CN110257517A - Primer, kit and method based on pyrosequencing detection MLH1 promoter gene methylation - Google Patents
Primer, kit and method based on pyrosequencing detection MLH1 promoter gene methylation Download PDFInfo
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Abstract
The invention discloses primer, kit and methods based on pyrosequencing detection MLH1 gene promoter methylation, the primer includes a pair of specificity amplification primer SEQ NO 1 and SEQ NO 2, wherein 5 ' the end label biotins of SEQ NO1, SEQ NO2 are used as sequencing primer simultaneously.The present invention not only can be achieved at the same time for the qualitative and quantitative of site methylation, while relatively other common detection methods have that high accuracy, high detection rate, high throughput, detection cycle be short and the advantages such as low pollution risk.Testing result can be conducive to the diagnosis, treatment and prognosis evaluation that clinician preferably carries out related disease.
Description
Technical field
The invention belongs to life sciences and field of biotechnology, are a kind of sides of MLH1 gene promoter methylation detection
Method.
Background technique
DNA methylation (DNA methylation) is one of important modification of epigenetic, and methylation generally betides
CG connected dinucleotides position (CpGs), and gene expression is sent out by changing chromatin Structure, DNA construction and stability etc.
Wave important regulating and controlling effect.With the development of table science of heredity, it was recognized that tumour is not only genetic disease, while also with
The epigenetics such as the extremely caused gene regulation of DNA methylation is not normal are closely related.In normal cell, the CpG of promoter region
Island is in non-methylation state, and the most of island CpG dinucleotides for being dispersed in distribution mostly occurs methylation.Often with gene in tumour
The whole methylation level of group reduces and certain CpG island regions methylation level increases (such as tumor suppressor gene) extremely, and this
Two kinds of variations can occur simultaneously in a kind of tumour.The reduction of genome entirety methylation level can lead to Protooncogene Activated etc.,
Further promote the generation of tumour.The island CpG of gene promoter area is abnormal hyper-methylation and can lead to Silenced gene transcription,
So that the expression such as important gene such as tumor suppressor gene is extremely reduced or is not expressed, and then also promotes the formation of tumour cell.
Research finds cancer in hereditary nonpolyposis colorectal cancer (hereditary non-polyposis colorectal
Cancer, HNPCC), gastric cancer, the generation of the kinds of tumors such as carcinoma of endometrium and microsatellite instability (Microsatellite
Instability, MSI) it is closely related, and MSI is due to DNA mismatch reparation (Mismatch Repair, MMR) system function
Caused by defect.MLH1 is studied most in human DNA mismatch repair gene (Mismatch Repair genes, MMR genes)
One of more gene, expression product can recognize and repair gene mutation, safeguards the stability of genome.Function occurs for MLH1 gene
Energy obstacle can cause the high frequency of genome to be mutated, so that normally functioning albumen can not be synthesized, and then lose original mispairing and repair
Multiple function, changes the stability of genome, and then the formation of induced tumor.Numerous studies discovery is present in HNPCC, sporadic knot
MSI in the carcinoma of the rectum and gastric cancer is often as caused by MLH1 gene promoter region methylates.Haydon's etc. grinds
Study carefully discovery has 15%-20% that MLH1 promoter methylation occurs in MSI Sporadic Colorectal Carcinoma.Fang etc. is studied using MSP
The discovery of MLH1 gene promoter zone methylation level has more than 16% gastric cancer there are MLH1 promoter hyper-methylations in gastric cancer.This
A little research prompt MLH1 promoter methylations and the MSI of tumour are closely related, and can induce the occurrence and development of tumour.Detect MLH1
Methylation foundation can be provided for the early diagnosis of colorectal cancer, while reference can provide to its therapeutic scheme, facilitates precisely
The development of medical treatment, and the prognosis of patient can be assessed.
Have at present for the conventional method of MLH1 promoter methylation detection: methylation status of PTEN promoter (MSP), sulfurous acid
PCR (BSP), methylation sensitive high-resolution melting curve analysis (MS-HRM), fluorescent quantitation is sequenced in hydrogen salt.Wherein MSP
Method is to carry out judgement sample using PCR amplification detection with the presence or absence of methylation, and the method is practical and universal, but cannot accomplish quantitative inspection
It surveys, and there are higher false positive risks；BSP method mainly combines sanger sequencing technologies by PCR to detect methylation
State, but since its is cumbersome, be not suitable for mass detection, while the number of selected clone may will affect detection knot
Fruit, therefore BSP can only be semiquantitative method；MS-HRM is the difference by the way that the difference of single base sequence to be transformed into melting curve
Different, to judge whether there is methylation, requirement of this method to instrument is quite high, needs the quantitative fluorescent PCR with HRM module
Instrument, at the same the method can only analysis detection segment entirety methylation state, and the methylation state in each site CpG cannot be specified；
Fluorescent quantitation is mainly to joined TaqMan probe in the detection, to ensure that higher based on the technology of MSP exploitation
Sensitivity and accuracy, but its detection for multiple methylation sites can only also accomplish that integration is analyzed, while probe cost
It is higher, therefore the method is relatively suitable for a small number of site great amount of samples detections.
Summary of the invention
In view of the drawbacks of the prior art, the invention discloses one kind detects MLH1 gene promoter based on pyrosequencing
Primer, kit and the method for methylation have detection extracting rate height, and detection cycle is short, and flux is high, accuracy height and low dirt
Contaminate the advantages such as risk.Testing result will be helpful to the diagnosis, treatment and prognosis evaluation of related disease.
The detection method that the present invention uses combines pyrosequencing techniques for PCR.Currently, pyrosequencing has been considered
It is the goldstandard of DNA methylation assay.Pyrosequencing techniques are the enzyme cascade chemistry in the same reaction system by 4 kinds of enzymatics
Luminescence-producing reaction, principle are: after primer and template DNA annealing, in archaeal dna polymerase (DNA polymerase), ATP sulphation
Enzyme, 4 kinds of enzymes of luciferase and apyrase synergistic effect under, by the polymerization of each dNTP on primer and one
The release coupling of secondary fluorescence signal is got up, and by detecting the release and intensity of fluorescence, achievees the purpose that the real time measure DNA sequence dna.
According to this principle, when detecting, only the site need to can determine according to the fluorescence signal intensity of C and T in single examined site
Methylation state so that testing result is more intuitive.Therefore this method not only can be qualitative, is also able to achieve quantitatively, or even combines
Related software can accurately analyze the methylation state of examined segment area.Meanwhile pyrosequencing instrument can be in 30min or so
The sequencing of interior achievable 96 samples, therefore have the advantages such as accuracy is high, detection flux is high, detection cycle is short.
Since DNA sequence dna is after sulphite is handled, part C base can be changed into T, and CG in sequence area is caused to contain
Amount and the generation of TM value largely make a variation, and then influence conventional primer design software and obtain ideal primer sequence in its sequence
Column.Therefore, the amplimer and sequencing primer that PCR is used in the present invention are designed, designed, wherein the knot at 3 end of sequencing primer
Coincidence point is located immediately at the previous base for examining first site, to further shorten detection cycle.Meanwhile amplification is drawn
The use ratio of object has higher amplification efficiency by repeatedly optimization experiment.Further, since in sulphite treatment process, sample
This DNA will receive a degree of degradation, so that late detection is influenced, thus the present invention uses two-wheeled PCR, improves detection
Rate.
Based on this pyrosequencing techniques, the present invention provides the primer of detection MLH1 gene promoter methylation, special
Sign is that the primer includes a pair of specificity amplification primer SEQ NO1 and SEQ NO 2, wherein 5 ' the end labels of SEQ NO1
Biotin, SEQ NO2 are used as sequencing primer simultaneously, and base sequence is as follows:
SEQ NO1:5'Bio-AAGGGTGGGGTTGGATGG-3'；
SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'.
Further, the ratio between use concentration of SEQ NO1:SEQ NO 2 is 0.5 μM: 0.5 μM.
The present invention also provides the kit based on pyrosequencing detection MLH1 gene promoter methylation, features
Be, the kit includes nucleic acid extracting reagent, bisulf iotate-treated reagent, PCR amplification system, PCR product it is single-stranded
Purified reagent, pyrosequencing reagent；The PCR amplification system includes drawing for expanding the specificity of MLH1 gene DNA fragment
Object, base sequence are as follows:
SEQ NO1:5'Bio-AAGGGTGGGGTTGGATGG-3'；
SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'；
The pyrosequencing reagent includes the primer that pyrosequencing is carried out to amplified nucleic acid fragment obtained, alkali
Basic sequence are as follows:
SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'.
Further, the sulphite reagent treatment include sulphite mixed liquor (Bisulfite Mix), without enzyme without
Bacterium water (RNase free water), DNA protection buffer (DNA Protect Buffer).The PCR amplification reagent includes
10*PCR buffer, 2mM dNTP, 5*Q dissolve buffer (Solution Buffer), 5U/ul Taq enzyme, 10 μM of amplimers
(SEQ NO1, SEQ NO 2) and aqua sterilisa.The single-stranded purified reagent includes the coated magnetic bead of streptavidin, 70% (V/V)
Ethyl alcohol, 8mg/ml NaOH solution, 1 × Wash Buffer, combination buffer, annealing buffer.The sequencing reagent includes DNA
Polymerase, ATP sulfurylase, luciferase, apyrase, substrate A PS, fluorescein, dNTP and sequencing primer
(SEQ NO2)。
The present invention also provides the method based on pyrosequencing detection MLH1 gene promoter methylation, feature exists
In, comprising the following steps:
(1) DNA in sample is extracted；
(2) template DNA is subjected to sulphite processing, and purification and recovery；
(3) using the purification and recovery product in step (2) as template, specificity amplification primer (SEQ NO1, SEQ NO are used
2) it carries out PCR amplification and obtains pcr amplification product；
SEQ NO1:5'Bio-AAGGGTGGGGTTGGATGG-3'；
SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'；
(4) it after pcr amplification product being carried out single-stranded purifying, places it in pyrosequencing instrument and is sequenced；Sequencing primer
Sequence are as follows: SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'；
(5) according to the pyrosequencing in step (4) as a result, obtaining the MLH1 promoter methylation state of sample.
Further, in step (2), sulphite processing and method for purifying and recycling according toBisulfite
The operation of (QIAGEN, the U.S.) method specification, the specific steps are as follows:
An example sample sulphite processing reaction system be 140 μ L:10 μ L DNA, 10 μ L RNase free water,
85μL Bisulfite Mix、35μL DNA Protect Buffer。
Response procedures are as follows: 95 DEG C of 5min, 60 DEG C of 25min, 95 DEG C of 5min, 60 DEG C of 85min, 95 DEG C of 5min, 60 DEG C of 175min.
The method for purifying and recycling of an example sample is as follows: 310 μ L Buffer being added in Xiang Shangshu sulphite reaction mixture
BL (wherein including 10 μ g/mL carrier RNA), oscillation mixes；250 μ L dehydrated alcohols are added, vibrate 15s, low-speed centrifugal is mixed
It is even；Above-mentioned mixed liquor is moved into EpiTect spin columns, 1min is centrifuged at full speed, abandons waste liquid；500 μ L Buffer are added
BW is centrifuged 1min at full speed, abandons waste liquid；500 μ L Buffer BD are added, is placed at room temperature for 15min, is centrifuged 1min at full speed, abandon waste liquid；
500 μ L Buffer BW are added, are centrifuged 1min at full speed, abandon waste liquid；Centrifugation 1min at full speed abandons waste liquid；Centrifugal column lid is opened, and
It is placed in metal bath, 56 DEG C of 5min；Centrifugal column is placed on a clean 1.5mL centrifuge tube, 20 μ L are added dropwise to film center
Buffer EB, 12000rpm 1min, the liquid collected carry out next step PCR or -20 DEG C of placement preservations.
PCR amplification method in step (3) is as follows:
The PCR system of an example sample is 50 μ L:10*PCR Buffer, 5 μ L, 5 μ L of 2mM dNTP, 5*Q Solution
10 μ L of Buffer, 10 μM of 1 μ L of primer SEQ NO1,10 μM of 1 μ L of SEQ NO2,0.25 μ L of 5U/ μ L Taq enzyme, aqua sterilisa
25.75 μ L, 3 μ L of template.
PCR amplification condition is 95 DEG C of 15min initial denaturations；94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 1min, 40 circulations, 72 DEG C
10min, 4 DEG C of preservations.
The specific steps of step (4) are as follows: delay 50 obtained μ L PCR products in conjunction with 3 μ L streptavidin bead and 47 μ L
Fliud flushing mixing, is incubated at room temperature 10~15min, during which shakes 2~3 times, in case bead sinks.Then Vaccuum prep is used
Vaccuum prep tool in workstation draws bead, and the Vaccuum prep tool with bead is successively put
Into cleaning 15s or so in 70% (V/V) ethyl alcohol, Denaturation Solution, 1 × Wash Buffer, then by Vaccuum
Prep tool is put into 96 orifice plates containing 41 μ L annealing buffers, 2 μ L DMSO and 2 μ L sequencing primers (SEQ NO2), release
96 orifice plate is placed in 80 DEG C of 2min by bead, is cooled to room temperature to get to single-stranded purified product needed for sequencing reaction.
The interpretation of result method of step (5) specifically: using sulphite treated MLH1 promoter sequence as standard sequence
Column, and it is inputted DNA methylation assay software PyroMark CpG, the MLH1 promoter first for obtaining sample is analyzed by the software
Base state.
The utility model has the advantages that currently, for detect MLH1 promoter methylation state most reliable method be exactly pyrosequencing,
And main technological route of the invention is that PCR combines pyrosequencing, by primer amplified destination region, and utilizes coke
Phosphoric acid sequencing analysis sample MLH1 promoter methylation state.This method not only can be achieved at the same time for the qualitative of site methylation
With it is quantitative, while relatively other common detection methods have high accuracy, high detection rate, high throughput, detection cycle it is short and
The advantages such as low pollution risk.Testing result can be conducive to the diagnosis, treatment and prognosis that clinician preferably carries out related disease
Assessment.
Detailed description of the invention
Fig. 1 is MLH1 gene promoter through sulphite treated partial sequence, and wherein box mark is examined by 5
Methylation sites.
Fig. 2 is agarose gel electrophoresis results figure after tumor specimen 1-12PCR amplification.
Fig. 3 is the pyrosequencing result of tumor sample 1.The methylation percentage that the sample examines 5 sites is respectively
5%, 3%, 5%, 6% 4%, (dash area is five detection sites in Fig. 3 Sequencing chromatogram, above dash area in corresponding frame
Numerical value be its methylate percentage)；The MLH1 promoter methylation percentage of the sample is 4.6%.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be noted that unaccounted routine in embodiment
Condition and method usually routinely use method according to fields experimenter: for example, " the essence of Ao Sibai and James Kingston chief editor
Partial numerator biological experiment guide " fourth edition, or according to step proposed by manufacturer and condition.
Embodiment 1
MLH1 promoter methylation detection method, comprising: a pair of of specificity amplification primer SEQ NO1 and SEQ NO 2,
5 ' the end label biotins of middle SEQ NO1, SEQ NO 2 are used as sequencing primer simultaneously, and base sequence is as follows:
SEQ NO1:5'Bio-AAGGGTGGGGTTGGATGG-3'；
SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'；
Further, the ratio between use concentration of SEQ NO1:SEQ NO 2 is 0.5 μM: 0.5 μM.
Specifically, MLH1 gene promoter methylation detection method includes following reagent:
(1) sulphite reagent treatment: Bisulfite Mix, RNase free water, DNA Protect
Buffer；
(2) PCR amplification reagent: 10*PCR Buffer, 2mM dNTP, 5*Q Solution Buffer, 5U/ul Taq
Enzyme, 10uM amplimer (SEQ NO1, SEQ NO 2) and aqua sterilisa；
(3) single-stranded purified reagent: streptavidin beads, 70% (V/V) ethyl alcohol, 8mg/ml NaOH solution, 1 × Wash
Buffer, combination buffer, annealing buffer；
(4) sequencing reagent: archaeal dna polymerase, ATP sulfurylase, luciferase, apyrase, substrate
APS, fluorescein and dNTP and sequencing primer (SEQ NO2).
(5) positive reference substance: containing through sulphite treated MLH1 promoter DNA；Negative controls: without containing warp
Sulphite treated MLH1 promoter DNA.
Embodiment 2:MLH1 promoter methylation detection method testing process
The application method following steps of 1.MLH1 promoter methylation detection method:
(1) DNA in sample is extracted；
(2) template DNA is subjected to sulphite processing, and purification and recovery；
(3) using the purification and recovery product in step (2) as template, according to the specificity amplification primer (SEQ NO1,
SEQ NO 2) carry out two-wheeled PCR amplification；
(4) it after PCR product being carried out single-stranded purifying, places it in pyrosequencing instrument and is sequenced；
(5) the MLH1 promoter methylation state of sample is obtained according to related software.
2. step (1) DNA is extracted: can choose and extract reagent progress DNA extracting known to those skilled in the art, can also make
DNA extracting is carried out with the method that biotech company develops.
3. step (2) sulphite processing and method for purifying and recycling according toBisulfite (QIAGEN, the U.S.)
The operation of method specification, specific as follows:
An example sample sulphite processing reaction system be 140 μ L:10 μ L DNA, 10 μ L RNase free water,
85μL Bisulfite Mix、35μL DNA Protect Buffer。
Response procedures are as follows: 95 DEG C of 5min, 60 DEG C of 25min, 95 DEG C of 5min, 60 DEG C of 85min, 95 DEG C of 5min, 60 DEG C of 175min.
The method for purifying and recycling of an example sample is as follows: 310 μ L Buffer being added in Xiang Shangshu sulphite reaction mixture
BL (wherein including 10 μ g/mL carrier RNA), oscillation mixes；250 μ L dehydrated alcohols are added, vibrate 15s, low-speed centrifugal is mixed
It is even；Above-mentioned mixed liquor is moved into EpiTect spin columns, 1min is centrifuged at full speed, abandons waste liquid；500 μ L Buffer are added
BW is centrifuged 1min at full speed, abandons waste liquid；500 μ L Buffer BD are added, is placed at room temperature for 15min, is centrifuged 1min at full speed, abandon waste liquid；
500 μ L Buffer BW are added, are centrifuged 1min at full speed, abandon waste liquid；Centrifugation 1min at full speed abandons waste liquid；Centrifugal column lid is opened, and
It is placed in metal bath, 56 DEG C of 5min；Centrifugal column is placed on a clean 1.5mL centrifuge tube, 20 μ L are added dropwise to film center
Buffer EB, 12000rpm 1min, the liquid collected carry out next step PCR or -20 DEG C of placement preservations.
4. PCR amplification method described in step (3) is as follows:
The PCR system of an example sample is 50 μ L:10*PCR Buffer, 5 μ L, 5 μ L of 2mM dNTP, 5*Q Solution
10 μ L of Buffer, 10 μM of 1 μ L of primer SEQ NO1,10 μM of 1 μ L of SEQ NO2,0.25 μ L of 5U/ μ L Taq enzyme, aqua sterilisa
25.75 μ L, 3 μ L of template.
PCR amplification condition is 95 DEG C of 15min initial denaturations；94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 1min, 40 circulations, 72 DEG C
10min, 4 DEG C of preservations.
5. specific steps described in step (4) are as follows: by 50 obtained μ L PCR products and 3 μ L streptavidin bead and 47 μ
The mixing of L combination buffer, is incubated at room temperature 10~15min, during which shakes 2~3 times, in case bead sinks.Then Vaccuum is used
Vaccuum prep tool in prep workstation draws bead, by the Vaccuum prep tool with bead according to
It is secondary to put 70% (V/V) ethyl alcohol, Denaturation Solution into, clean 10s or so in 1 × Wash Buffer, then will
Vaccuum prep tool is put into 96 holes containing 41 μ L annealing buffers, 2 μ L DMSO and 2 μ L sequencing primers (SEQ NO 2)
In plate, bead is discharged, which is placed in 80 DEG C of 2min, is cooled to room temperature to get to single-stranded needed for sequencing reaction
Purified product.
6. interpretation of result described in step (5), specifically: using sulphite treated MLH1 promoter sequence as standard
Sequence, and it is inputted DNA methylation assay software PyroMark CpG, the MLH1 promoter for obtaining sample is analyzed by the software
Methylation state.
Embodiment 3: clinical sample detection
12, clinical tissue sample to be checked is taken, is detected using method of the invention.According to the method in embodiment 2
Carry out sample DNA extraction, sulfiting, purification and recovery, PCR amplification, pyrosequencing and interpretation of result, 12 samples
MLH1 gene promoter methylation state outcome is as shown in table 1:
1 tumor sample testing result of table
Note: the numerical value on each sample methylation one column of percentage in table 1 indicates in MLH1 promoter the flat of 5 detection sites
Methylate percent value, which provides after being analyzed by software PyroMark CpG.
Fig. 2 is agarose gel electrophoresis results figure after PCR amplification of the present invention.
Fig. 3 is the pyrosequencing result of tumor sample 1.
By the result of embodiment 3 it is found that using PCR joint pyrosequencing of the invention detection method, can succeed and
The methylation state of accurate detection sample MLH1 gene promoter.Therefore, detection method of the invention can be achieved at the same time for position
Point methylates qualitative and quantitative, while by the primer usage ratio after optimization, ensure that higher PCR amplification success rate.
Further, since pyrosequencing techniques are used in combination, so that this detection method has high accuracy, high throughput, detection
Period is short and the advantages such as low pollution risk, it is final analyze result and can be conducive to clinician preferably carry out examining for related disease
Disconnected, treatment and prognosis prediction.
Sequence table
<110>Beijing Ai Dikang Laboratory of medical test Co., Ltd
<120>primer, kit and method based on pyrosequencing detection MLH1 promoter gene methylation
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<170> SIPOSequenceListing 1.0
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aagggtgggg ttggatgg 18
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gcgatacgat tcaccactat ctc 23
Claims (10)
1. the primer based on pyrosequencing detection MLH1 gene promoter methylation, which is characterized in that the primer includes
A pair of of specificity amplification primer SEQ NO 1 and SEQ NO 2, wherein 5 ' the end label biotins of SEQ NO 1, SEQ NO 2 are same
Shi Zuowei sequencing primer, base sequence are as follows:
SEQ NO 1:5'Bio-AAGGGTGGGGTTGGATGG-3'；
SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'.
2. primer as described in claim 1, which is characterized in that the ratio between use concentration of SEQ NO 1:SEQ NO 2 is 0.5 μM
∶0.5μM。
3. the kit based on pyrosequencing detection MLH1 gene promoter methylation, which is characterized in that the kit
Including nucleic acid extracting reagent, bisulf iotate-treated reagent, PCR amplification system, the single-stranded purified reagent of PCR product, pyrophosphoric acid
Sequencing reagent；The PCR amplification system includes the specific primer for expanding MLH1 gene DNA fragment, base sequence are as follows:
SEQ NO 1:5'Bio-AAGGGTGGGGTTGGATGG-3'；
SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'；
The pyrosequencing reagent includes the primer that pyrosequencing is carried out to amplified nucleic acid fragment obtained, base sequence
It is classified as:
SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'.
4. kit as claimed in claim 3, which is characterized in that the sulphite reagent treatment includes sulphite mixing
Liquid (Bisulfite Mix) protects buffer (DNA Protect without enzyme sterile water (RNase free water), DNA
Buffer)。
5. kit as claimed in claim 3, which is characterized in that the PCR amplification reagent includes 10*PCR buffer, 2mM
DNTP, 5*Q dissolve buffer (Solution Buffer), 5U/ul Taq enzyme, 10 μM of amplimers (SEQ NO 1, SEQ NO
And aqua sterilisa 2).
6. kit as claimed in claim 3, which is characterized in that the single-stranded purified reagent includes the coated magnetic of streptavidin
Pearl, 70% (V/V) ethyl alcohol, 8mg/ml NaOH solution, 1 × Wash Buffer, combination buffer, annealing buffer.
7. kit as claimed in claim 3, which is characterized in that the sequencing reagent includes archaeal dna polymerase, ATP sulphation
Enzyme, luciferase, apyrase, substrate A PS, fluorescein, dNTP and sequencing primer (SEQ NO 2).
8. the method based on pyrosequencing detection MLH1 gene promoter methylation, which comprises the following steps:
(1) DNA in sample is extracted；
(2) template DNA is subjected to sulphite processing, and purification and recovery；
(3) it using the purification and recovery product in step (2) as template, uses specificity amplification primer (SEQ NO 1, SEQ NO 2)
It carries out PCR amplification and obtains pcr amplification product；
SEQ NO 1:5'Bio-AAGGGTGGGGTTGGATGG-3'；
SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'；
(4) it after pcr amplification product being carried out single-stranded purifying, places it in pyrosequencing instrument and is sequenced；Sequencing primer sequence
Are as follows: SEQ NO 2:5'-GCGATACGATTCACCACTATCTC-3'；
(5) according to the pyrosequencing in step (4) as a result, obtaining the MLH1 promoter methylation state of sample.
9. method as claimed in claim 3, which is characterized in that the amplification condition of step (3) are as follows: 95 DEG C of 15min initial denaturations；94
DEG C 30s, 53 DEG C of 30s, 72 DEG C of 1min, 49 circulations, 72 DEG C of 10min, 4 DEG C of preservations.
10. method as claimed in claim 3, which is characterized in that step (5) is with sulphite treated MLH1 promoter sequence
It is classified as standard sequence, and is inputted DNA methylation assay software PyroMark CpG, is analyzed by the software and obtains sample
MLH1 promoter methylation state.
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| US20150218642A1 (en) * | 2011-01-11 | 2015-08-06 | Beijing Institute For Cancer Research | Primer group for detecting cpg island methylation of p16 gene using methylation- specific fluorescence technique |
| CN105567797A (en) * | 2014-11-11 | 2016-05-11 | 香港中文大学深圳研究院 | PCR primer, method and kit for detecting CpG island methylation of DACT2 gene promoter area |
| CN106755316A (en) * | 2016-11-11 | 2017-05-31 | 济南艾迪康医学检验中心有限公司 | The primer and detection method of MLH1 gene promoter methylations detection |
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| US20150218642A1 (en) * | 2011-01-11 | 2015-08-06 | Beijing Institute For Cancer Research | Primer group for detecting cpg island methylation of p16 gene using methylation- specific fluorescence technique |
| CN105567797A (en) * | 2014-11-11 | 2016-05-11 | 香港中文大学深圳研究院 | PCR primer, method and kit for detecting CpG island methylation of DACT2 gene promoter area |
| CN104328201A (en) * | 2014-11-18 | 2015-02-04 | 宁波大学 | Detection kit and detection method for auxiliary diagnosis of Alzheimer's disease |
| CN106755316A (en) * | 2016-11-11 | 2017-05-31 | 济南艾迪康医学检验中心有限公司 | The primer and detection method of MLH1 gene promoter methylations detection |
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Application publication date: 20190920 |