CN105018592A

CN105018592A - Pyrosequencing technology-based detection of methylation of MGMT gene promoter

Info

Publication number: CN105018592A
Application number: CN201510175687.7A
Authority: CN
Inventors: 陈华; 刘小青
Original assignee: Cage Biological Engineering (shenzhen) Co Ltd
Current assignee: Cage Biological Engineering (shenzhen) Co Ltd
Priority date: 2015-04-13
Filing date: 2015-04-13
Publication date: 2015-11-04
Anticipated expiration: 2035-04-13
Also published as: CN105018592B; WO2016165591A1

Abstract

The invention relates to pyrosequencing technology-based detection of methylation of MGMT gene promoter. Specifically speaking, the invention relates to a sequencing primer used for pyrosequencing technology-based detection of methylation of the MGMT gene promoter. The invention also relates to a kit and a micro-array which include the sequencing primer and are used for pyrosequencing technology-based detection of methylation of the MGMT gene promoter. The invention further relates to application of the sequencing primer in preparation of the kit and the micro-array that are used for pyrosequencing technology-based detection of methylation of the MGMT gene promoter. The invention also relates to a method for detecting methylation of the MGMT gene promoter in a sample by using the sequencing primer.

Description

Mgmt gene promoter methylation based on pyrosequencing techniques detects

Technical field

The mgmt gene promoter methylation that the present invention relates to based on pyrosequencing techniques detects.Specifically, the present invention relates to the sequencing primer detecting mgmt gene promoter methylation based on pyrosequencing techniques.The invention still further relates to test kit and the microarray for the detecting mgmt gene promoter methylation based on pyrosequencing techniques that comprise sequencing primer.The invention still further relates to sequencing primer for the preparation of the purposes detected based on pyrosequencing techniques in the test kit of mgmt gene promoter methylation and microarray.The invention still further relates to the method using sequencing primer to detect mgmt gene promoter methylation in sample.

Background technology

Pyrosequencing techniques is that a kind of cascade reaction based on enzyme-to-substrate carries out detecting and quantitative gene sequence analysis.In whole reaction system, using the PCR primer of strand as template, sequencing primer and its annealed combination, four kinds of dNTP are added in reaction system according to set order.If the base complementrity of the dNTP added on template strand is combined, the tetra-sodium group PPi of the mole number such as release and incorporation under the effect of archaeal dna polymerase.The PPi of sulfurylase catalysis release and adenosine-5'-phosphosulfate form adenosine triphyosphate (the Adenosine Triphosphate of equivalent, ATP), ATP drives the fluorescein of luciferase mediation to oxidation state, concurrent third contact of a total solar or lunar eclipse signal.The optical signal that instrument detects then is reflected in the mode at fluorescent signal peak in the Sequencing chromatogram occurred in real time.DNTP as added can not combine with template complementation, then directly degraded by bisphosphatase, reaction continues to enter next round.By the circulation of said process, the PCR primer of strand obtains complementation and extends, and judges base type by the presence or absence of each base positions fignal center, is judged the number of base by the height of fignal center and area.Manganic pyrophosphate complex initiation method is suitable for carrying out quantitative analysis to 10-100bp length sequences, and its repeatability and accurate performance and Sanger check order and match in excellence or beauty, and detection speed then improves greatly.Multiple pyrosequencing techniques, while maintenance pyrosequencing techniques advantage, strengthens it for the detectivity at a distance of more than 100bp site.Two even multiple sequencing reactions carry out at same Kong Weizhong simultaneously, and the order added by layout four kinds of dNTP of science obtains special detection sequence, thus detect while realizing multiple site apart from each other.

O6-MG-DNA-methyltransgerase (O6-methylguanine-DNA-methyltransferase, MGMT) be a kind of DNA repair protein of ubiquitous uniqueness, can in the presence without any cofactor or albumen by methyl from the O6 bit transition of 6 methyl guanines to the cysteine residues of self, guanine is reduced, and Temozolomide (temozolomide, TMZ) in vivo can by Methyl transporters to the methylation sites on DNA after hydrolytic action, the critical sites of 6 methyl guanines Temozolomide cytotoxicity just.MGMT makes DNA alkylation damage be repaired, and is the common drug nitrosourea of cells for therapeutic administration malignant glioma and the major cause of Temozolomide generation resistance.Studies have found that, by causing MGMT inactivation to be that tumour is to TMZ chemosensitivity important factor to methylating of mgmt gene promotor.Research finds that the chemotherapy of the negative glioma person of MGMT to BCNU class (chloroethylnitrosoure, CENU) is efficient apparently higher than MGMT positive.Therefore MGMT makes the height of MGMT expression level in tissue can reflect the sensitivity differences of Different Individual to alkylating agent medicine to the repair that alkylating agent damages.Mgmt gene promoter zone methylation degree can MGMT expression level in indication body, and then predicts the lifetime that glioblastoma patient accepts alkylating agent chemotherapy.Therefore before formulating chemotherapy regimen, detect the expression of mgmt protein in glioma cells in tissue, nitrosourea antitumor drug and TMZ are not selected to the tumour patient of MGMT high expression level, can resistance be avoided, be expected to improve chemotherapeutic efficacy.U.S. FDA is also recommended using the patient of Temozolomide to carry out the analysis of MGMT promoter methylation, to instruct patient medication.

Now existing different kinds of molecules Biological Detection technology is applied to the detection in mgmt gene promoter methylation site, as real-time quantitative PCR, LDR, RFLP, HRM, gene chip, direct Sequencing etc.Wherein non-order-checking mode needs to rely on enzyme and cuts or the genotype of mode interpretation patient of cluster, and there is the probability of larger somatotype mistake, this is undoubtedly clinical diagnosis and avoids greatly.Although direct Sequencing accuracy is high, the DNA fragmentation that sequencing reaction produces needs, through capillary electrophoresis separation, to detect fluorescent signal thereafter by detection system, consuming time longer and testing cost is higher.

Manganic pyrophosphate complex initiation method can carry out real-time quantitative analysis to sequence in certain length, and its repeatability and accurate performance and Sanger check order and match in excellence or beauty, and detection speed improves greatly, and testing cost obviously reduces.In addition, the detection sensitivity of pyrosequencing techniques also checks order far above Sanger.This technology has been widely used in the aspect such as microorganism identification and point medical jurisprudence mirror, genetic analysis, SNP detection at present.Therefore, the application that pyrosequencing techniques detects at mgmt gene promoter methylation will have great value and potentiality with popularization.

But, the scheme still not having commercialization to realize pyrosequencing techniques at present to detect at mgmt gene promoter methylation.

Summary of the invention

One aspect of the present invention relates to the sequencing primer for detecting mgmt gene promoter methylation based on pyrosequencing techniques.In one embodiment, described sequencing primer comprises the nucleotide sequence as shown in SEQ ID NO:3.In another embodiment, described sequencing primer has the nucleotide sequence as shown in SEQ ID NO:3, or by or be substantially made up of the such as nucleotide sequence shown in SEQ ID NO:3.In one embodiment, described sequencing primer for target sequence be human genome No. 10 chromosomal 131,265, the sequence (SEQ ID NO:6) of 519 to 131,265,537.

The present invention relates to a kind of test kit or microarray for detecting mgmt gene promoter methylation based on pyrosequencing techniques on the other hand.In one embodiment, described test kit or microarray comprise the sequencing primer for Manganic pyrophosphate complex initiation, and described sequencing primer comprises the nucleotide sequence as shown in SEQ ID NO:3.In another embodiment, described sequencing primer has the nucleotide sequence as shown in SEQ ID NO:3, or by or be substantially made up of the such as nucleotide sequence shown in SEQ ID NO:3.In one embodiment, described sequencing primer for target sequence be human genome No. 10 chromosomal 131,265, the sequence (SEQ ID NO:6) of 519 to 131,265,537.

In one embodiment, described test kit or microarray also comprise following primer sets, described primer sets comprises and has by the primer of the nucleotide sequence shown in SEQ ID NO:1 and having by the primer of the nucleotide sequence shown in SEQ ID NO:2, or by or be substantially made up of the primer of the nucleotide sequence shown in SEQ ID NO:2 the primer of the nucleotide sequence shown in SEQ ID NO:1 and having by having.

In one embodiment, described test kit or microarray also comprise show analytical sequence by shown in SEQ ID NO:4 and/or allocation order by the specification sheets shown in SEQ ID NO:5.

The present invention relates on the other hand sequencing primer for the preparation of the purposes detected based on pyrosequencing techniques in the test kit of the mgmt gene promoter methylation in sample or microarray.In one embodiment, described test kit or microarray comprise the sequencing primer for Manganic pyrophosphate complex initiation, and described sequencing primer comprises the nucleotide sequence as shown in SEQ ID NO:3.In another embodiment, described sequencing primer has the nucleotide sequence as shown in SEQ ID NO:3, or by or be substantially made up of the such as nucleotide sequence shown in SEQ ID NO:3.In one embodiment, described sequencing primer for target sequence be human genome No. 10 chromosomal 131,265, the sequence (SEQ ID NO:6) of 519 to 131,265,537.

In one embodiment, described test kit or microarray also comprise show Manganic pyrophosphate complex initiation analytical sequence used by shown in SEQ ID NO:4 and/or allocation order by the specification sheets shown in SEQ ID NO:5.

In one embodiment, described sample is biological sample, preferred described sample is humoral sample or tissue sample, and more preferably described sample is selected from biopsy samples, cell culture, whole blood, blood plasma, serum, saliva, brains liquid, sweat, urine, ight soil, peritoneal fluid, juice and tear.

The invention still further relates to a kind of method detecting the mgmt gene promoter methylation in sample based on pyrosequencing techniques, described method comprises:

(1) extract and optional purification of samples in DNA,

(2) DNA of extraction and optional purifying is increased, thus obtains amplified production,

(3) Manganic pyrophosphate complex initiation is carried out to the amplified production of step (2).

In one embodiment, described Manganic pyrophosphate complex initiation uses following sequencing primer to carry out, and described sequencing primer comprises the nucleotide sequence as shown in SEQ ID NO:3.In another embodiment, described sequencing primer has the nucleotide sequence as shown in SEQ ID NO:3, or by or be substantially made up of the such as nucleotide sequence shown in SEQ ID NO:3.In one embodiment, described sequencing primer for target sequence be human genome No. 10 chromosomal 131,265, the sequence (SEQ ID NO:6) of 519 to 131,265,537.

In one embodiment, described amplification is pcr amplification, preferred described pcr amplification uses following primer sets, described primer sets comprises and has by the primer of the nucleotide sequence shown in SEQ ID NO:1 and having by the primer of the nucleotide sequence shown in SEQ ID NO:2, or by or be substantially made up of the primer of the nucleotide sequence shown in SEQ ID NO:2 the primer of the nucleotide sequence shown in SEQ ID NO:1 and having by having.

In one embodiment, described Manganic pyrophosphate complex initiation also uses by the analytical sequence shown in SEQ ID NO:4 and/or by the allocation order shown in SEQ ID NO:5.

Accompanying drawing explanation

fig. 1for a techniqueflow and each principal element schematic diagram of Manganic pyrophosphate complex initiation.

fig. 2for Manganic pyrophosphate complex initiation detects mgmt gene promoter zone methylation schematic diagram.

fig. 3for mgmt gene promoter zone methylation detected result sectional drawing, wherein X-coordinate represents assigned sequence, and ordinate zou represents the fluorescence signal intensity that instrument collects.

fig. 4for the result sectional drawing using different sequencing primer to detect mgmt gene promoter zone methylation, wherein X-coordinate represents assigned sequence, and ordinate zou represents the fluorescence signal intensity that instrument collects.

fig. 5for using the graphic representation of detected value compared with theoretical value of the ratio that methylates detected by the order-checking system comprising sequencing primer of the present invention.

Embodiment

With reference to for illustration of example application several aspect of the present invention is described hereinafter.Should be understood that, state many details, relation and method to provide and fully understand of the present invention.But, will readily appreciate that those of ordinary skill in the related art, can the present invention be implemented when not containing one or more detail or the present invention can be implemented with additive method.

The object of the invention is to solve the shortcoming to the detection in mgmt gene promoter methylation site in prior art, the probability of such as larger somatotype mistake, consuming time longer or testing cost is higher.The pyrosequencing techniques of the sequencing primer of the application of the invention of the present invention and solve the problems referred to above.

The pyrosequencing techniques of sequencing primer of the present invention is used to have the following advantages:

1) instrument and reagent are by system optimization, can reach optimum Detection results, and easily stdn;

2) detected result is provided by software automatically, avoids result to judge subjectivity;

3) operating process is easy and save time;

4) linear lag methylated in scope at 0%-100% is high, can be used for detection by quantitative.

Unless otherwise stated, all scientific and technical terminologies used herein have the implication that one skilled in the art of the present invention understand usually.The definition of the general term in Cytobiology and molecular biology can be see: the gene VIII that Yu Long etc. translate, International Standard Book Number: ISBN:978-7-03-014597-0, and Science Press publishes (2005); The cell that Zhang Jinfeng etc. translate and molecular biology, Zhong Xin press publishes (2004), ISBN:978-7-50-860075-8; With the biological chemistry that Wang Jingyan etc. compiles, Higher Education Publishing House publishes (2002), ISBN:978-7-04-011088-3; The people such as Kendrew, J. (volume), The Encyclopedia of Molecular Biology, Blackwell Science Ltd. publishes (1994), ISBN 0-632-02182-9; And Meyers, R.A. (volume), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, VCH Publishers, Inc. (1995) are published, ISBN 1-56081-5698.Although can adopt when implementing of the present invention similar or be equal to any method and the material of methods described herein and material, this document describes concrete materials and methods.

sample

Term used herein " sample " comprises any sample containing nucleic acid molecule.Sample can derive from biogenetic derivation (" biological sample "), such as organize (such as biopsy samples), extract or comprise cell (such as tumour cell), the cell culture of cell lysate and biological or physiological fluid, such as whole blood, blood plasma, serum, saliva, brains liquid, sweat, urine, ight soil, juice, milk, peritoneal fluid etc.Available from the sample in source or can directly use with the sample after improving sample characteristic (such as prepare blood plasma from blood, dilute mucus etc.) in pre-treatment.Of the present invention in some, sample is human physiology fluid, such as human serum.Of the present invention in some, sample is the tumor tissues that such as obtains through tissue examination of biopsy samples or cell.Of the present invention in some, sample is pernicious or normal tissue sample.

The sample that can carry out analyzing and/or use according to the present invention comprises the polynucleotide of clinical source, such as DNA or RNA.

The method extracting nucleic acid from sample is well-known in the art, and available such as phenol and chloroform carry out DNA extraction, or uses commercially available DNA extraction reagent to extract.Such as, post test kit (such as GENERATION (registered trademark) Capture Column Kit Gentra) can be used to extract.

It should be understood that nucleic acid carrys out purifying by the many conventional purification process in this area, such as, use PrepSEQ test kit (from Applied Biosystems) and U.S. Patent number 5,234, method in 809 etc.The present invention preferably adopts box such as EpiTect Plus LyseAll Bisulfite Kit (QIAGEN company) in commercial reagent to carry out Isolation and purification to nucleic acid.After Isolation and purification nucleic acid, bisulf iotate-treated can be carried out to nucleic acid such as DNA and react for PCR.

primer

" primer " used herein is often referred to the linear oligonucleotide with target complement sequence and annealing.The lower limit of primer length is pressed hybridization ability and determines, because very short primer (being such as less than 5 Nucleotide) does not form thermodynamically stable duplex under most of hybridization conditions.Primer length changes usually in 8-50 Nucleotide.In certain embodiments, primer is between about 15-25 Nucleotide.Term used herein " forward primer " refers to and target DNA oligonucleotide that specific chains is annealed.Term used herein " reverse primer " refers to and the oligonucleotide that the opposite strand of target DNA is annealed.In a word, forward primer and reverse primer are oriented on target DNA sequence in the mode being similar to PCR primer usually, make its 3' end than its 5' end closer to target sequence.Naturally occurring Nucleotide (especially guanine, VITAMIN B4, cytosine(Cyt) and thymus pyrimidine, " G ", " A ", " C " and " T " hereinafter referred to as) and nucleotide analog, primer all used in the present invention.Term used herein " sequencing primer " refers to for the initial Oligonucleolide primers to the sequencing reaction that nucleic acid carries out.

" amplified production " used herein refers to from nucleic acid-templated, the nucleic acid of the amplification produced by nucleic acid amplification.

" template DNA " used herein or " template ribonucleic acid " refer to as the nucleic acid for target needed for increasing.Such as, template ribonucleic acid is reversed record for cDNA, and this template cDNA is for generation of amplified production.

Term used herein " nucleotide analog " refers to the compound structurally similar to naturally occurring Nucleotide.Nucleotide analog can have the phosphate backbones of change, sugar moieties, core base or its combination.The nucleotide analog usually with the core base of change especially gives different base pairings and base stacking characteristic.The nucleotide analog (such as peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA)) with the phosphoric acid-sugared skeleton of change especially changes chain characteristic usually, and such as secondary structure is formed.

Example for detecting the PCR primer of mgmt gene promoter methylation, sequencing primer and target series based on pyrosequencing techniques sees the following form 1.

PCR primer, sequencing primer and corresponding target sequence that table 1. the present invention is used

The nucleotide sequence of PCR primer of the present invention and sequencing primer also comprises its modified forms, as long as the amplification of described primer or order-checking effect are not subject to significantly affecting.Described modification can for such as in nucleotide sequence or two ends add one or more nucleotide residue, in nucleotide sequence, lack one or more nucleotide residue or the one or more nucleotide residues in sequence replaced to other nucleotide residue, such as A is replaced to T, C is replaced to G etc.It will be apparent to those skilled in the art that the primer of described modified forms is also encompassed within the present invention, within the protection domain of particularly claim.In one embodiment, the modified forms of the nucleotide sequence of PCR primer and sequencing primer is as chemical reinforcing type primer disclosed in CN103270174A.

Such as general DNA synthesizer (394 types such as manufactured by Applied Biosystems) can be used, synthesize each Nucleotide in primer of the present invention through chemical process.Any other method well-known in the art also can be adopted to carry out synthetic oligonucleotide, such as PCR primer and sequencing primer.

Use the genomic dna extracted from sample as template, and use PCR primer pair mgmt gene to carry out amplified reaction, to obtain amplified production.Amplified reaction includes but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR) (LCP), automatically maintains sequence replicating (3SR), amplification (NASBA) based on nucleotide sequence, strand displacement amplification (SDA), multiple displacement amplification (MDA) and rolling circle amplification (RCA), it is disclosed in below with reference in document (being hereby incorporated by reference): Mullis etc., United States Patent (USP) the 4th, 683, No. 195; 4th, 965, No. 188; 4th, 683, No. 202; 4th, 800,159 (PCR) number; Gelfand etc., United States Patent (USP) the 5th, 210, No. 015 (with " Taqman " or " Taq " [registered trademark] probe PCR in real time of carrying out); Wittwer etc., United States Patent (USP) the 6th, 174, No. 670; Kacian etc., United States Patent (USP) the 5th, 399, No. 491 (" NASBA "); Lizardi, United States Patent (USP) the 5th, 854, No. 033; Aono etc., No. 4-262799, Japanese Patent Publication JP (rolling circle amplification); Etc..

Preferred use PCR method increases to target nucleotide.PCR method itself is well-known in the art.Term " PCR " comprises the derivative form of this reaction, and it includes but not limited to reverse transcription PCR, PCR in real time, nested PCR, multiplex PCR and quantitative fluorescent PCR etc.Preferred use fluorescence quantitative PCR method carries out quantitative amplification to target nucleotide.

Under primer, template DNA and hot resistant DNA polymerase exist, use the primer (reverse primer) of hybridizing with sense strand and the primer (forward primer) of hybridizing with antisense strand, by making annealing, be cycled to repeat about 30 times ~ 50 times (such as 45 times) of extension and denaturing step carry out PCR.In one embodiment, PCR is real-time fluorescence quantitative PCR.PCR employs following primer sets in one embodiment, described primer sets comprises and has by the primer of the nucleotide sequence shown in SEQ ID NO:1 and having by the primer of the nucleotide sequence shown in SEQ ID NO:2, or by or be substantially made up of described primer.Those skilled in the art it is understood that other PCR method and primer sets also can be used, as long as can target fragment be amplified.Those skilled in the art can select PCR method as required routinely, and conventional design goes out required PCR primer.

In PCR of the present invention, the hot resistant DNA polymerase of various routine can be used to increase, include but not limited to FastStart Taq archaeal dna polymerase (Roche), Ex Taq (registered trademark, Takara), Z-Taq, AccuPrime Taq archaeal dna polymerase and HotStarTaq Plus archaeal dna polymerase.

The method selecting suitable PCR reaction conditions based on primer Tm is well-known in the art, and those of ordinary skill in the art according to primer length, GC content, desired specificities and sensitivity, the polysaccharase character etc. that uses, can select top condition.Such as, following condition can be used to carry out quantitative fluorescent PCR reaction: 95 DEG C 5 minutes, 95 DEG C 20 seconds, 60 DEG C 30 seconds, 72 DEG C 20 seconds, circulate 45 times.Reaction system can be 50 μ L.

After acquisition PCR primer, can process PCR primer, to obtain the single stranded PCR products combined with sequencing primer complementation.The generation of single stranded PCR products and purifying are undertaken by the method that this area is known.The method of common generation and purification of single stranded PCR primer includes but not limited to the T7 reverse transcription method (people such as Hughes, Nat. Biotechnol., 2001, 19:342-347), Exonucleolytic enzyme process (Higuchi and Ochman, Nucleic. Acids Res., 1989, 17:5865), denaturing high-performance liquid chromatography (denaturing high-performance liquid chromatography, DHPLC) (Dickman and Hornby, Anal. Biochem., 2000, 284:164-167) and the magnetic capture method (people such as Espelund, Nucleic. Acids Res., 1990, 18:6157-6158) etc.In one embodiment, the present invention obtains the single stranded PCR products combined with sequencing primer complementation by magnetic capture method.In another embodiment, the present invention is processed PCR primer according to the specification sheets of manufacturers by PyroMark Q24 vacuum work station, to obtain the single stranded PCR products combined with sequencing primer complementation.

In addition, asymmetric PCR method also can be used directly to prepare single stranded PCR products, thus eliminate carry out extra process after PCR.Asymmetric PCR can prepare DNA single chain while pcr amplification.Conventional asymmetric PCR uses the primer of two inequalities, normally increases in the circulation started.Along with the increase of circulation, measure few primer and exhausted gradually, and the primer of excess can continue straight line amplification generation DNA single chain (Gyllensten and Erlich, Proc. Natl. Acad. Sci. U.S.A., 1988,85:7652-7656).

After acquisition single stranded PCR products, sequencing primer of the present invention can be adopted to carry out Manganic pyrophosphate complex initiation.In one embodiment, described sequencing primer can comprise the nucleotide sequence as shown in SEQ ID NO:3.In another embodiment, described sequencing primer can have the nucleotide sequence as shown in SEQ ID NO:3, or by or be substantially made up of the such as nucleotide sequence shown in SEQ ID NO:3.In one embodiment, described sequencing primer for target sequence be human genome No. 10 chromosomal 131,265, the sequence (SEQ ID NO:6) of 519 to 131,265,537.In one embodiment, can as shown in SEQ ID NO:7,10 or 13 for sequencing primer of the present invention.

In one embodiment, adopt PyroMark Gold Q24 Reagents test kit and the real-time quantitative tetra-sodium sequenator (model: PyroMark Q24 MDx) of QIAGEN company, method carries out Manganic pyrophosphate complex initiation to specifications.Following hybridization conditions can be used to carry out Manganic pyrophosphate complex initiation: 80 DEG C of heating 2min, annealing at room temperature 20 minutes.In one embodiment, Manganic pyrophosphate complex initiation also uses by the analytical sequence shown in SEQ ID NO:4 and/or by the allocation order shown in SEQ ID NO:5.Analytical sequence is use when having carried out interpretation of result after order-checking has been tested, and this analytical sequence and sequencing result are compared by the software of analyser.Such as analytical sequence is YGAYGTTYGTAGGTTTTYG, and software will have the site of Y to calculate the ratio of C/T to mark; In actual applications, ratio value can change along with sample, and this ratio value then indicates methylated degree.Allocation order is that instrument is in the order of carrying out the nucleotides substrate sprayed in sequencing procedure; During order-checking, nucleotides substrate adds in reaction tank according to allocation order by instrument in order, if fluorescent signal detected when spraying into A, is A, by that analogy with regard to representing the sequencing result in this site.Those skilled in the art can Choice and design analytical sequence and/or allocation order routinely as required, and uses different analytical sequences and/or allocation order according to the situation of concrete target sequence in actual applications.In one embodiment, Manganic pyrophosphate complex initiation can use analytical sequence as shown in table 3 and/or allocation order.

test kit

The present invention relates to a kind of test kit for detecting mgmt gene promoter methylation based on pyrosequencing techniques, it contains the combination of sequencing primer of the present invention or sequencing primer and primer sets.The invention still further relates to the purposes be combined in for the preparation of detecting based on pyrosequencing techniques in the test kit of mgmt gene promoter methylation of sequencing primer of the present invention or sequencing primer and primer sets.In one embodiment, described sequencing primer can comprise the nucleotide sequence as shown in SEQ ID NO:3.In another embodiment, described sequencing primer can have the nucleotide sequence as shown in SEQ ID NO:3, or by or be substantially made up of the such as nucleotide sequence shown in SEQ ID NO:3.In one embodiment, described primer sets comprises and has by the primer of the nucleotide sequence shown in SEQ ID NO:1 and having by the primer of the nucleotide sequence shown in SEQ ID NO:2, or by or be substantially made up of described primer.

Test kit can comprise implements the inventive method material used or reagent (comprising sequencing primer and primer sets).Test kit can comprise depot reaction reagent (primer such as in suitable vessel, dNTP, enzyme etc.) and/or support material (such as the specification sheets etc. of damping fluid, examinations).Such as, test kit can comprise one or more container (such as box) containing respective reaction reagent and/or support material.Such content can be delivered to set recipient together or separately.Such as, first container can containing enzyme for measuring, and second container contains primer sets and the 3rd container contains sequencing primer.Described test kit also can containing being applicable to the compartment holding described reagent or container.As an example, test kit can contain sequencing primer, primer sets, PCR reaction buffer, working instructions.Test kit also can contain polysaccharase and dTNP etc.Test kit also can contain UNG, for mark, the positive and negative control etc. in Quality Control.Test kit also can comprise for the reagent from sample preparation nucleic acid such as DNA.Test kit of the present invention also can comprise other any sequencing primer and/or primer sets except sequencing primer of the present invention and/or primer sets, such as, effectively can detect sequencing primer and/or the primer sets of mgmt gene promoter methylation.Above example can not be interpreted as that restricted application is in test kit of the present invention and content thereof.

In one embodiment, the specification sheets in test kit indicate Manganic pyrophosphate complex initiation analytical sequence used by shown in SEQ ID NO:4 and/or allocation order by shown in SEQ ID NO:5.

microarray

The present invention relates to a kind of microarray for detecting mgmt gene promoter methylation based on pyrosequencing techniques, it contains the combination of sequencing primer of the present invention or sequencing primer and primer sets.The invention still further relates to the purposes be combined in for the preparation of detecting based on pyrosequencing techniques in the microarray of mgmt gene promoter methylation of sequencing primer of the present invention or sequencing primer and primer sets.In one embodiment, described sequencing primer can comprise the nucleotide sequence as shown in SEQ ID NO:3.In another embodiment, described sequencing primer can have the nucleotide sequence as shown in SEQ ID NO:3, or by or be substantially made up of the such as nucleotide sequence shown in SEQ ID NO:3.In one embodiment, described primer sets comprises and has by the primer of the nucleotide sequence shown in SEQ ID NO:1 and having by the primer of the nucleotide sequence shown in SEQ ID NO:2, or by or be substantially made up of described primer.

Microarray refers to the solid support with flat surfaces, it has nucleic acid array, each member in array comprises the identical copy of oligonucleotide on the region or site that are fixed on and spatially determine or polynucleotide, and described region or site are not overlapping with the region of other member in array or site; That is, described region or site are spatially discrete.In addition, the hybridization site spatially determined can be " addressable ", because the identity of its position and immobilized oligonucleotide thereof is known or predetermined (being such as known or predetermined before its use).Usual oligonucleotide or polynucleotide are strand, and usually held by 5'-or 3'-end with solid support covalently bound.The density of the nucleic acid containing non-overlapped district in microarray is greater than 100/cm usually ², more preferably greater than 1000/cm ².Microarray technology is disclosed in such as below with reference in document: the Microarrays:A Practical Approach (IRL Press, Oxford, 2000) that Schena edits; Southern, Current Opin. Chem. Biol., 2:404-410,1998, its full content is incorporated herein by reference.

Although be described above various embodiments of the present invention, it should be understood that, it only provides by way of example, and and unrestricted.Can carry out according to disclosure herein many changes of disclosed embodiment, and can not the spirit or scope of the present invention be deviated from.Therefore, range of the present invention and scope should not be subject to any above-mentioned embodiment and limited.

The all documents mentioned herein are all incorporated herein by reference.All publications that the application quotes and patent document all combine by reference for all objects, and degree of quoting is as pointed out each publication or patent document individually.

Embodiment

Unless otherwise indicated, otherwise all commercial acquisition of material that embodiment is used herein, various specific experiment methods for carrying out testing are the experimental technique of this area routine (see " fine works molecular biology experiment guide " (1999) that such as F. Ao Sibai etc. edits, Science Press, " Molecular Cloning: A Laboratory guide (third edition) " (2002) of the chief editor such as ISBN 7-03-006408-9 and J. Pehanorm Brooker, Science Press, ISBN 7-03-010338-6) or the step of advising according to manufacturers and condition, and can be determined routinely as required by those skilled in the art.Below some materials and methods is described in detail.

embodiment 1: use the Manganic pyrophosphate complex initiation that sequencing primer of the present invention carries out

material:

method:

As shown in Figure 1, first adopt PCR Master Mix, a HS Taq and two PCR primer pair bisulfite conversion the human genome DNA of purifying increase on Rotor-Gene Q platform, comprise fluorescence dye in PCR Master Mix, can embed in amplification procedure in ever-increasing PCR double-stranded products.Therefore the process of whole product enrichment can carry out Real-Time Monitoring by the software of Rotor-Gene Q, thus guarantees that the PCR primer of reliable in quality is analyzed for follow-up Manganic pyrophosphate complex initiation.By PyroMark Q24 vacuum work station, PCR primer is processed, the single stranded PCR products that final acquisition combines with sequencing primer complementation.Specific Manganic pyrophosphate complex initiation program is run afterwards on PyroMark Q24 Manganic pyrophosphate complex initiation instrument, instrument the allocation order according to following table 2 can add four kinds of dNTP successively, by the cascade reaction of enzyme-to-substrate, the effective extension of dNTP in single stranded product is received by instrument with the form of optical signal, and finally appears in real time in software interface with the form of fignal center.Postrun result carries out automatic analysis by software, decreases burden and the error of manual analysis.

The analytical sequence of table 2. PyroMark Q24 software design patterns and allocation order

Experimentation is as follows:

One. get the clinical sample that comes from clinical hospitals, the step of advising according to manufacturers with the EpiTect Plus LyseAll Bisulfite Kit of QIAGEN company and condition carry out Tissue Lysis, sulfitation conversion, nucleic acid purification;

Two. get the nucleic acid PCR primer after purifying 1,2 and carry out pcr amplification (adopting the real-time fluorescence quantitative PCR analyser (model: Rotor-Gene Q) of QIAGEN company);

PCR condition is as follows: 95 DEG C: 5 minutes; 95 DEG C: 20 seconds, 60 DEG C: 30 seconds, 72 DEG C: 20 seconds, totally 45 circulations.

Three. Manganic pyrophosphate complex initiation

Fig. 2 is that Manganic pyrophosphate complex initiation detects mgmt gene promoter zone methylation schematic diagram.

Adopt PyroMark Gold Q24 Reagents test kit and the real-time quantitative tetra-sodium sequenator (model: PyroMark Q24 MDx) of QIAGEN company, operate according to manufacturer specification method and condition, sequencing primer is sequencing primer shown in SEQ ID NO:3, the hybridization conditions of sequencing primer is: 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 Manganic pyrophosphate complex initiation and expected sequence (i.e. CGACGCCCGCAGGTCCTCG, human genome No. 10 chromosomal 131,265,519 to 131,265, the sequence of 537) consistent, also consistent with detection " gold standard " Sanger sequencing result, and order-checking background is low.

embodiment 2: the comparison of different sequencing primer

Material used in the present embodiment, instrument and method and condition all with embodiment 1, but employ the combination of the sequencing primer shown in following table 3 and analytical sequence and allocation order in Manganic pyrophosphate complex initiation process.

Table 3: the combination of different sequencing primers and 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 condition is: 95 DEG C: 5 minutes; 95 DEG C: 20 seconds, 60 DEG C: 30 seconds, 72 DEG C: 20 seconds, totally 45 circulations.

The hybridization conditions of sequencing primer is: 80 DEG C of heating 2min, annealing at room temperature 20 minutes.

Experimental result is shown in Fig. 4.The result of result display sequencing primer 1 is best, and region to be analyzed is shown as blueness (representing that result is passed through), with expected sequence (i.e. CGACGCCCGCAGGTCCTCG, human genome No. 10 chromosomal 131,265,519-131, the sequence of 265,537) consistent, and order-checking background is low; The region to be analyzed of sequencing primer 2,3,4 is shown as yellow or redness (representing that result is uncertain or failed), not too consistent with expected sequence, and order-checking background is high.Therefore select sequencing primer 1 as sequencing primer of the present invention.

embodiment 3: the accuracy of sequencing primer of the present invention, precision and linear properties assessment

material:

method:

Choose each portion of standard clinical sample that the known ratio of methylating is respectively 0%, 25%, 50%, 75%, 100%, the order-checking system that employing comprises sequencing primer of the present invention (SEQ ID NO:3) detects, each sample duplicate detection 8 times, the detected value of the ratio that methylates of statistics 4 detection site, and compare with theoretical value.

Experiment material, instrument and experimentation that detection uses and condition are all with embodiment 1; Sequenator draws the percentage ratio of the C base on each methylation sites automatically, is the detected value of the ratio that methylates in this site.

The detected result of the ratio that methylates is as shown in table 4 below, and result shows: detected value and the theoretical value of the ratio that methylates are close, and detection accuracy is higher; The standard deviation in each site is less than 2.2, and mean value is 0.81, demonstrates good precision; Detected value and the theoretical value of the ratio that methylates in 4 sites present good linear relationship, and the relation conefficient of linear fit is all greater than 0.99(and sees Fig. 5).Therefore, sequencing primer of the present invention can be applicable to detection by quantitative and detection accuracy is high.

Table 4: the theoretical value of the ratio that methylates and detected value

Claims

1., for detecting a sequencing primer for mgmt gene promoter methylation based on pyrosequencing techniques, described sequencing primer comprises the nucleotide sequence as shown in SEQ ID NO:3.

2. the sequencing primer of claim 1, wherein said sequencing primer has the nucleotide sequence as shown in SEQ ID NO:3.

3. the sequencing primer of claim 1 or 2, wherein said sequencing primer for target sequence be human genome No. 10 chromosomal 131,265, the sequence of 519 to 131,265,537: SEQ ID NO:6.

4., for detecting test kit or the microarray of mgmt gene promoter methylation based on pyrosequencing techniques, it comprises the sequencing primer any one of claim 1-3.

5. the test kit of claim 4 or microarray, it also comprises following primer sets, and described primer sets comprises and has by the primer of the nucleotide sequence shown in SEQ ID NO:1 and have by the primer of the nucleotide sequence shown in SEQ ID NO:2.

6. the test kit of claim 4 or 5 or microarray, its also comprise show analytical sequence by shown in SEQ ID NO:4 and/or allocation order by the specification sheets shown in SEQ ID NO:5.

7. the sequencing primer any one of claim 1-3 is for the preparation of the purposes detected based on pyrosequencing techniques in the test kit of the mgmt gene promoter methylation in sample or microarray.

8. the purposes of claim 7, wherein said test kit or microarray also comprise following primer sets, and described primer sets comprises and has by the primer of the nucleotide sequence shown in SEQ ID NO:1 and having by the primer of the nucleotide sequence shown in SEQ ID NO:2.

9. the purposes of claim 7 or 8, wherein said test kit or microarray also comprise show Manganic pyrophosphate complex initiation analytical sequence used by shown in SEQ ID NO:4 and/or allocation order by the specification sheets shown in SEQ ID NO:5.

10. the purposes of claim 7 or 8, wherein said sample is biological sample, preferred described sample is humoral sample or tissue sample, and more preferably described sample is selected from biopsy samples, cell culture, whole blood, blood plasma, serum, saliva, brains liquid, sweat, urine, ight soil, peritoneal fluid, juice and tear.