CN108220405A - The method and kit of microsatellite stability state-detection - Google Patents
The method and kit of microsatellite stability state-detection Download PDFInfo
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Abstract
The present invention relates to a kind of method and kit of microsatellite stability state-detection, the method is detected for the quasi- monomorphism mononucleotide repeats bits point of 5 high sensitivities and specificity, and 5 quasi- monomorphism mononucleotides repeat site and are：NR 21, NR 24, BAT 25, BAT 26 and MONO 27.The method of the present invention further includes the pentanucleotide good to 2 polymorphisms and repeats site：Penta D and Penta E synchronize detection, identical to ensure to detect samples sources, and detection sample is avoided to obscure.
Description
Technical field
The invention belongs to field of biological detection, are related to a kind of Multiple detection system and reagent, and in particular to a kind of microsatellite
The method and kit, the method and kit of more particularly to described microsatellite stability state-detection of stability status detection exist
Application in immunization therapy and Jessica Lynch's syndrome auxiliary diagnosis.
Background technology
Wyman in 1980 etc. proposes microsatellite sequence first, which is made of 2-6 nucleotide, with dinucleotides
Repetitive sequence (CA/GT) n it is most commonly seen.Normal cell has complete DNA mismatch repair system in breeding, can
To find microsatellite sequence copy error in time and correct rapidly so that microsatellite sequence high-fidelity replicates, and is defended so as to remain micro-
Star is stablized.Partial tumors are in generating process since DNA mismatch repair function defect (dMMR) cannot find microsatellite sequence in time
The mistake of row in a replication process is inserted into or is lacked so as to cause recurring unit the change for causing microsatellite sequence length and then leads
Cause Microsatellite instability (MSI).Current research trend is thought, due to cell mismatch repair system function
It is impaired that microsatellite sequence is caused to repeat to mix or lack, microsatellite sequence length is caused to change, so as to show MSI.
MSI detects gene loci, and BAT-25 (mononucleotide repetition), BAT-26 (monokaryon glycosides are recommended in NCI in 1997
Acid repeats), D2S123 (repetition of two nucleotide), D5S346 (repetition of two nucleotide), 5 bases of D17S250 (repetition of two nucleotide)
Because of detection site, define simultaneously：2 and 2 or more gene loci variations are known as high microsatellite instability (MSI-H), 1 gene
Mutation is known as low microsatellite instability (MSI-L), and being known as microsatellite without gene loci variation stablizes (MSS), also both
Bethesda standards.It is found in the scientific research and clinical practice of several later years, selects the gene loci that two nucleotide repeat easy
It generates archaeal dna polymerase and slips product (English original text is stutter), specificity and sensibility do not have mononucleotide duplicate loci
It is good.So at 2002, Bethesda standards are corrected, and MSI detections recommend mononucleotide duplicate loci,
2004, Jeffery of Promega companies et al. carried out the analysis of system to the MSI nucleotide duplicate locis detected,
As a result it shows：In terms of specificity and sensitivity, mononucleotide duplicate loci is better than two nucleotide duplicate locis
With Trinucleotide repeats gene loci.
The incidence of China's colorectal cancer increases year by year, its incidence occupies the 4th in malignant tumour, and in America and Europe
Developed country occupies malignant tumour second.Lynch syndromes, also known as hereditary nonpolyposis colorectal cancer (HNPCC) are
Dominant hereditary disease caused by MMR germlines are mutated.More than 90% Lynch syndromes have MSI features, and sporadic knot
There was only about 15% in the carcinoma of the rectum, so can clinically carry out the screening of Lynch syndromes with MSI detections.Lynch syndromes are suffered from
A variety of Lynch syndromes related neoplasms such as person and its common colorectal cancer of family member, carcinoma of endometrium, gastric cancer, oophoroma, because
This MSI detections are significant for sufferers themselves and its family member.Bethesda standards suggest doubtful Lynch syndromes
Patient carry out MSI detections, MSI detection become screen Lynch syndromes essential items for inspection.MSI colorectal cancer patients
It is insensitive to the chemotherapy based on 5 FU 5 fluorouracil, while MSI colorectal cancer patients are good compared with the outcome of MSS patient, illustrate MSI
Medication guide and prognosis prediction of the detection available for colorectal cancer.
Programmed death 1 (PD-1) are the novel immune therapies that immune system is escaped by releasing tumour cell,
The mechanism of action of PD-1 immunotherapies is to design specific protein antibody for PD-1 or PD-L1, prevents PD-1's and PD-L1
Identification process, T cell function is restored in part, so as to which T cell be allow to kill tumour cell.Wherein PD-1 antibody medicine Keytruda
As first " broad-spectrum anti-cancer drug ", for treating the solid tumor of " MSI-H/dMMR hypotypes ", this is that U.S. FDA is based on tumour for the first time
Biomarker rather than tumour home position have approved a new drug.
In view of microsatellite stability state-detection is in Analysis of Genetic Background, medicine response, immunotherapy prognosis etc.
Significance selects appropriate microsatellite stability Multiple detection body repetition site development and improvement, easily, highly sensitive
System and detection kit are very necessary.
Invention content
Since the meaning and application value of microsatellite stability state-detection are more and more brighter and clearer, particularly controlled in tumour immunity
It treats in (PD-1/PD-L1) and Jessica Lynch's syndrome and shows more and more important potential value, the present invention is based on current societies
It may require that, provide a kind of multiple fluorescence PCR-capillary electrophoresis method to detect microsatellite state.By detecting the swollen of patient
Tumor tissue and normal structure can quickly identify its microsatellite state, guidance provided for follow-up visit.
Although the result that the detection method of the present invention obtains is possibly used for the medication guide and prognosis prediction of disease, this hair
Bright detection method is not related to the diagnose and treat of any disease in itself, do not seek yet to the diagnose and treat of clinical disease into
Any kind of limitation of row.I.e.：The method of detection microsatellite stability state of the present invention be in itself for non-diagnostic and/or
Non-treatment purpose.
Specifically, the present invention provides embodiments below.
On the one hand, the present invention provides a kind of method for detecting microsatellite stability state, the described method comprises the following steps：
(1) 5 quasi- monomorphism mononucleotides repeat site in PCR amplification sample；
(2) capillary electrophoresis detection is carried out to pcr amplification product；
(3) fluoroscopic examination, with the size and fluorescence intensity of gene sequencer detection amplified production；
(4) data collection and analysis；
It is characterized in that, 5 quasi- monomorphism mononucleotides repeat site for NR-21, NR-24, BAT-25, BAT-26 and
MONO-27；It further includes 2 polymorphism pentanucleotides of synchronous amplification and detection and repeats site, 2 polymorphisms pentanucleotide weight
Reduction point is Penta D and Penta E.
It is of the present invention detection microsatellite stability state method, it is characterised in that NR-21, NR-24, BAT-25,
The nucleotide sequence of BAT-26 and MONO-27 is respectively SEQ ID NO:1-5.
The method of detection microsatellite stability state of the present invention, it is characterised in that 5 quasi- monomorphism monokaryon glycosides
Acid repeat site PCR amplification primer be：SEQ ID NO:8-9、SEQ ID NO:10-11、SEQ ID NO:12-13、SEQ
ID NO:14-15、SEQ ID NO:16-17。
The method of detection microsatellite stability state of the present invention, it is characterised in that the Penta D's and Penta E
Nucleotide sequence is respectively SEQ ID NO:6-7.
The method of detection microsatellite stability state of the present invention, the primer pair of synchronous amplification Penta D and Penta E
Respectively SEQ ID NO:18-19、SEQ ID NO:20-21.
The method of detection microsatellite stability state of the present invention, it is characterised in that the amplimer is respectively by three kinds
The fluorescein label of different colours, the combination of three groups of fluorescent markers are respectively：First group of NR-21, BAT-26, MONO-27；Second
Group：BAT-25 and Penta D；Third group：NR-24 and Penta E.
The method of detection microsatellite stability state of the present invention, three kinds of fluoresceins labeled as FAM, HEX and
ROX, the amplimer in preferably first group repetition site is marked with FAM, the amplimer in second group of repetition site is marked with HEX,
The amplimer that third group repeats site is marked with ROX.
On the other hand, the present invention provides a kind of reagent for the method for being used to carry out the detection microsatellite stability state
Box, it is characterised in that including following primer pair：SEQ ID NO:8-9、SEQ ID NO:10-11、SEQ ID NO:12-13、SEQ
ID NO:14-15、SEQ ID NO:16-17；And the primer pair SEQ ID NO of amplification Penta D:18-19, extension Penta
The primer pair SEQ ID NO of E:5 ' the ends of 20-21, wherein sense primer are marked with fluorescein.
Kit of the present invention, it is characterised in that comprising primer mixed liquor in kit, in the primer mixed liquor
Each primer pair concentration is as follows：SEQ ID NO:8-9 is 5 μM, SEQ ID NO:10-11 is 3.75 μM, SEQ ID NO:12-13 is
5μM、SEQ ID NO:14-15 is 25 μM, SEQ ID NO:16-17 is 5 μM, SEQ ID NO:18-19 is 1.25 μM, SEQ ID
NO:20-21 is 10 μM.
Kit of the present invention, it is characterised in that further included in kit dNTP, 10 × Buffer, archaeal dna polymerase,
DdH2O etc..
For the technology contents of the clearer description present invention, the present invention is described in further detail technical solution.
In one embodiment, the present invention specifically provides one group for detecting the primer sets of microsatellite instability state
Object is closed, the amplimer composition can expand 5 quasi- monomorphism mononucleotides and repeat site and 2 five repetition nucleotide simultaneously
Repeat site：NR-21, NR-24, BAT-25, BAT-26, MONO-27, Penta D and Penta E, wherein amplimer base
Sequence is：
NR-21 primers：
Sense primer:GGAGTCGCTGGCACAGTTCTA(SEQ ID NO:8)
Downstream primer:TCTGGTCACTCGCGTTTACA(SEQ ID NO:9)
BAT-25 primers：
Sense primer:CTCGCCTCCAAGAATGTAAGT(SEQ ID NO:10)
Downstream primer:ATTCTGCATTTTAACTATGGCTCT(SEQ ID NO:11)
BAT-26 primers：
Sense primer:TGAAATTGGATATTGCAGCAGTCAG(SEQ ID NO:12)
Downstream primer:CTCCTTTATAAGCTTCTTCAGTATATGTC(SEQ ID NO:13)
NR-24 primers：
Sense primer:AGGTCTGCCTTAACGTGA(SEQ ID NO:14)
Downstream primer:AGATTGTGCCATTGCATT(SEQ ID NO:15)
MONO-27 primers：
Sense primer:GGCAGGGAAATGGTGGGAACC(SEQ ID NO:16)
Downstream primer:AAGGGTGGATCAAATTTCACTTGG(SEQ ID NO:17)
Penta D primers：
Sense primer:ACTTGAGCCTGGAAGGTCGA(SEQ ID NO:18)
Downstream primer:TTGCCTAACCTATGGTCATAACGATTTTTTTG(SEQ ID NO:19)
Penta E primers
Sense primer:GGCTGAAACAGGAGAATCAC(SEQ ID NO:20)
Downstream primer:TGTGTAAAGTGCTTAGTATCAT(SEQ ID NO:21)
The amplimer expands site and is marked respectively by the fluorescein of three kinds of different colours, and three groups of combinations are respectively：
First group of NR-21, BAT-26, MONO-27；Second group：BAT-25 and PentaD；Third group：NR-24 and Penta E.
FAM, HEX and ROX can be respectively labeled as in described three groups.
Described NR-21, BAT-26, MONO-27 are marked for FAM, BAT-25 the and Penta D are marked for HEX；It is described
NR-24 and Penta E are marked for ROX.
5 ' ends of the primer pair middle and upper reaches primer are marked with fluorescein.
In another embodiment, the present invention provides a kind of amplification system for detecting microsatellite instability state, including
Aforementioned Primer composition.
The total volume of the amplification system is 20 μ l, wherein 2 0.2 μ l of μ l, dNTP of buffer solution, 7.0 μ l Primer compositions,
0.2 μ l of Taq enzyme, 8.6 μ l of deionized water add in template amount as 2 μ l, and the primer concentration in the primer mixture is respectively 25 μM
NR-24 primer pairs, 5 μM of NR-21 primer pairs, BAT-26 primer pairs, MONO-27 primer pairs, 3.75 μM of BAT-25,10 μM
Penta E primer pairs, 1.25 μM of Penta D primer pairs.
The amplification condition of the amplification system is：95 DEG C of thermal startings, 5-10min；94 DEG C of denaturation, 30s, 62 DEG C of annealing,
90s；70 DEG C of extensions, 90s；60 DEG C of last extensions, 30min.
The compositions such as Primer composition of the kit by fluorescent marker of the present invention, PCR buffer solutions, dNTP, archaeal dna polymerase,
Based on Multiplex fluorescent PCR, for detecting the microsatellite state in tissue.Fluorescent marker composition in kit is by 7 pairs
Primer forms：5 quasi- monomorphism mononucleotides repeat site (NR-21, NR-24, BAT-25, BAT-26 and MONO-27) and 2
Five repeat the amplimer that nucleotide repeats site (Penta D and Penta E).Pcr amplification product using ABI 3130 (or
3730 grade Genetic Analysers of ABI 3130XL, ABI 3500DX, ABI) Capillary Electrophoresis is carried out, as a result using GeneMapper
(or GeneMarker) software is analyzed.
The present invention achieves following beneficial technique effect：
1st, the present invention provides one group of new quasi- monomorphism mononucleotides to repeat site as detection target and with reference to target,
The MSI detection methods of Promega companies are improved and have developed, testing result figure spectrum shows that the selection for repeating site group causes
Testing result is sensitive and accurate.Polymorphism good Penta D and Penta E are employed simultaneously obscures mistake for reducing sample
Occur so that the method adapts to Large-scale Screening and detection.
2nd, the present invention is realized by using three kinds of fluorescent marker designs and the optimization of amplification system in same amplification body
Detection is carried out at the same time to 7 sites in system so that primer length, amplified fragments 7 target detections of different sizes can be at one
It is completed in system.
3rd, the present invention adjusts the phase of primer pair between 7 target spots for the difference of 7 kinds of repetition site targets abundance in the sample
It to concentration, is respectively positioned in the range of same or similar fluorescence intensity level, realizes same so as to the fluorescence intensity for making 7 kinds of amplified productions
Step detection avoids multiplicating and carries out fluoroscopic examination.
Description of the drawings
The parting collection of illustrative plates that Fig. 1 is obtained using present invention detection normal structure：NR-21, BAT-26, MONO-27 are marked with FAM
Note；BAT-25 and Penta D are marked with HEX；NR-24 and Penta E are marked with ROX.
The parting collection of illustrative plates that Fig. 2 is obtained using present invention detection tumor tissues：NR-21, BAT-26, MONO-27 are marked with FAM
Note；BAT-25 and Penta D are marked with HEX；NR-24 and Penta E are marked with ROX.
Good normal distribution is presented in the amplified production clip size that five quasi- monomorphism mononucleotides repeat site.Fig. 1
It is consistent with the testing result peak type of Penta E with Penta D in Fig. 2, show that the normal sample and tumor sample derive from phase
Same individual.Fig. 1 is consistent with the amplified fragments peak type in five in Fig. 2 quasi- monomorphism mononucleotides repetition sites to show the individual
Microsatellite is in stable condition in middle normal structure and tumor tissues.
Specific embodiment
Can also the present invention further be understood by embodiment, wherein the embodiment illustrates some preparations or user
Method.It is to be appreciated, however, that these embodiments do not limit the present invention.The change of the present invention of currently known or further exploitation
Change is considered within the scope of the invention described herein and claimed below.
Embodiment 1DNA is extracted
DNA is carried out using tissue DNA extracts kit (Tiangeng is biochemical) to the cancerous tissue of patient and normal structure respectively to carry
It takes, according to specification, DNA extractions are finished to be quantified, and be diluted to 1ng/ul operating procedure with ultraviolet specrophotometer.
Embodiment 2PCR is expanded
1st, PCR reaction systems
7 pairs of primers are dissolved respectively and are made into the working solution of a concentration of 10uM, being then made into primer by 1 volume ratio of table afterwards mixes
Close liquid：
NR-21 primers：
Sense primer:GGAGTCGCTGGCACAGTTCTA(SEQ ID NO:8)
Downstream primer:TCTGGTCACTCGCGTTTACA(SEQ ID NO:9)
BAT-25 primers：
Sense primer:CTCGCCTCCAAGAATGTAAGT(SEQ ID NO:10)
Downstream primer:ATTCTGCATTTTAACTATGGCTCT(SEQ ID NO:11)
BAT-26 primers：
Sense primer:TGAAATTGGATATTGCAGCAGTCAG(SEQ ID NO:12)
Downstream primer:CTCCTTTATAAGCTTCTTCAGTATATGTC(SEQ ID NO:13)
NR-24 primers：
Sense primer:AGGTCTGCCTTAACGTGA(SEQ ID NO:14)
Downstream primer:AGATTGTGCCATTGCATT(SEQ ID NO:15)
MONO-27 primers：
Sense primer:GGCAGGGAAATGGTGGGAACC(SEQ ID NO:16)
Downstream primer:AAGGGTGGATCAAATTTCACTTGG(SEQ ID NO:17)
Penta D primers：
Sense primer:ACTTGAGCCTGGAAGGTCGA(SEQ ID NO:18)
Downstream primer:TTGCCTAACCTATGGTCATAACGATTTTTTTG(SEQ ID NO:19)
Penta E primers
Sense primer:GGCTGAAACAGGAGAATCAC(SEQ ID NO:20)
Downstream primer:TGTGTAAAGTGCTTAGTATCAT(SEQ ID NO:21)
Above-mentioned primer is 5 ' -3 ', prepares primer mixed liquor, and the addition of each primer is as shown in table 1.
Each primer pair addition in 1 primer mixed liquor of table
| Primer pair title (F+R) | Concentration (μM) |
| NR-21 primer pairs | 5 |
| BAT-25 primer pairs | 3.75 |
| BAT-26 primer pairs | 5 |
| NR-24 primer pairs | 25 |
| MONO-27 primer pairs | 5 |
| Penta D primer pairs | 1.25 |
| Penta E primer pairs | 10 |
Prepare amplification system.It will match after each reaction reagent (buffer solution, primer mixed liquor and dNTP etc.) oscillation mixing by table 2
Into PCR reaction mixtures, 18ul is in PCR reaction tubes for packing, last to add in 2ul templates toward each reaction tube, under entering after centrifugation
One step.
Each primer final concentration is respectively 2.5 μM of NR-24,5 μM NR-21, BAT-26, MONO-27 in PCR amplification system
With Penta D, 10 μM of Penta E and BAT-25.
Table 2PCR amplification systems
| Component | 20 μ L systems (μ L) |
| Primer mixed liquor | 7 |
| dNTP(25mM) | 0.2 |
| 10×buffer | 2 |
| Archaeal dna polymerase (5U/ μ L) | 0.2 |
| ddH2O | 8.6 |
| DNA profiling | 2 |
2nd, PCR response procedures
PCR reaction tubes are placed in PCR amplification instrument, are run by the program of table 3.
Table 3PCR amplification programs
3 capillary electrophoresis detection of embodiment
The 1 μ l point samples of pcr amplification product that Example 2 obtains are on 1.5-2.0% Ago-Gels, after electrophoresis 20min
Observation is as a result, the detection that is available on the machine if occurring bright ladder strip band at 70-300bp.
According to the band brightness of the sample on running gel figure, sample extension rate is determined, by extension rate by amplified production
Dilution is for use.By internal standard (ROX500) and formamide in proportion 15：1000 mixing, take 9 μ l mixtures to be added in 96 orifice plates, then add
Enter the 1 μ l of sample diluted, mixing stands several minutes, writes plate number, be put into after centrifugation on ABI 3130XL sequenators, prepares inspection
It surveys.
ABI 3130XL sequenator data acquisition softwares, the plate mark of the upper machine testing of editor are opened, importing detects program.
Operation is clicked, that is, starts to detect.After detection, by data copy to CD.
4 data processing of embodiment
Initial data is imported, in the File menu selection Add sample to project of homepage, finds sample text
Part, filesselected folder, clicks add to list, clicks add, and sample file is shown in Project windows；
Selection analysis parameter.Define analysis method, panel and size standard.Browse sample electrophoresis
Initial data, a wilful sample file name, " raw data " is selected under " sample " menu.Moving tracing line, stops cursor
On the right side of primer peak (before first red internal standard peak), the numerical value shown using in window lower left corner X-axis at this time as
Starting point in analysismethod analytical parameters；
Green analysis button is clicked, save project dialog boxes occurs, is preserved after name, software, that is, start to process number
According to the lower left corner shows analysis completed after the completion of analysis.
The data analyzed using GeneMapper softwares simultaneously generate collection of illustrative plates, see Fig. 1 and 2.
5 interpretation of result of embodiment
1) MSI criterion：
Compared with normal structure, there is band and increases or shift in amplified production in tumor tissues, then judges site shakiness
It is fixed.There are two five sites or more site changes and is judged as the high unstable state of microsatellite (MSI-H), only changes there are one site
Become then into the low unstable state of microsatellite (MSI-L), change without site and then stablize (MSS) state for microsatellite.
2) the MSI result judgements of sample：
The normal structure of above-mentioned sample and tumor tissues sample CE collection of illustrative plates are subjected to genetic fragment analysis, got the bid according to collection of illustrative plates
The genetic fragment size shown is synchronous with photoluminescence peak peak shape work to be compared, and as shown in Figure 1 to Figure 2, abscissa is genetic fragment base
Numerical value, ordinate are fluorescence values.
Wherein, Fig. 1 normal tissue genotypes fragment analysis figure, Fig. 2 are tumor tissues sample fragment analysis figure.As a result it shows：
Penta D and Penta E bands in normal structure sample and tumor tissues sample without shifting phenomena, illustrate normal structure sample
It is same person with tumor tissues samples sources.As a result it shows：BAT-26, BAT-25, NR-24, MONO- of tumor tissues sample
27 and NR-21 genetic fragments do not occur band shifting phenomena compared to normal structure sample standard deviation, so detected person's tumour is defended to be micro-
Star stablizes (MSS) state.
The content of present invention merely illustrates some claimed specific embodiments, one of them or more skill
Recorded technical characteristic can be combined with arbitrary one or more technical solutions in art scheme, these are combined and obtain
Technical solution also in the application protection domain, technical solution is disclosed in the present invention just as obtained from these are combined
It is specifically recorded in content the same.
Sequence table
<110>Shanghai Hui Zhen bio tech ltd
<120>The method and kit of microsatellite stability state-detection
<130>Nothing
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 621
<212> DNA
<213> NR-21
<400> 1
ggtaaaggca gtctcctgtt ttattagggg gagaggtgaa gggaaatcca ggctcacttt 60
ctgaataagc cactgcctgg tgcacagagc agaaccatcc tggtttctga agacacatcc 120
ctttcagcag aattccagcc ggagtcgctg gcacagttct atttttatat ttaaatgtat 180
gtctcccctg gccttttttt tttttttttt ttttagcaac acttttcttg tttgtaaacg 240
cgagtgacca gaaagtgtga atgcggagta ggaatatttt tcgtgttctc ttttatctgc 300
ttgccttttt tagagagtag cagtggttcc tatttcggaa aaggacgttc taattcaaag 360
ctctctccca atatatttac acgaatacgc atttagaaag ggaggcagct tttgaggttg 420
caatcctact gagaaggatg gaagaaggag ccaggcaccg aaacaacacc gaaaagaaac 480
acccaggtgg gggcgagtcg gacgccagcc ccgaggctgg ttccggaggg ggcggagtag 540
ccctgaagaa agagatcgga ttggtcagtg cctgtggtat catcgtaggg aacatcatcg 600
gctctggaat ctttgtctcg c 621
<210> 2
<211> 346
<212> DNA
<213> BAT-25
<400> 2
cacaggctca tacatagaaa gagatgtgac tcccgccatc atggaggatg acgagttggc 60
cctagactta gaagacttgc tgagcttttc ttaccaggtg gcaaagggca tggctttcct 120
cgcctccaag aatgtaagtg ggagtgattc tctaaagagt tttgtgtttt gtttttttga 180
tttttttttt tttttttttt tttttgagaa cagagcattt tagagccata gttaaaatgc 240
agaatgtcat ttaaaacaaa agtattggat tttttataat ataagcaaca ctatagtatt 300
aaaaagttag ttttcactct ttacaagtta aaatgaattt aaatgg 346
<210> 3
<211> 312
<212> DNA
<213> BAT-26
<400> 3
ccagtggtat agaaatcttc gatttttaaa ttcttaattt taggttgcag tttcatcact 60
gtctgcggta atcaagtttt tagaactctt atcagatgat tccaactttg gacagtttga 120
actgactact tttgacttca gccagtatat gaaattggat attgcagcag tcagagccct 180
taaccttttt caggtaaaaa aaaaaaaaaa aaaaaaaaaa agggttaaaa atgttgaatg 240
gttaaaaaat gttttcattg acatatactg aagaagctta taaaggagct aaaatatttt 300
gaaatattat ta 312
<210> 4
<211> 345
<212> DNA
<213> NR-24
<400> 4
catcactgcc cttcctcaag acctaaaata gctccctatt tagtgaaaaa ttatctgaat 60
atttaaggtc tgccttaacg tgatccccat tgctgaattt tacctcctga ctccaaaaac 120
tcttctcttc cctgggccca gtcctatttt tttttttttt tttttttttt gtgagacaga 180
gtctcactct gtcacccagg ttggaatgca atggcacaat ctccgctcac tgcaacgtcc 240
gcctcccggg ttcacgccat tctcctgcct cagcctcccg aatagctggg actacaggtc 300
gccgccaaca cgcctggcta attttttgta tttttagtag agacg 345
<210> 5
<211> 345
<212> DNA
<213> MONO-27
<400> 5
tcagccgggc gtggtggagg gcgcctgtag tcccagctac tcgggaggct gaggcaggga 60
aatggtggga acccaggggg tggagattgc agtgagctga gattgcgcca ctgcactcca 120
gcgtgggaga cagagcaaga ctctgcctca aaaaaaaaaa aaaaaaaaaa aaaaaatcct 180
ggttttactt ttttttcttt tttagttggc caagtgaaat ttgatccacc cttaagaaag 240
gagacagaac cacatcatga acttgtaagt agtatagcct tagaatgtta gcactgaaaa 300
taaatgtttt aatttgtttt tgtgtgatgt attagtactg accaa 345
<210> 6
<211> 353
<212> DNA
<213> Penta-D
<400> 6
ttaaatagcc aggcatggtg aggctgaagt aggatcactt gagcctggaa ggtcgaagct 60
gaagtgagcc atgatcacac cactacactc cagcctaggt gacagagcaa gacaccatct 120
caagaaagaa aaaaaagaaa gaaaagaaaa gaaaagaaaa gaaaagaaaa gaaaagaaaa 180
gaaaaaacga aggggaaaaa aagagaatca taaacataaa tgtaaaattt ctcaaaaaaa 240
tcgttatgac cataggttag gcaaatattt cttagatatc acaaaatcat gacctattaa 300
aaaataataa taaagtaagt ttcatcaaaa cttaaaagtt ctactcttca aaa 353
<210> 7
<211> 505
<212> DNA
<213> Penta-E
<400> 7
gccgatgcag gtgtattacc tgagctcagg agatcaagac cagcctgggc aacatggtga 60
aaccccgtct ctactaaaat acaaaaaatt agctgggtgt ggtggtaggc acctgtaatc 120
ccagctactc tggaggctga aacaggagaa tcacttgaac ccaggaggtg gagattgaag 180
tgagccgaga tcacgccatt gcactccagc ctgggcgact gagcaagact cagtctcaaa 240
gaaaagaaaa gaaaagaaaa gaaaattgta aggagttttc tcaattaata acccaaataa 300
gagaattctt tccatgtatc aatcatgata ctaagcactt tacacacatg tatgttatgt 360
aatcattata tcatgcatgc aaggtaatga gtattatttt cctcatttta taaaagagga 420
aactgatgtt tgaggctact ttgcttaaga ccacagaact agcaaaggaa aagagaagtg 480
aatgtatccc tgatcccctt taaca 505
<210> 8
<211> 21
<212> DNA
<213> NR-21 P1
<400> 8
ggagtcgctg gcacagttct a 21
<210> 9
<211> 20
<212> DNA
<213> NR-21 P2
<400> 9
tctggtcact cgcgtttaca 20
<210> 10
<211> 21
<212> DNA
<213> BAT-25 P1
<400> 10
ctcgcctcca agaatgtaag t 21
<210> 11
<211> 24
<212> DNA
<213> BAT-25 P2
<400> 11
attctgcatt ttaactatgg ctct 24
<210> 12
<211> 25
<212> DNA
<213> BAT-26 P1
<400> 12
tgaaattgga tattgcagca gtcag 25
<210> 13
<211> 29
<212> DNA
<213> BAT-26 P2
<400> 13
ctcctttata agcttcttca gtatatgtc 29
<210> 14
<211> 18
<212> DNA
<213> NR-24 P1
<400> 14
aggtctgcct taacgtga 18
<210> 15
<211> 18
<212> DNA
<213> NR-24 P2
<400> 15
agattgtgcc attgcatt 18
<210> 16
<211> 21
<212> DNA
<213> MONO-27 P1
<400> 16
ggcagggaaa tggtgggaac c 21
<210> 17
<211> 24
<212> DNA
<213> MONO-27 P2
<400> 17
aagggtggat caaatttcac ttgg 24
<210> 18
<211> 20
<212> DNA
<213> Penta D P1
<400> 18
acttgagcct ggaaggtcga 20
<210> 19
<211> 32
<212> DNA
<213> Penta D P2
<400> 19
ttgcctaacc tatggtcata acgatttttt tg 32
<210> 20
<211> 20
<212> DNA
<213> Penta E P1
<400> 20
ggctgaaaca ggagaatcac 20
<210> 21
<211> 22
<212> DNA
<213> Penta E P2
<400> 21
tgtgtaaagt gcttagtatc at 22
Claims (10)
1. a kind of method for detecting microsatellite stability state, the method includes：
(1) 5 quasi- monomorphism mononucleotides repeat site in PCR amplification sample；
(2) capillary electrophoresis detection is carried out to pcr amplification product；
(3) fluoroscopic examination, with the size and fluorescence intensity of gene sequencer detection amplified production；
(4) data collection and analysis；
It is characterized in that, 5 quasi- monomorphism mononucleotides repeat site for NR-21, BAT-25, BAT-26, NR-24 and
MONO-27；It further includes 2 polymorphism pentanucleotides of synchronous amplification and detection and repeats site, 2 polymorphisms pentanucleotide weight
Reduction point is Penta D and Penta E.
2. the method for microsatellite stability state is detected according to claim 1, it is characterised in that NR-21, BAT-25, BAT-
26th, the nucleotide sequence of NR-24 and MONO-27 is respectively SEQ ID NO:1-5.
3. the method for microsatellite stability state is detected according to claim 1, it is characterised in that 5 quasi- monomorphism monokaryon glycosides
Acid repeat site PCR amplification primer be：SEQ ID NO:8-9、SEQ ID NO:10-11、SEQ ID NO:12-13、SEQ
ID NO:14-15、SEQ ID NO:16-17。
4. according to claim 1 detect microsatellite stability state method, it is characterised in that the Penta D and
The nucleotide sequence of Penta E is respectively SEQ ID NO:6-7.
5. the method for microsatellite stability state is detected according to claim 4, synchronous amplification Penta D and Penta E's
Primer pair is respectively SEQ ID NO:18-19、SEQ ID NO:20-21.
6. the method for microsatellite stability state is detected according to claim 1-5, it is characterised in that the amplimer point
It is not marked by the fluorescein of three kinds of different colours, the combination of three groups of fluorescent markers is respectively：First group of NR-21, BAT-26,
MONO-27；Second group：BAT-25 and Penta D；Third group：NR-24 and Penta E.
7. detecting the method for microsatellite stability state according to claim 6, three kinds of fluoresceins are labeled as FAM, HEX
And ROX, the amplimer in preferably first group repetition site is marked with FAM, the amplimer in second group of repetition site is marked with HEX
The amplimer that note, third group repeat site is marked with ROX.
8. a kind of kit for being used to carry out claim 1-7 the methods, it is characterised in that including following primer pair：SEQ ID
NO:8-9、SEQ ID NO:10-11、SEQ ID NO:12-13、SEQ ID NO:14-15、SEQ ID NO:16-17；And expand
Increase the primer pair SEQ ID NO of Penta D:The primer pair SEQ ID NO of 18-19, extension Penta E:20-21, middle and upper reaches
5 ' ends of primer are marked with fluorescein.
9. kit as claimed in claim 8, it is characterised in that primer mixed liquor, the primer mixed liquor are included in kit
In each primer pair concentration it is as follows：SEQ ID NO:8-9 is 5 μM, SEQ ID NO:10-11 is 3.75 μM, SEQ ID NO:12-13
For 5 μM, SEQ ID NO:14-15 is 25 μM, SEQ ID NO:16-17 is 5 μM, SEQ ID NO:18-19 is 1.25 μM, SEQ
ID NO:20-21 is 10 μM.
10. kit as claimed in claim 8 or 9, it is characterised in that dNTP, 10 × Buffer, DNA are further included in kit
Polymerase.
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