CN104830855A - Probe for detecting SEPT9 gene methylation and application of probe - Google Patents
Probe for detecting SEPT9 gene methylation and application of probe Download PDFInfo
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Abstract
The invention relates to a probe and kit for detecting SEPT9 gene methylation and discloses an optimized probe for detecting SEPT9 gene methylation. 2-3 deoxyinosine nucleotide bases (I) are introduced at a specific position on the methylation probe so that the methylation probe can be fully combined with a methylated sample DNA and the existence of a non-methylated site in a combination zone can be tolerated. Therefore, the detection efficiency of SEPT9 gene methylation is effectively increased. The probe can be applied to the kit for detecting SEPT9 gene methylation in combination with SEPT9 and GAPDH gene amplification primers.
Description
Technical field
The invention belongs to field of gene detection, more specifically, the present invention relates to the probe and application thereof that detect SEPT9 gene methylation.
Background technology
DNA methylation is one of apparent modification mode of gene, may be present in all higher organisms.DNA methylation can close the activity of some gene, demethylation reactivating and expressing then induction of gene.In Mammals, methylating of DNA only betides on the cytosine(Cyt) of CpG, the cytosine(Cyt) making CpG dinucleotides 5 ' hold under the effect of DNA methylation transferring enzyme (DNMTs) change into 5 ' methylcystein (
mc).This DNA modification mode does not change gene order, but it can the expression of regulatory gene.DNA methylation studies epigenetics mechanism the most deep, and it plays an important role in maintenance normal cell function, chromatin Structure modification, x chromosome inactivation, genomic imprinting, fetal development and human tumor occur.
Colorectal cancer is a kind of malignant tumour of serious threat human health, and the annual neopathy number of cases in the whole world is about 1,200,000, and number of dying of illness in year is more than 600,000.Sickness rate is only second to lung cancer and mammary cancer, and occupy malignant tumour the 3rd, case fatality rate occupies the 4th.In recent years, its M & M is in the trend risen year by year.WHO statistic data shows, 5 years survival rates of colorectal cancer patients are about 60% in north america, and China only has 32%.On diagnosis of colorectal carcinoma, treatment level, also there is larger gap in China compared with developed countries.Research is thought, within 5 years, survival rate has obvious dependency with the severity of disease during diagnosis, and 5 years survival rates of progressive stage are less than 10%, and 5 years survival rates of the colorectal cancer patients of early diagnosis then can reach more than 90%.Therefore, early discovery, early diagnosis, early treatment are significant for raising treatment of colorectal cancer effect.
Septin albumen is that a class has the active protein family of guanosine triphosphate kinases (GTPase), take part in a series of important physiological process, as intracellular matter transport, cell fission and apoptosis etc. in human body.In human body, the gene of coding septin albumen has 13 kinds.Wherein septin 9 gene (SEPT9) is positioned at No. 17 chromosomal 17q25 position, total length 219kb, and one has 18 kinds of different montage products, 15 peptide species of can encoding.The distribution of these variation spliceosomes has tissue specificity.Nearest result of study display, the generation of the methylating of SEPT9 gene (being especially positioned at methylating of SEPT9 gene V2 spliceosome CpG island, promoter region) and colorectal cancer and develop closely related.It can as of a colorectal cancer early diagnosis specific molecular marker.Therefore, detect the methylation state of SEPT9 gene, significant to the aspect such as diagnosis, treatment, Index for diagnosis of colorectal cancer.
The methylated method of current researching DNA mainly contains: 1. methylation sensitive inscribe enzyme process; 2. bisulfite (Bisulfite) modifies method; 3. specific recognition methylates antibody assay; 4. mass spectrum or red, orange, green, blue, yellow (ROGBY) etc.Wherein bisulfite modifies method is the CpG methylation detecting method be most widely used at present.The method adopts Na
2s
2o
5, methylated C will be there is not and will be converted into U, and occur methylated in process sample DNA
mc remains unchanged; After pcr amplification, U changes T into, and
mc changes C into, thus makes methylating on genome/non-methylating change C/T (Y) polymorphism into, by detecting the C/T state on target site, can determine whether there is methylating of gene.
The methylation detecting method modified based on bisulfite is varied, such as BSP clone sequencing, fluorescent PCR method, chip hybridization methods etc.Wherein fluorescent PCR method and chip hybridization methods all need the methylation state by using the probe that methylates to identify target site.Under normal circumstances, methylate probe based on exhaustive methylation gene order and design, namely all in detector segments CpG sites all adopt G base and methylated C base pair complementarity, so effectively can only detect the sample of exhaustive methylation.The generation of tumour, development are very complicated processes, and tumour cell has obvious heterogeneous feature.Different tumour cells is in the different etap, causes the methylation of SEPT9 gene in tumour cell constantly to change with developmental process.When the SEPT9 gene in tumour cell is in partial methylation state, the joint efficiency of typical probe and template DNA is lower, has influence on the sensitivity of detection.Under extreme case, in detector segments, there is 1 non-methylation sites, just may cause probe and template DNA cannot be in conjunction with, obtain false negative result.The probe that methylates designed routinely is not suitable for detecting the methylated sample of SEPT9 Gene Partial.
Therefore, the new SEPT9 gene methylation probe of exploitation and test kit is needed, to detect the methylation state of gene more fully and effectively.
Summary of the invention
The object of the present invention is to provide the probe and application thereof that detect SEPT9 gene methylation.
In a first aspect of the present invention, provide a kind of probe for detecting SEPT9 gene methylation, the nucleotide sequence of described probe is as shown in SEQ ID NO:3 and SEQ ID NO:4.
In a preference, described probe carries detectable.
In another preference, described detectable is fluorescent marker.
In another aspect of this invention, provide a kind of test kit for detecting SEPT9 gene methylation, described test kit comprises described probe.
In a preference, also comprise in described test kit: the primer of pcr amplification SEPT9 gene; Preferably, the nucleotide sequence of described primer is as shown in SEQ ID NO:6 and SEQ ID NO:7.
In another preference, also comprise in described test kit: the primer of pcr amplification GAPDH gene, and the probe of specific detection GAPDH gene, the detectable that this probe carries is different from for the detectable entrained by the probe of SEPT9 gene.
In another preference, the nucleotide sequence of the primer of pcr amplification GAPDH gene is as shown in SEQ IDNO:14 and SEQ ID NO:15; The probe nucleotide sequence of specific detection GAPDH gene is as shown in SEQ ID NO:16.
In another preference, 5 ' end of the probe of specific detection GAPDH gene arranges JOE fluorescent mark, and 3 ' end arranges BHQ1 fluorescent mark; 5 ' the end detecting the probe of SEPT9 gene methylation arranges FAM, and 3 ' end arranges BHQ1 fluorescent mark.
In another aspect of this invention, detect while a kind of nondiagnostic is provided the method for SEPT9 gene methylation, described method comprises: with the cdna sample after bisulfite process, adopt PCR method, gene chips or membrane hybridization detect described in probe and this cdna sample in conjunction with situation, thus the SEPT9 methylation status of acquisition testing gene sample.
In a preference, described detectable is fluorescent marker, utilize the pcr amplification primer shown in SEQ ID NO:6 and SEQ ID NO:7 and the arbitrary described probe of claim 1-3 to carry out the detection of fluorescent PCR method, thus obtain the methylation status of SEPT9 gene.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, typical probe detect the amplified fluorescence curve of different methylation level sample.
The amplified fluorescence curve of the different methylation level sample of Fig. 2, probe in detecting of the present invention.
Fig. 3, test kit of the present invention detect the amplification curve of SEPT9 methylation positive sample.
Fig. 4, test kit of the present invention detect SEPT9 and to methylate the amplification curve of negative sample
Embodiment
The present inventor is through deep research, disclose a kind of probe of detection SEPT9 gene methylation of optimization, deoxyinosine nucleotide base (I) is introduced by the specific position at probe, the SEPT9 DNA methylation assay probe be optimized, it can either combine with the sample DNA of exhaustive methylation, can tolerate again in conjunction with section internal memory in 1 non-methylated site, thus effectively improve the detection efficiency to the sample that not exclusively methylates.
As used herein, " sample " or " sample " need carry out the nucleic acid substances of methylation state of DNA detection, it can come from individuality (as human or animal), also can be other source, more such as through amplification or the laboratory nucleic acid material without amplification, or the test article of synthetic.Should be understood that and not only diagnostic purpose is related to the detection of " sample " or " sample ", also can relate to other non-diagnostic object.
The sequence (as 5 ' ATCG 3 ' → GCTA) in as used herein, the sequence of " complementation " typically refers to 5 '-3 ' sequence in direction be converted to its 3 '-5 ' direction, and then get its complementary sequence (as GCTA → 5 ' CGAT 3 ').
" complementation " comprises " substantially complementary ", and described " substantially complementary " refers to that the sequence of two sections of Nucleotide is enough complementary, can interact, as formed secondary structure (as loop-stem structure) in the foreseeable mode of one.Usually, the nucleotide sequence of two " substantially complementary " mutually between have at least the base of 70% to be complementary; Preferably, the base of 80% is had at least to be complementary; Preferred, have at least the base of 90% to be complementary; Preferred further, have at least the base of 95% to be complementary; As 96%, 98%.
As used herein, there is the base of 100% to be complementary between the nucleotide sequence that " complete complementary " refers to two " complementations ".
As used herein, " pairing " of base refers to that in two sequences, corresponding base constitutes the connection of key (as hydrogen bond).Abundant in two sequences (as more than the base of 70%, 80% or 90% being pairing) " pairing " of base makes two sequences that complementation occur.
The present invention is based on the methylation detecting method that bisulfite is modified, DNA sample is through Na
2s
2o
5after process, there is not methylated C and be converted into U, and occur methylated
mc remains unchanged, and after pcr amplification, U changes T into, and
mc changes C into, thus makes to methylate/non-methylating change C/T (Y) polymorphism into, by detect methylate probe and bisulfite modified outcome in conjunction with situation, the methylation state of sample DNA can be specified.
Technical scheme of the present invention is as follows: according to the methylate regional sequence of SEPT9 gene after bisulfite process, selects one section of region (5 '-T comprising 5 CpG sites
yGgATTT
yGYGgTTAA
yGYG-3 ', wherein YG represents CpG site) as methylating probe-binding region; Design two with the probe that methylates of target section complementation:
Probe A:5 '-C
c
tTAACC
c
aAATCC
a-3 ' (SEQ ID NO:3)
Probe B:5 '-C
c
tTAACC
c
aAATCC
a-3 ' (SEQ ID NO:4)
In probe A, with the basecount for methylation sites, start at from the 1st for the base of methylation sites, guanine and G and deoxyinosine Nucleotide and I press the arrangement of I-G-I-G-I order.
In probe B, with the basecount for methylation sites, start at from the 1st for the base of methylation sites, guanine and G and deoxyinosine Nucleotide and I press the arrangement of G-I-G-I-G order.Article two, the length of probe is consistent, and difference is the position that base I occurs.After the probe of wherein and DNA profiling combine, can produce another probe and repel, so in hybridization process, the probe that base pair complementarity is stronger can preferentially be combined on target template DNA.
Inosinic acid base (I) is the analogue of a kind of guanine base (G), can combine with cytosine(Cyt) (C) pairing.Combined by 3 hydrogen bond formation between guanine base (G) and cytosine(Cyt) (C), and xanthoglobulin base (I) lacks an amido, 2 hydrogen bonds can only be formed with cytosine(Cyt) (C), although the bonding force of G: C base pair is higher than the bonding force of I: C base pair, but methylate in probe at GC rich content, replace a part of G with I, effectively can reduce the Tm value of crossbred, improve the specificity of DNA methylation assay.Another characteristic of inosinic acid base (I) can combine with A, G, C and T tetra-kinds of base pairings, and wherein the bonding force of I: C base pair is the strongest, and the bonding force of I: T, I: A and I: G base pair then will be weaker than I: C base pair.
In two probes of the present invention, G and I alternately occurs by specific arrangement mode, G and I all energy and methylated C base pairing combines, and can detect the sample of exhaustive methylation.For non-methylated sample, the base that G and I is corresponding is thymus pyrimidine (T), can not match combination between G, T, and the bonding force between I: T is more weak, so can not produce hybridization signal between probe and non-methylate DNA.
Probe according to the present invention's design is specially adapted to detect incomplete methylated sample.When the 1st, 3 or 5 CpG position in target section existing 1 non-methylation sites, probe A and target section can effectively combine.When in target section the 2nd or 4 CpG positions on exist 1 non-methylation sites time, probe B and target section can effectively combine.
After obtaining probe of the present invention, obviously, it can be applied to and anyly to realize in the method for DNA methylation assay by probe by those skilled in the art.Described method includes but not limited to: the multiple methylation detecting methods such as fluorescent PCR detection, genechip detection, film hybridization check.
After obtaining probe of the present invention, for the ease of using and commercialization, described probe sets can be formed in a test kit.Therefore, present invention includes the test kit comprising probe of the present invention.Such as when this test kit is a kind of fluorescent PCR kit, wherein also can comprise the primer pair in the specific region that methylates of specific amplification SEPT9 gene, for primer and the probe of the external control gene that increases, and the reagent such as PCR damping fluid.
Preferably, for the PCR primer of the SEPT9 gene methylation fragment that increases in described test kit, sequence is as follows:
Upstream primer: 5 '-GTTGTTTTTCGCGCGATTC-3 ' (SEQ ID NO:6);
Downstream primer: 5 '-CCACCTTCGAAATCCGAAAT-3 ' (SEQ ID NO:7);
Preferably, external control in described test kit adopts glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), PCR primer and probe sequence as follows:
Upstream primer: 5 '-TGGGTGGTTATTGTGAAAAGTT-3 ' (SEQ ID NO:14);
Downstream primer: 5 '-CTCCAATCCCTAACCCTACCTT-3 ' (SEQ ID NO:15);
Fluorescent probe: 5 '-CCTTTCCTACTAAAACCCAAAACCAAAC-3 ' (SEQ ID NO:16).
After obtaining probe of the present invention and/or primer, for the ease of using and commercialization, described probe and/or primer collection can be formed in a test kit.Therefore, present invention includes the test kit comprising probe of the present invention and/or primer.
Preferably, other detection reagent coordinating described probe and/or primer can also be comprised in described test kit.Such as when this test kit is a kind of fluorescent PCR kit, wherein also can comprise the primer pair in the specific region that methylates of specific amplification testing gene, and the reagent such as PCR damping fluid.
Preferably, in described test kit, also can comprising the working instructions of the using method of the probe described in explanation, correctly using for instructing those skilled in the art.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.
The methylation level detection example of embodiment 1, SEPT9 gene
In the present embodiment, adopt Fluorescence PCR assay, test SEPT9 gene methylation probe is to the detection case of different methylation level sample.
SEPT9 gene V2 spliceosome promoter region sequence to be measured is as follows:
TCTGCACTGCAGGAGCGCGGGCGCGGCGCCCCAGCCAGCGCGCAGGGCCCGGGCCCCGCCGGGGGCGCTTCCTCGCCGCTGCCCTCCGCGCGACCCGCTGCCCACCAGCCATCATG
CAGCTGGATGGGATCATTTCGGACTTCGAAGGTGGGTGCTGGGCTGGCTGCTGCGGCCGCGGACGTGCTGGAGAGGACCCTGCGGGTGGGCCTGGCGCGGGACGGGGGTG(SEQ ID NO:1)
Square frame section wherein contains 5 CpG methylation sites, is the present embodiment middle probe binding site.
Above-mentioned SEPT9 gene order is through bisulfite and Na
2s
2o
5sequence after process is as follows:
TTTGTATTGTAGGAGYGYGGGYGYGGYGTTTTAGTTAGYGYGTAGGGTTYGGGTTTYGTYGGGGGYGTTTTTTYGTYGTTGTTTTTYGYGYGATTYGTTGTTTATTAGTTATTATG
TAGTTGGATGGGATTATTTYGGATTTYGAAGGTGGGTGTTGGGTTGGTTGTTGYGGTYGYGGAYGTGTTGGAGAGGATTTTGYGGGTGGGTTTGGYGYGGGAYGGGGGTG(SEQID NO:2)
Wherein
section contains 5 methylation sites, is called successively: 1 ~ site, site 5 according to 5 ' ~ 3 ' order.
1, probe and design of primers
According to SEPT9 gene promoter area fragment through bisulfite and Na
2s
2o
5sequence after process, design PCR probe and primer as follows:
SEPT9-P-1:5 '-C
cGTTAACC
cGAAATCC
a-3 ' (probe A, SEQ IDNO:3 of the present invention);
SEPT9-P-2:5 '-CGC
tTAACCGC
aAATCCGA-3 ' (probe B, SEQ IDNO:4 of the present invention);
SEPT9-P-C:5 '-CGCGTTAACCGCGAAATCCGA-3 ' (typical probe SEQ ID NO:5);
SEPT9-F:5 '-GTTGTTTTTCGCGCGATTC-3 ' (amplimer, SEQ ID NO:6);
SEPT9-R:5 '-CCACCTTCGAAATCCGAAAT-3 ' (amplimer, SEQ ID NO:7).
Whole probe all adopts 5 '-FAM and 3 '-BHQ1 fluorescent mark.
I represents deoxyinosine nucleotide base.
2, SEPT9 gene methylation fluorescent PCR detection system
Adopt fluorescent PCR method to detect the methylation state of target site, prepare two fluorescent PCR systems altogether, wherein first reaction system uses typical probe SEPT9-P-C, and second reaction system uses the combination of probe SEPT9-P-1 and SEPT9-P-2 of the present invention.
The preparation of template DNA:
The gene fragment of synthetic is adopted to test SEPT9 gene methylation fluorescent PCR system.The SEPT9 gene fragment that synthesis 7 kinds is different altogether, its difference is that the methylation state in probe-binding region section 5 CpG sites is different.Synthesis fragment adopts ultraviolet spectrophotometer to measure concentration, and is converted into corresponding gene copy number (copies).Adopt TE damping fluid that synthesis fragment is diluted to 100copies/uL, as the DNA profiling that the fluorescent PCR that methylates detects.
Comprise in 30uL PCR reaction system: 1 × PCR buffer, 2.5mM MgCl
2, 200 μMs of dNTP, upstream and each 0.5uM of downstream amplification primer, each 0.2uM of fluorescent probe SEPT9-P-1 and SEPT9-P-2, or SEPT9-P-C 0.2uM, 1.25U Taq archaeal dna polymerase and 5uL template DNA.
Run following amplification program:
First stage: 95 DEG C 3 minutes, 1 circulation;
Subordinate phase: 95 DEG C 15 seconds, 60 DEG C 60 seconds, 45 circulations;
Signal collection: collect FAM fluorescent signal during subordinate phase 60 DEG C, each circulating collection first order fluorescence.
3, two kinds of fluorescent PCR systems are to the amplification of different methylation level sample
Adopt two kinds of fluorescent PCR systems, increase respectively:
exhaustive methylation sample (probe binding site: TCGGATTTCGCGGTTAACGCG (SEQ ID NO:8), wherein 5 CpG are and methylate),
the non-sample that the methylates (probe binding site: T in site 1
gGATTTCGCGGTTAACGCG (SEQID NO:9)),
the non-sample that the methylates (probe binding site: TCGGATTT in site 2
gCGGTTAACGCG (SEQ ID NO:10)),
the non-sample that the methylates (probe binding site: TCGGATTTCG in site 3
gGTTAACGCG (SEQ ID NO:11)),
the non-sample that the methylates (probe binding site: TCGGATTTCGCGGTTAA in site 4
gCG (SEQ ID NO:12)),
the non-sample that the methylates (probe binding site: TCGGATTTCGCGGTTAACG in site 5
g (SEQ ID NO:13)) and
the completely non-sample that methylates (5 sites all do not methylate).
Result shows, and typical probe effectively can only detect the sample of exhaustive methylation, and not good to the expanding effect containing 1 non-methylation sites sample, Ct value is (Fig. 1) to the rear obviously.And adopt probe combinations of the present invention, for exhaustive methylation sample, and the Detection results basically identical (Fig. 2) of sample containing 1 non-methylation sites.
Above-mentioned experimental result shows, the SEPT9 probe in the present invention effectively can improve the detection efficiency of SEPT9 gene methylation.
Embodiment 2, the application of SEPT9 gene methylation detection kit in colorectal cancer pattern detection
SEPT9 gene promoter methylation is a specific molecular marker of colorectal cancer early diagnosis.Under normal circumstances, in the tumor tissues of colorectal cancer patients or blood sample, SEPT9 gene is methylation state, and in healthy tissues or blood sample, SEPT9 gene is non-methylation state.Adopt the SEPT9 in the present invention to methylate probe, combined with fluorescent PCR detection method, can develop the methylation detection kit for SEPT9 gene, can detect the methylation state of SEPT9 gene in sample fast, delicately.The contrast agents of GAPDH gene amplification can also be added in test kit, for the internal reference of reaction tubes, make detected result more accurately, reliably.
1, the PCR primer in SEPT9 gene methylation detection kit and probe
(A) for SEPT9 gene methylation detect primer and probe as follows:
SEPT9-F:5 '-GTTGTTTTTCGCGCGATTC-3 ' (amplimer, SEQ ID NO:6);
SEPT9-R:5 '-CCACCTTCGAAATCCGAAAT-3 ' (amplimer, SEQ ID NO:7);
SEPT9-P-1:5 '-C
cGTTAACC
cGAAATCC
a-3 ' (probe A, SEQID NO:3 of the present invention);
SEPT9-P-2:5 '-CGC
tTAACCGC
aAATCCGA-3 ' (probe B, SEQ ID NO:4 of the present invention);
The SEPT9 probe that methylates all adopts 5 '-FAM+3 '-BHQ1 fluorescent mark.
(B) for the primer (GAPDH-F and GAPDH-R) of GAPDH gene amplification and probe (GAPDH-P) as follows:
GAPDH-F：5’-TGGGTGGTTATTGTGAAAAGTT-3’(SEQ ID NO:14)；
GAPDH-R：5’-CTCCAATCCCTAACCCTACCTT-3’(SEQ ID NO:15)；
GAPDH-P：5’-CCTTTCCTACTAAAACCCAAAACCAAAC-3’(SEQ ID NO:16)；
GAPDH gene probe adopts 5 '-JOE+3 '-BHQ1 fluorescent mark.
2, the extraction of sample DNA
Get the Colorectal Carcinoma sample of clinical definite, and routine through the Carcinoma side normal tissue check sample each 50 of pathological identification, add 180 μ l DNA extraction liquid and 20 μ l Proteinase K Solution, 56 DEG C of digestion 1-3 hour; Add 300 μ l GDT damping fluids, mix latter 70 DEG C and hatch 10 minutes; Add 300 μ l dehydrated alcohols, be transferred to after mixing in centrifugal column, centrifugal 1 minute, abandon the filtrate in collection tube; Add 700 μ l washings PW, 8000 revs/min centrifugal 1 minute, abandons filtrate; In centrifugal column, add 700 μ l washings WA, 8000 revs/min centrifugal 1 minute, abandons filtrate; Dry residual liquid (12000 revs/min centrifugal 2 points) in centrifugal column; Add 100 μ lDNA elutriants to the film central authorities in centrifugal column, room temperature to place after 1 minute 8000 revs/min centrifugal 2 minutes, gained sample is tumor tissues DNA sample.After measuring OD value, sample DNA being diluted to concentration is that 20ng/uL is for subsequent use.
3. the bisulfite of sample DNA is modified
Get in 30uL sample to be tested DNA to PCR reaction tubes, add the consoluet bisulfite solution of 60uL, then add 10uL conversion protection liquid; After covering tightly pipe lid, reaction solution is centrifugal is in short-term collected at the bottom of pipe by reaction solution in vibration mixing; Reaction tubes is placed in PCR instrument, 95 DEG C 5 minutes, 60 DEG C 1 hour, amount to 5 circulations.Reaction terminates rear taking-up reaction tubes, adds 330uL MB damping fluid and 1uL CarrierRNA, then adds 270uL dehydrated alcohol, vibration mixing.Proceed in purification column by the solution in reaction tubes, the centrifugal 1min of 8000rpm, abandons waste liquid.Add 500 μ l washingss (the 1st time) in purification column, the centrifugal 1min of 8000rpm, abandons waste liquid.Add 500 μ l doctor solutions in purification column, room temperature places the centrifugal 1min of 15min, 8000rpm, abandons waste liquid.Add 500 μ l washingss (the 2nd time) in purification column, the centrifugal 1min of 8000rpm, abandons waste liquid.Purification column is proceeded in new collection tube, the centrifugal 1min of 13000rpm, remove residual solution.Proceeded to by purification column in 1.5mL centrifuge tube, add the central authorities of 30 μ l DNA elutriants to film, room temperature places the centrifugal 1min of 3min, 8000rpm, and gained solution is used for fluorescent PCR amplification.
4, SEPT9 gene and GAPDH gene composite fluorescence PCR detection system
Test kit adopts the composite fluorescence amplification system of SEPT9 gene and GAPDH gene: comprise in 30uL PCR reaction system: 1 × PCR buffer, 3mM MgCl
2, 200 μMs of dNTP, SEPT9 upstream region of gene and each 0.5uM of downstream amplification primer, fluorescent probe SEPT9-P-1 and SEPT9-P-2 each 0.2uM, GAPDH upstream and downstream amplification primer each 0.3uM, 0.3uM GAPDH-P fluorescent probe, 1.25U Taq DNA polymerase and 20ng template DNAs.
Run following amplification program:
First stage: 95 DEG C 3 minutes, 1 circulation;
Subordinate phase: 95 DEG C 15 seconds, 60 DEG C 60 seconds, 45 circulations;
Signal collection: collect FAM and JOE fluorescent signal during subordinate phase 60 DEG C, each circulating collection first order fluorescence.
Fluorescent PCR detected result shows, SEPT9 and GAPDH gene composite fluorescence PCR detection system effectively can detect the methylation state of SEPT9 gene in sample.The amplification of FAM and JOE fluorescent signal detected simultaneously, show that sample is the SEPT9 gene methylation positive (Fig. 3); The amplification of JOE fluorescent signal can only be detected, show that sample is SEPT9 gene methylation feminine gender (Fig. 4).FAM and JOE two kinds of fluorescent signals all can't detect, and show that detection system is abnormal, result is insincere, carries out fluorescent PCR detection again after again need preparing sample.
As a result, in 50 routine Colorectal Carcinoma samples, positive 48 examples of SEPT9 gene methylation detected altogether, positive rate is 96%.In 50 routine cancer beside organism check samples, detected result is all that SEPT9 gene methylation is negative.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. for detecting a probe for SEPT9 gene methylation, it is characterized in that, the nucleotide sequence of described probe is as shown in SEQ ID NO:3 and SEQ ID NO:4.
2. probe as claimed in claim 1, it is characterized in that, described probe carries detectable.
3. probe as claimed in claim 2, it is characterized in that, described detectable is fluorescent marker.
4. for detecting a test kit for SEPT9 gene methylation, it is characterized in that, described test kit comprises the arbitrary described probe of claim 1-3.
5. test kit as claimed in claim 4, is characterized in that, also comprise in described test kit: the primer of pcr amplification SEPT9 gene; Preferably, the nucleotide sequence of described primer is as shown in SEQ ID NO:6 and SEQ ID NO:7.
6. test kit as claimed in claim 4, it is characterized in that, also comprise in described test kit: the primer of pcr amplification GAPDH gene, and the probe of specific detection GAPDH gene, the detectable that this probe carries is different from for the detectable entrained by the probe of SEPT9 gene.
7. test kit as claimed in claim 6, it is characterized in that, the nucleotide sequence of the primer of pcr amplification GAPDH gene is as shown in SEQ ID NO:14 and SEQ ID NO:15; The probe nucleotide sequence of specific detection GAPDH gene is as shown in SEQ ID NO:16.
8. test kit as claimed in claim 7, is characterized in that, 5 ' end of the probe of specific detection GAPDH gene arranges JOE fluorescent mark, and 3 ' end arranges BHQ1 fluorescent mark;
5 ' the end detecting the probe of SEPT9 gene methylation arranges FAM, and 3 ' end arranges BHQ1 fluorescent mark.
9. the method for a nondiagnostic ground detection SEPT9 gene methylation, it is characterized in that, described method comprises: with the cdna sample after bisulfite process, adopt PCR method, gene chips or membrane hybridization test right require the arbitrary described probe of 1-3 and this cdna sample in conjunction with situation, thus the SEPT9 methylation status of acquisition testing gene sample.
10. method as claimed in claim 8, it is characterized in that, described detectable is fluorescent marker, utilize the pcr amplification primer shown in SEQ ID NO:6 and SEQ ID NO:7 and the arbitrary described probe of claim 1-3 to carry out the detection of fluorescent PCR method, thus obtain the methylation status of SEPT9 gene.
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