine borane reaction, then carries out purification process and obtain
DNA sample after conversion.
8. sample-pretreating method according to claim 7, which is characterized in that TET oxydasis described in step (1) it is anti-
Answering condition is 37 ± 2 DEG C of 80 ± 5min of incubation；And/or TET oxydasis reaction system is as follows:
DNA sample to be measured,
TET oxidation buffer liquid, 15 ± 1 μ l,
1.5mM Fe2+, 3.33 ± 0.03 μ l,
TET enzyme, 4 ± 0.3 μM, adding water to total volume is 50 μ l.
9. sample-pretreating method according to claim 7, which is characterized in that the processing of Proteinase K described in step (1)
Are as follows: 1 ± 0.1 μ l concentration is added directly into oxidation reaction system after the completion of the oxidation reaction of DNA sample to be measured and TET enzyme is
The Proteinase K of 0.8U is placed in 50 ± 2 DEG C of 1 ± 0.1h of incubation.
10. according to the described in any item sample-pretreating methods of claim 7-9, which is characterized in that pyridine borane in step (2)
Reaction condition is to be incubated for 16 ± 2h in 37 ± 2 DEG C；
And/or reaction system includes: DNA sample after oxidation, the 3M sodium acetate solution of 10 ± 1 μ l, the 10M pyridine boron of 5 ± 0.5 μ l
Alkane, 50 μ l of total volume.
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