CN105018592B - Mgmt gene promoter methylation detection based on pyrosequencing techniques - Google Patents
Mgmt gene promoter methylation detection based on pyrosequencing techniques Download PDFInfo
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Abstract
The present invention relates to the mgmt gene promoter methylation detection based on pyrosequencing techniques.It specifically, the present invention relates to the sequencing primer based on pyrosequencing techniques detection mgmt gene promoter methylation.The invention further relates to be used for kit and microarray based on pyrosequencing techniques detection mgmt gene promoter methylation comprising sequencing primer.The invention further relates to sequencing primer to prepare the purposes in being used to detect the kit and microarray of mgmt gene promoter methylation based on pyrosequencing techniques.The invention further relates to the method using mgmt gene promoter methylation in sequencing primer detection sample.
Description
Technical field
The present invention relates to the mgmt gene promoter methylation detection based on pyrosequencing techniques.Specifically, this hair
It is bright to be related to for the sequencing primer based on pyrosequencing techniques detection mgmt gene promoter methylation.The invention further relates to bag
It is used for kit and microarray based on pyrosequencing techniques detection mgmt gene promoter methylation containing sequencing primer.This
Invention further relates to sequencing primer and is preparing for the reagent based on pyrosequencing techniques detection mgmt gene promoter methylation
Purposes in box and microarray.The invention further relates to the side using mgmt gene promoter methylation in sequencing primer detection sample
Method.
Background technology
Pyrosequencing techniques are that a kind of cascade reaction based on enzyme-to-substrate is detected and quantitative sequence analysis skill
Art.In whole reaction system, using single-stranded PCR product as template, sequencing primer and its annealed combination, four kinds of dNTP according to
Set order is added in reaction system.If the dNTP added is combined with the base complementrity on template strand, in archaeal dna polymerase
The lower release of effect and the pyrophosphoric acid group PPi of incorporation equimolar number.The PPi and adenosine 5'-phosphate of sulfurylase catalysis release
Sulfuric anhydride forms the adenosine triphyosphate (Adenosine Triphosphate, ATP) of equivalent, ATP driving luciferases
The fluorescein of mediation is converted to oxidation state, concurrent third contact of a total solar or lunar eclipse signal.The optical signal that instrument detects is then in a manner of fluorescence signal peak
It is reflected in the Sequencing chromatogram occurred in real time.DNTP such as addition then cannot directly be dropped with the complementary combination of template by bisphosphatase
Solution, reaction go successively to next round.By the circulation of the above process, single-stranded PCR product obtains complementation and extends, and passes through each alkali
The presence or absence of base location signal peak judges base type, judges the number of base by the height and area of signal peak.Pyrophosphoric acid is surveyed
Sequence method is suitable for carrying out quantitative analysis to 10-100bp length sequences, its repeatability and accurate performance match in excellence or beauty with Sanger sequencings, and
Detection speed then greatly improves.It is right to strengthen its while pyrosequencing techniques advantage is kept for multiple pyrosequencing techniques
In the detectability at a distance of more than 100bp sites.Two are carried out at the same time even more than sequencing reaction in same Kong Weizhong, pass through section
The order that four kinds of dNTP of layout are added obtains special detection sequence, while so as to fulfill multiple sites apart from each other
Detection.
O6-MG-DNA- transmethylases（O6-methylguanine-DNA-methyltransferase,
MGMT）It is a kind of unique DNA repair protein of generally existing, can be incited somebody to action in the presence of no any confactor or albumen
Methyl is reduced guanine from the O6 bit transitions of 6 methyl guanines to the cysteine residues of itself, and Temozolomide
Methyl can be transferred to the methylation sites on DNA, 6 methyl by (temozolomide, TMZ) after hydrolysis in vivo
Guanine is exactly the critical sites of Temozolomide cytotoxicity.MGMT is repaired DNA alkylation damages, is cell to controlling
The main reason for treating the common drug nitrosourea and Temozolomide generation drug resistance of malignant glioma.Studies have found that pass through
MGMT inactivations are caused to be a key factor of the tumour to TMZ chemosensitivities methylating for mgmt gene promoter.Research hair
Existing MGMT feminine gender glioma persons are bright to the chemotherapy effective percentage of chloroethyl nitrosourea class (chloroethylnitrosoure, CENU)
It is aobvious to be higher than MGMT positives.Therefore the repair that MGMT damages alkylating agent causes the high low energy of MGMT expressions in tissue
Enough reflect sensitivity differences of the Different Individual to alkylating agent medicine.Mgmt gene promoter zone methylation degree can be with indication body
Interior MGMT expressions, so the life cycle for receiving glioblastoma patient alkylating agent chemotherapy be predicted.Therefore in formulationization
The expression of mgmt protein in glioma cells in tissue is detected before treatment scheme, nitrosourea is not selected to the tumor patient of MGMT high expression
Antitumor drug and TMZ, can be expected to improve chemotherapeutic efficacy to avoid drug resistance.U.S. FDA is also recommended to the trouble using Temozolomide
Person carries out MGMT promoter methylation analyses, to instruct patient medication.
It there is now the detection that different kinds of molecules Biological Detection technology is applied to mgmt gene promoter methylation site, strictly according to the facts
When quantitative PCR, LDR, RFLP, HRM, genetic chip, direct Sequencing etc..Wherein non-sequencing mode needs to rely on digestion or cluster
The genotype of mode interpretation patient, there are the probability of larger parting mistake, this is undoubtedly clinical diagnosis and avoids greatly.Though direct Sequencing
Right accuracy is high, but the DNA fragmentation that sequencing reaction produces needs, by capillary electrophoresis separation, to be detected by detecting system thereafter glimmering
Optical signal, it is higher to take longer and testing cost.
Pyrosequencing method can in certain length sequence carry out real-time quantitative analysis, its repeatability and accurate performance and
Sanger sequencings match in excellence or beauty, and detection speed greatly improves, and testing cost substantially reduces.In addition, the detection of pyrosequencing techniques
Sensitivity is also sequenced far above Sanger.The technology is widely used to microbial identification and point-score medicine mirror, science of heredity at present
Analysis, SNP detections etc..Therefore, the application and popularization that pyrosequencing techniques are detected in mgmt gene promoter methylation
To have great value and potentiality.
However, still realize pyrosequencing techniques in the side that mgmt gene promoter methylation detects without commercialization at present
Case.
The content of the invention
One aspect of the present invention is related to for the sequencing based on pyrosequencing techniques detection mgmt gene promoter methylation
Primer.In one embodiment, the sequencing primer includes such as SEQ ID NO:Nucleotide sequence shown in 3.At another
In embodiment, the sequencing primer has such as SEQ ID NO:Nucleotide sequence shown in 3, or by or substantially by such as SEQ
ID NO:Nucleotide sequence composition shown in 3.In one embodiment, the target sequence that the sequencing primer is directed to is behaved
131,265,519 to 131,265,537 sequence (the SEQ ID NO of No. 10 chromosome of genome: 6).
Another aspect of the present invention is related to a kind of be used for based on pyrosequencing techniques detection mgmt gene promoter methylation
Kit or microarray.In one embodiment, the kit or microarray include the sequencing for pyrosequencing
Primer, the sequencing primer include such as SEQ ID NO:Nucleotide sequence shown in 3.In another embodiment, the survey
Sequence primer has such as SEQ ID NO:Nucleotide sequence shown in 3, or by or substantially by such as SEQ ID NO:Core shown in 3
Nucleotide sequence forms.In one embodiment, the target sequence that the sequencing primer is directed to is No. 10 dyeing of human genome
Sequence (the SEQ ID NO of the 131,265,519 to 131,265,537 of body: 6).
In one embodiment, the kit or microarray also include following primer sets, and the primer sets include tool
Have by SEQ ID NO:The primer of nucleotide sequence shown in 1 and with by SEQ ID NO:Nucleotide sequence shown in 2
Primer, or by or substantially by with by SEQ ID NO:The primer of nucleotide sequence shown in 1 and with by SEQ ID NO:
The primer composition of nucleotide sequence shown in 2.
In one embodiment, the kit or microarray, which also include, shows analytical sequence by SEQ ID NO:4 institutes
Show and/or allocation order is by SEQ ID NO:Specification shown in 5.
Another aspect of the present invention is related to sequencing primer and prepared for being detected based on pyrosequencing techniques in sample
Purposes in the kit or microarray of mgmt gene promoter methylation.In one embodiment, the kit or micro-
Array includes the sequencing primer for pyrosequencing, and the sequencing primer includes such as SEQ ID NO:Nucleotides sequence shown in 3
Row.In another embodiment, the sequencing primer has such as SEQ ID NO:Nucleotide sequence shown in 3, or by or
Substantially by such as SEQ ID NO:Nucleotide sequence composition shown in 3.In one embodiment, the sequencing primer is directed to
Target sequence is 131,265,519 to 131,265,537 sequence (the SEQ ID NO of No. 10 chromosome of human genome: 6).
In one embodiment, the kit or microarray also include following primer sets, and the primer sets include tool
Have by SEQ ID NO:The primer of nucleotide sequence shown in 1 and with by SEQ ID NO:Nucleotide sequence shown in 2
Primer, or by or substantially by with by SEQ ID NO:The primer of nucleotide sequence shown in 1 and with by SEQ ID NO:
The primer composition of nucleotide sequence shown in 2.
In one embodiment, the kit or microarray are also comprising the analytical sequence shown used in pyrosequencing
By SEQ ID NO:Shown in 4 and/or allocation order is by SEQ ID NO:Specification shown in 5.
In one embodiment, the sample is biological sample, and preferably described sample is humoral sample or tissue sample,
More preferably described sample is selected from biopsy samples, cell culture, whole blood, blood plasma, serum, saliva, Cerebrospinal fluid, sweat
Liquid, urine, excrement, peritoneal fluid, juice and tear.
The invention further relates to the mgmt gene promoter methylation in a kind of detection sample based on pyrosequencing techniques
Method, the described method includes：
(1) DNA in extraction and optional purification of samples,
(2) DNA for extracting and optionally purifying is expanded, so that amplified production is obtained,
(3) pyrosequencing is carried out to the amplified production of step (2).
In one embodiment, the pyrosequencing is carried out using following sequencing primer, and the sequencing primer includes
Such as SEQ ID NO:Nucleotide sequence shown in 3.In another embodiment, the sequencing primer has such as SEQ ID
NO:Nucleotide sequence shown in 3, or by or substantially by such as SEQ ID NO:Nucleotide sequence composition shown in 3.At one
In embodiment, target sequence that the sequencing primer is directed to for No. 10 chromosome of human genome 131,265,519 to
131,265,537 sequence (SEQ ID NO: 6).
In one embodiment, the amplification is PCR amplification, and preferably described PCR amplification uses following primer sets, described
Primer sets, which include, to be had by SEQ ID NO:The primer of nucleotide sequence shown in 1 and with by SEQ ID NO:Shown in 2
The primer of nucleotide sequence, or by or substantially by with by SEQ ID NO:The primer of nucleotide sequence shown in 1 and have
By SEQ ID NO:The primer composition of nucleotide sequence shown in 2.
In one embodiment, the pyrosequencing is also used by SEQ ID NO:Analytical sequence shown in 4 and/
Or by SEQ ID NO:Allocation order shown in 5.
In one embodiment, the sample is biological sample, and preferably described sample is humoral sample or tissue sample,
More preferably described sample is selected from biopsy samples, cell culture, whole blood, blood plasma, serum, saliva, Cerebrospinal fluid, sweat
Liquid, urine, excrement, peritoneal fluid, juice and tear.
Brief description of the drawings
Fig. 1 is a techniqueflow of pyrosequencing and each essential element schematic diagram.
Fig. 2 detects mgmt gene promoter zone methylation schematic diagram for pyrosequencing.
Fig. 3 is mgmt gene promoter zone methylation testing result sectional drawing, and wherein abscissa represents assigned sequence, ordinate
Represent the fluorescence signal intensity that instrument is collected.
Fig. 4 is the result sectional drawing that mgmt gene promoter zone methylation is detected using different sequencing primers, wherein abscissa
Assigned sequence is represented, ordinate represents the fluorescence signal intensity that instrument is collected.
Fig. 5 is the detection using the ratio that methylates detected by the sequencing system including the sequencing primer of the present invention
Curve map of the value compared with theoretical value.
Embodiment
Several aspects of the present invention are described below with reference to the example application for explanation.It should be appreciated that statement
Many details, relation and method fully understand the present invention to provide.However, in those of ordinary skill in the related art
Will readily appreciate that, can implement in the case of without one or more details the present invention or can with other methods come
Implement the present invention.
The shortcomings that present invention aims at detection to mgmt gene promoter methylation site in the prior art is solved, example
Probability, time-consuming longer or testing cost such as larger parting mistake is higher.By the present invention in that the sequencing primer with the present invention
Pyrosequencing techniques and solve the above problem.
Had the following advantages using the pyrosequencing techniques of the sequencing primer of the present invention：
1）Instrument and reagent can reach optimal detection result, and easily standardization by system optimization；
2）Testing result is provided by software automatically, avoids result from judging subjectivity；
3）Operating process is easy and time saving；
4）The linearity in the range of 0%-100% methylates is high, can be used to quantitatively detect.
Unless otherwise stated, all scientific and technical terminologies used herein have one skilled in the art of the present invention
Normally understood implication.The definition of general term in Cytobiology and molecular biology may refer to：The gene that Yu Long etc. is translated
VIII, International Standard Book Number：ISBN:978-7-03-014597-0, Science Press publish（2005）；Cell that Zhang Jinfeng etc. is translated with
Molecular biology, Zhong Xin publishing houses publish（2004）, ISBN:978-7-50-860075-8；With the bioid of the volume such as Wang Jingyan
Learn, Higher Education Publishing House publishes（2002）, ISBN:978-7-04-011088-3；Kendrew, J. et al. (eds.),
The Encyclopedia of Molecular Biology, Blackwell Science Ltd. publish (1994),
ISBN 0-632-02182-9;And Meyers, R.A. (eds.), Molecular Biology and Biotechnology:
A Comprehensive Desk Reference, VCH Publishers, Inc. publish (1995), ISBN 1-
56081-5698.Although can be used when implementing the present invention similar or equivalent to methods described herein and any method of material and
Material, but this document describes specific material and method.
Sample
Terms used herein " sample " includes any sample containing nucleic acid molecules.It is (" raw that sample can derive from biological source
Thing sample "), such as tissue (such as biopsy samples), extract or including cell (such as tumour cell), cell cracking
Cell culture including thing and biology or physiological fluid, for example, whole blood, blood plasma, serum, saliva, Cerebrospinal fluid, sweat, urine,
Excrement, juice, milk, peritoneal fluid etc..Obtained from the sample in source or in pretreatment to improve sample characteristic (such as from blood system
Standby blood plasma, dilution mucus etc.) after sample can be used directly.In certain aspects of the invention, sample is human physiology fluid, such as
Human serum.In certain aspects of the invention, sample be biopsy samples for example through tissue examination obtain tumor tissues or
Cell.In certain aspects of the invention, sample is pernicious or normal tissue sample.
Can include the polynucleotides of clinical source according to the present invention sample being analyzed and/or used, for example, DNA or
RNA。
The method that nucleic acid is extracted from sample is well-known in the art, can carry out DNA with such as phenol and chloroform and carry
Take, or extracted using commercially available DNA extracts reagents.For example, column kit (such as GENERATION (registrars can be used
Mark) Capture Column Kit Gentra) extracted.
It should be understood that nucleic acid can be purified by many conventional purification process in this area, such as use PrepSEQ
Method in kit (coming from Applied Biosystems) and U.S. Patent number 5,234,809 etc..The present invention preferably adopts
Nucleic acid is extracted with commercial reagent box such as EpiTect Plus LyseAll Bisulfite Kit (QIAGEN companies)
And purifying.Extracting with after purification of nucleic acid, bisulf iotate-treated can carried out to nucleic acid such as DNA and be reacted for PCR.
Primer
" primer " used herein is often referred to the linear oligonucleotide with target sequence complementation and annealing.The lower limit of primer length
Depending on hybridization ability, because very short primer (being, for example, less than 5 nucleotide) does not form heat under most of hybridization conditions
The duplex that mechanics is stablized.Primer length usually changes in 8-50 nucleotide.In certain embodiments, primer is between big
Between about 15-25 nucleotide.Terms used herein " forward primer " refers to the few core with a specific chain annealing of target DNA
Thuja acid.Terms used herein " reverse primer " refers to the oligonucleotides with the annealing of the opposite strand of target DNA.In short, forward primer
Usually it is oriented in reverse primer in a manner of similar to PCR primer on target DNA sequence so that its 3' end is than its 5' end more
Close to target sequence.(especially guanine, adenine, cytimidine and thymidine, are hereinafter referred to as naturally occurring nucleotide
" G ", " A ", " C " and " T ") and nucleotide analog, it can be used in the primer of the present invention.Terms used herein " draw by sequencing
Thing " refers to the Oligonucleolide primers for originating the sequencing reaction carried out to nucleic acid.
" amplified production " used herein refers to from nucleic acid-templated, by nucleic acid amplification and the nucleic acid of amplification that produces.
" template DNA " or " template ribonucleic acid " used herein refer to the nucleic acid as the required target for amplification.For example,
Template ribonucleic acid is reverse transcribed into cDNA, and template cDNA is used to produce amplified production.
Terms used herein " nucleotide analog " refers to the compound being structurally similar with naturally occurring nucleotide.
Nucleotide analog can have the phosphate backbones changed, sugar moieties, nucleobase or its combination.Usually there is the nucleobase changed
Nucleotide analog especially assign different base pairings and base stacking characteristic.The core of phosphoric acid with change-sugar skeleton
Thuja acid analog (such as peptide nucleic acid (PNA), lock nucleic acid (LNA)) usually especially changes chain characteristic, such as secondary structure is formed.
For PCR primer, sequencing primer and target based on pyrosequencing techniques detection mgmt gene promoter methylation
The example of series see the table below 1.
PCR primer, sequencing primer and corresponding target sequence used in 1. present invention of table
The PCR primer of the present invention and the nucleotide sequence of sequencing primer further include its modified forms, as long as the primer
Amplification or sequencing effect be not significantly affected.The modification can be for example in nucleotide sequence or both ends addition
One or more nucleotide residues, lack in nucleotide sequence one or more nucleotide residues or by one in sequence
A or multiple nucleotide residues are substituted for other nucleotide residue, such as A is substituted for T, and C is substituted for G etc..This area skill
Art personnel understand that the primer of the modified forms is also covered by within the present invention, is particularly within scope of the claims.
In one embodiment, the modified forms of the nucleotide sequence of PCR primer and sequencing primer are institute in such as CN103270174A
Disclosed chemical reinforcing type primer.
For example general DNA synthesizer (such as 394 types manufactured by Applied Biosystems) can be used, through chemistry
Method synthesizes each nucleotide in primer of the present invention.Also widow can be synthesized using any other method well-known in the art
Nucleotide, such as PCR primer and sequencing primer.
Using the genomic DNA extracted from sample as template, and mgmt gene expand instead using PCR primer
Should, to obtain amplified production.Amplified reaction includes but not limited to PCR (PCR), ligase chain reaction
(LCP), sequence replicating (3SR), the amplification (NASBA) based on nucleotide sequence, strand displacement amplification (SDA), more replacements are maintained automatically
Amplification (MDA) and rolling circle amplification (RCA) are changed, it is disclosed in below with reference in document (being hereby incorporated by reference)：Mullis etc., the U.S.
Patent the 4,683,195th；No. 4,965,188；No. 4,683,202；4,800,159th (PCR) number；Gelfand etc.,
U.S. Patent No. 5,210,015 (with " Taqman " or " the real-time PCR that carries out of Taq " [registration mark] probe)；Wittwer
Deng, U.S. Patent No. 6,174, No. 670；Kacian etc., U.S. Patent No. 5,399, No. 491 (" NASBA ")；Lizardi, the U.S.
Patent the 5,854,033rd；Aono etc., Japanese Patent Publication JP No. 4-262799 (rolling circle amplification)；Etc..
Target nucleotide is expanded preferably using PCR methods.PCR methods are well-known in the art in itself.Term " PCR "
Derivative form including the reaction, it includes but not limited to reverse transcription PCR, real-time PCR, nested PCR, multiplex PCR and fluorescence
Quantitative PCR etc..Quantitative amplification is carried out to target nucleotide preferably using fluorescence quantitative PCR method.
In the presence of primer, template DNA and hot resistant DNA polymerase, the primer (reverse primer) hybridized with sense strand is used
With the primer (forward primer) hybridized with antisense strand, by make the circulating repetition about 30 times of annealing, extension and denaturing step~
50 times (such as 45 times) carry out PCR.In one embodiment, PCR is real-time fluorescence quantitative PCR.In an embodiment
Middle PCR has used following primer sets, and the primer sets, which include, to be had by SEQ ID NO:The primer of nucleotide sequence shown in 1
With with by SEQ ID NO:The primer of nucleotide sequence shown in 2, or by or be made of substantially the primer.This area
Technical staff, it is understood that other PCR methods and primer sets can also be used, as long as it is amplifiable go out target fragment.Ability
Field technique personnel can routinely select PCR methods as needed, and conventional design goes out required PCR primer.
It in the PCR of the present invention, various conventional hot resistant DNA polymerases can be used to be expanded, include but not limited to
FastStart Taq archaeal dna polymerases (Roche), Ex Taq (registration mark, Takara), Z-Taq, AccuPrime Taq
Archaeal dna polymerase and HotStarTaq Plus archaeal dna polymerases.
The method that suitable PCR reaction conditions are selected based on primer Tm is well-known in the art, the common skill in this area
Art personnel can select most according to primer length, G/C content, desired specificities and sensitivity, used polymerization enzymatic property etc.
Good condition.For example, the following conditions can be used to carry out quantitative fluorescent PCR reaction：95 DEG C 5 minutes, 95 DEG C 20 seconds, 60 DEG C 30
Second, 72 DEG C 20 seconds, circulate 45 times.Reaction system can be 50 μ L.
After PCR product is obtained, PCR product can be handled, to obtain and the complementary single-stranded PCR combined of sequencing primer
Product.The generation and purifying of single stranded PCR products can be carried out by method well known in the art.Common generation and purification of single stranded
The method of PCR product includes but not limited to T7 reverse transcription methods（Hughes et al., Nat. Biotechnol., 2001,19:
342-347）, Exonucleolytic enzyme process（Higuchi and Ochman, Nucleic. Acids Res., 1989,17:5865）, become
Property high performance liquid chromatography（Denaturing high-performance liquid chromatography, DHPLC）
（Dickman and Hornby, Anal. Biochem., 2000,284:164-167）With magnetic capture method（Espelund etc.
People, Nucleic. Acids Res., 1990,18:6157-6158）Deng.In one embodiment, the present invention passes through magnetic
Pearl prize law obtains and the complementary single stranded PCR products combined of sequencing primer.In another embodiment, the present invention passes through
PyroMark Q24 vacuum works stations are handled PCR product according to the specification of manufacturer, with acquisition and sequencing primer
The single stranded PCR products that complementation combines.
In addition, it is possible to use asymmetric PCR method directly prepares single stranded PCR products, and volume is carried out after PCR so as to eliminate
Outer processing.It is single-stranded that asymmetric PCR can prepare DNA while PCR amplification.Conventional asymmetric PCR uses two inequalities
Primer, is normally expanded in the circulation of beginning.With the increase of circulation, measure few primer and gradually exhausted, and excess
It is single-stranded that primer can continue straight line amplification generation DNA（Gyllensten and Erlich, Proc. Natl. Acad. Sci.
U.S.A., 1988, 85:7652-7656）.
, can sequencing primer progress pyrosequencing using the present invention after single stranded PCR products are obtained.In an embodiment party
In case, the sequencing primer can include such as SEQ ID NO:Nucleotide sequence shown in 3.In another embodiment, it is described
Sequencing primer can have such as SEQ ID NO:Nucleotide sequence shown in 3, or by or substantially by such as SEQ ID NO:Shown in 3
Nucleotide sequence composition.In one embodiment, the target sequence that the sequencing primer is directed to is human genome the 10th
Sequence (the SEQ ID NO of the 131,265,519 to 131,265,537 of chromosome: 6).In one embodiment, for this
The sequencing primer of invention can be such as SEQ ID NO:7th, shown in 10 or 13.
In one embodiment, using the PyroMark Gold Q24 Reagents kits and reality of QIAGEN companies
Shi Dingliang pyrophosphoric acid sequenators（Model：PyroMark® Q24 MDx）, to specifications method carry out pyrosequencing.
Following hybridization conditions can be used to carry out pyrosequencing：80 DEG C of heating 2min, annealing at room temperature 20 minutes.In an embodiment
In, pyrosequencing is also used by SEQ ID NO:Analytical sequence shown in 4 and/or by SEQ ID NO:Distribution shown in 5
Sequentially.After the completion of analytical sequence sequencing experiment use during interpretation of result, the software of analyzer by this analytical sequence with
Sequencing result is compared.For example analytical sequence is YGAYGTTYGTAGGTTTTYG, software will count the site for being labeled with Y
Calculate the ratio of C/T；In practical applications, ratio value can change with sample, this ratio value then indicates the degree to methylate.
Allocation order is the order for the nucleotides substrate that instrument sprays in sequencing procedure is carried out；During sequencing, instrument is according to allocation order
Nucleotides substrate is added in reaction tank in order, if detecting fluorescence signal when spraying into A, just represents the sequencing in this site
As a result it is A, and so on.Those skilled in the art routinely can select and design as needed analytical sequence and/or distribution is suitable
Sequence, and different analytical sequence and/or allocation order are used according to the situation of specific target sequence in practical applications.One
In a embodiment, analytical sequence and/or allocation order as shown in table 3 can be used in pyrosequencing.
Kit
It is used for the kit based on pyrosequencing techniques detection mgmt gene promoter methylation the present invention relates to a kind of,
It contains the combination of the sequencing primer of the present invention or sequencing primer and primer sets.The invention further relates to sequencing primer of the present invention or
The combination of person's sequencing primer and primer sets is being prepared for based on pyrosequencing techniques detection mgmt gene promoter methylation
Kit in purposes.In one embodiment, the sequencing primer can include such as SEQ ID NO:Nucleosides shown in 3
Acid sequence.In another embodiment, the sequencing primer can have such as SEQ ID NO:Nucleotide sequence shown in 3, or
Person by or substantially by such as SEQ ID NO:Nucleotide sequence composition shown in 3.In one embodiment, the primer sets bag
Containing with by SEQ ID NO:The primer of nucleotide sequence shown in 1 and with by SEQ ID NO:Nucleotides sequence shown in 2
The primer of row, or by or be made of substantially the primer.
Kit can include the material or reagent (including sequencing primer and primer sets) implemented used in the method for the present invention.Reagent
It is (such as slow that box can include storage reaction reagent (such as primer in suitable vessel, dNTP, enzyme etc.) and/or support material
The specification etc. of fliud flushing, examinations).For example, kit, which can include one or more, contains respective reaction reagent and/or branch
Hold the container (such as box) of material.Such content can together or separately be delivered to set recipient.For example, first
Container can contain the enzyme for being useful for measure, and second container contains primer sets and the 3rd container contains sequencing primer.The reagent
Box is also containing the compartment for being adapted to accommodate the reagent or container.As an example, kit can contain sequencing primer, primer
Group, PCR reaction buffers, operation instructions.Kit can also contain polymerase and dTNP etc..Kit can also contain UNG, use
In internal standard, the positive and negative control of Quality Control etc..Kit can also include the reagent being used for from sample preparation nucleic acid such as DNA.
Kit of the present invention can also include except the present invention sequencing primer and/or primer sets in addition to other any sequencing primers and/
Or primer sets, such as can effectively detect the sequencing primer and/or primer sets of mgmt gene promoter methylation.Above example is not
It is understood that and is suitable for the invention kit and its content for limitation.
In one embodiment, the specification in kit indicates analytical sequence used in pyrosequencing by SEQ
ID NO:Shown in 4 and/or allocation order is by SEQ ID NO:Shown in 5.
Microarray
It is used for the microarray based on pyrosequencing techniques detection mgmt gene promoter methylation the present invention relates to a kind of,
It contains the combination of the sequencing primer of the present invention or sequencing primer and primer sets.The invention further relates to sequencing primer of the present invention or
The combination of person's sequencing primer and primer sets is being prepared for based on pyrosequencing techniques detection mgmt gene promoter methylation
Microarray in purposes.In one embodiment, the sequencing primer can include such as SEQ ID NO:Nucleosides shown in 3
Acid sequence.In another embodiment, the sequencing primer can have such as SEQ ID NO:Nucleotide sequence shown in 3, or
Person by or substantially by such as SEQ ID NO:Nucleotide sequence composition shown in 3.In one embodiment, the primer sets bag
Containing with by SEQ ID NO:The primer of nucleotide sequence shown in 1 and with by SEQ ID NO:Nucleotides sequence shown in 2
The primer of row, or by or be made of substantially the primer.
Microarray refers to the solid support with flat surfaces, it is with nucleic acid array, each member bag in array
Containing the oligonucleotides or the identical copy of polynucleotides on fixed spatially definite region or site, the region or position
Point is not overlapping with the region of other members in array or site；That is, the region or site are spatially discrete
's.In addition, spatially definite hybridization site can be " addressable ", because the body of the oligonucleotides of its position and its immobilization
Part is known or predetermined (such as being known or predetermined before its use).Usual oligonucleotides or polynucleotides
To be single-stranded, and usually it is covalently attached by 5'- ends or 3'- ends with solid support.Nucleic acid containing non-overlapped area in microarray
Density is typically larger than 100/cm2, more preferably greater than 1000/cm2.Microarray technology is disclosed in for example below with reference in document：
The Microarrays that Schena is edited: A Practical Approach (IRL Press, Oxford, 2000)；
Southern, Current Opin. Chem. Biol., 2:404-410,1998, entire contents are incorporated by reference into
Herein.
Although various embodiments of the present invention are described above, it should be understood that it is only by way of example
There is provided, and not limit.Many changes to disclosed embodiment can be carried out according to this disclosure, without carrying on the back
From the spirit or scope of the present invention.Therefore, range of the invention and scope should not be restricted by any of above embodiment and limited.
All documents being mentioned above are all incorporated herein by reference.The all publications and patents that the application quotes
File is all incorporated by reference for all purposes, and reference degree is as individually pointed out each publication or patent document one
Sample.
Embodiment
Unless otherwise stated, the otherwise acquisition purchased in market of the material used in embodiment hereof, for the various tools tested
Body experimental method is the experimental method of this area routine (see, for example, chief editors such as F. Ao Sibai《Fine works molecular biology is real
Test guide》(1999), the chief editor's such as Science Press, ISBN 7-03-006408-9 and J. Pehanorm Brookers《Molecular cloning is real
Test guide (third edition)》(2002), Science Press, ISBN 7-03-010338-6) or according to the step proposed by manufacturer
Rapid and condition, and can routinely be determined as needed by those skilled in the art.Some materials and method have been carried out in detail below
State.
Embodiment 1：The pyrosequencing carried out using sequencing primer of the present invention
Material：
Method：
As shown in Figure 1, first using PCR Master Mix, HS Taq and two PCR primer to bisulfite conversion
And the human genome DNA purified is expanded on Rotor-Gene Q platforms, contaminated in PCR Master Mix comprising fluorescence
Material, can be embedded in amplification procedure in ever-increasing PCR double-stranded products.Therefore the process of whole product enrichment can pass through
The software of Rotor-Gene Q is monitored in real time, so that it is guaranteed that the PCR product of reliable in quality is for follow-up pyrosequencing point
Analysis.PCR product is handled by PyroMark Q24 vacuum works station, it is final to obtain and the complementary list combined of sequencing primer
Chain PCR product.Specific pyrosequencing program is run on PyroMark Q24 pyrosequencing instrument afterwards, instrument can basis
Allocation order shown in table 2 below sequentially adds four kinds of dNTP, and by the cascade reaction of enzyme-to-substrate, dNTP is in single stranded product
Effectively extension is received in the form of optical signal by instrument, and is finally appeared in real time in software interface in the form of signal peak.Fortune
Result after row is automatically analyzed by software, reduces the burden and error of manual analysis.
The analytical sequence and allocation order of 2. PyroMark Q24 software design patterns of table
Experimentation is as follows：
One, takes a clinical sample for coming from clinical hospitals, with the EpiTect Plus LyseAll of QIAGEN companies
It is pure that Bisulfite Kit according to the step proposed by manufacturer and condition carry out Tissue Lysis, sulfiting conversion, nucleic acid
Change；
Two, take nucleic acid after purification to carry out PCR amplification with PCR primer 1,2（Using the real time fluorescent quantitative of QIAGEN companies
PCR analyzers（Model：Rotor-Gene Q））；
PCR conditions are as follows：95℃：5 minutes；95℃：20 seconds, 60 DEG C：30 seconds, 72 DEG C：20 seconds, totally 45 circulations.
Three, pyrosequencings
Fig. 2 detects mgmt gene promoter zone methylation schematic diagram for pyrosequencing.
Using the PyroMark Gold Q24 Reagents kits and real-time quantitative pyrophosphoric acid sequence of QIAGEN companies
Analyzer（Model：PyroMark® Q24 MDx）, operated according to manufacturer specification method and condition, sequencing primer is
SEQ ID NO:Sequencing primer shown in 3, the hybridization conditions of sequencing primer are：80 DEG C of heating 2min, annealing at room temperature 20 minutes.
Sequencing result is shown in Fig. 3.As can be seen from Figure 3, the result of pyrosequencing and expected sequence be (i.e.
CGACGCCCGCAGGTCCTCG, 131,265,519 to 131,265,537 sequence of No. 10 chromosome of human genome) one
Cause, it is also consistent with detection " goldstandard " Sanger sequencing results, and it is low that background is sequenced.
Embodiment 2：The comparison of different sequencing primers
Material, instrument and method used and condition are with embodiment 1, but during pyrosequencing in the present embodiment
The sequencing primer shown in table 3 below and the combination of analytical sequence and allocation order are used.
Table 3：Different sequencing primers and the combination of analytical sequence and allocation order
Sequencing primer 1：TTTAGAAAGTTTTGAGTTT (SEQ ID NO: 3)
Sequencing primer 2：TTTAGAAAGTTTTGAGTT (SEQ ID NO: 7)
Sequencing primer 3：GTTTTTAGAAAGTTTTG (SEQ ID NO: 10)
Sequencing primer 4：GATAGTTAGAGTTTTTAGA (SEQ ID NO: 13)
PCR conditions are：95℃：5 minutes；95℃：20 seconds, 60 DEG C：30 seconds, 72 DEG C：20 seconds, totally 45 circulations.
The hybridization conditions of sequencing primer are：80 DEG C of heating 2min, annealing at room temperature 20 minutes.
Experimental result is shown in Fig. 4.The result of the results show sequencing primer 1 is best, and region to be analyzed is shown as blueness（Represent knot
Fruit passes through）, with expected sequence (i.e. CGACGCCCGCAGGTCCTCG, the 131,265,519- of No. 10 chromosome of human genome
131,265,537 sequence) unanimously, and it is low that background is sequenced；The region to be analyzed of sequencing primer 2,3,4 is shown as yellow or red
（Represent that result does not know or fails）, it is less consistent with expected sequence, and it is high that background is sequenced.Therefore selection 1 conduct of sequencing primer
The sequencing primer of the present invention.
Embodiment 3：Accuracy, precision and the linear properties assessment of sequencing primer of the present invention
Material：
Method：
The ratio of methylating known to selection is respectively 0%, 25%, 50%, 75%, 100% each portion of standard clinical sample, is used
Include sequencing primer (the SEQ ID NO of the present invention:3) the sequencing system including is detected, and each sample repeats detection 8 times,
The detected value of the ratio that methylates of 4 detection sites is counted, and compared with theoretical value.
Experiment material, instrument and experimentation used in detection and condition are the same as embodiment 1；Sequenator is automatically derived
The percentage of C bases on each methylation sites, is the detected value of the ratio that methylates in the site.
The testing result for the ratio that methylates is as shown in table 4 below, the results show：The detected value for the ratio that methylates connects with theoretical value
Closely, detection accuracy is higher；The standard deviation in each site is less than 2.2, and average value 0.81, shows good precision；4
Good linear relationship is presented with theoretical value in the detected value of the ratio that methylates in site, and the related coefficient of linear fit is all higher than
0.99（See Fig. 5）.Therefore, sequencing primer of the invention can be applied to quantitative detection and detection accuracy is high.
Table 4：The theoretical value and detected value for the ratio that methylates
Claims (11)
1. a kind of be used for the sequencing primer based on pyrosequencing techniques detection mgmt gene promoter methylation, wherein the survey
Sequence primer is such as SEQ ID NO:Nucleotide sequence shown in 3.
2. the sequencing primer of claim 1, wherein the target sequence that the sequencing primer is directed to is No. 10 dyeing of human genome
The sequence of the 131,265,519 to 131,265,537 of body：SEQ ID NO: 6.
3. a kind of be used for kit or microarray based on pyrosequencing techniques detection mgmt gene promoter methylation, it is wrapped
The sequencing primer of any one of 1-2 containing claim.
4. the kit or microarray of claim 3, it also includes following primer sets, and the primer sets are by SEQ ID NO: 1
Shown primer and by SEQ ID NO:Primer shown in 2.
5. the kit or microarray of claim 3 or 4, it, which is also included, shows analytical sequence by SEQ ID NO:Shown in 4 and/
Or allocation order is by SEQ ID NO:Specification shown in 5.
6. the sequencing primer of any one of claim 1-2 is being prepared for being detected based on pyrosequencing techniques in sample
Purposes in the kit or microarray of mgmt gene promoter methylation.
7. the purposes of claim 6, wherein the kit or microarray also include following primer sets, the primer sets are served as reasons
SEQ ID NO:Primer shown in 1 and by SEQ ID NO:Primer shown in 2.
8. the purposes of claim 6 or 7, wherein the kit or microarray are also comprising the analysis shown used in pyrosequencing
Sequence is by SEQ ID NO:Shown in 4 and/or allocation order is by SEQ ID NO:Specification shown in 5.
9. the purposes of claim 6 or 7, wherein the sample is biological sample.
10. the purposes of claim 9, wherein the sample is humoral sample or tissue sample.
11. the purposes of claim 9, wherein the sample is selected from biopsy samples, cell culture, whole blood, blood plasma, blood
Clearly, saliva, Cerebrospinal fluid, sweat, urine, excrement, peritoneal fluid, juice and tear.
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| CN105779402A (en) * | 2016-03-30 | 2016-07-20 | 苏州偲聚生物材料有限公司 | Polypeptide for detecting MGMT (MethylGuanine Methyl Transferase) antibody in serum, detection device and detection kit |
| CN108570504A (en) * | 2018-06-15 | 2018-09-25 | 上海润达榕嘉生物科技有限公司 | A kind of MGMT promoter methylations detection primer and its detection method |
| CN109355364A (en) * | 2018-10-12 | 2019-02-19 | 领星生物科技（上海）有限公司 | The methylation detection method on the island MGMT promoter region CpG |
| CN112646808A (en) * | 2020-12-25 | 2021-04-13 | 嘉兴允英医学检验有限公司 | Primer, kit and method for detecting MGMT methylation |
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| EP1900825A1 (en) * | 2006-09-13 | 2008-03-19 | Rheinische Friedrich-Wilhelms-Universität Bonn | Sensitive and reproducible method for the determination of MGMT promoter methylation in clinical samples |
| CN102424857A (en) * | 2011-12-30 | 2012-04-25 | 陕西北美基因股份有限公司 | Taqman hydrolysis probe and method for quantitatively detecting methylation level of MGMT (O6-methylguanine-DNA-methyltransferase) |
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| CN103571958A (en) * | 2013-11-04 | 2014-02-12 | 北京海思特临床检验所有限公司 | Primer and kit for detecting methylation of MGMT (O<6>-methylguanine-DNA methyltransferase) genes and use method of primer |
| CN104726561A (en) * | 2015-02-12 | 2015-06-24 | 福州艾迪康医学检验所有限公司 | Reagent and method for detecting MGMT gene promoter methylation |
| CN105018592B (en) * | 2015-04-13 | 2018-05-08 | 凯杰生物工程（深圳）有限公司 | Mgmt gene promoter methylation detection based on pyrosequencing techniques |
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| EP1900825A1 (en) * | 2006-09-13 | 2008-03-19 | Rheinische Friedrich-Wilhelms-Universität Bonn | Sensitive and reproducible method for the determination of MGMT promoter methylation in clinical samples |
| CN102424857A (en) * | 2011-12-30 | 2012-04-25 | 陕西北美基因股份有限公司 | Taqman hydrolysis probe and method for quantitatively detecting methylation level of MGMT (O6-methylguanine-DNA-methyltransferase) |
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