CN109337985A - A kind of composite amplification system and kit of microsatellite instability detection in Gene Mutation - Google Patents
A kind of composite amplification system and kit of microsatellite instability detection in Gene Mutation Download PDFInfo
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Abstract
The invention belongs to field of biotechnology more particularly to a kind of composite amplification systems and kit of microsatellite instability detection in Gene Mutation.The present invention provides a kind of composite amplification system of microsatellite instability detection in Gene Mutation, composite amplification system simultaneously expands mononucleotide microsatellite gene loci and dinucleotide microsatellite gene loci；The mononucleotide microsatellite gene loci includes NR-21, NR-22, NR-24, NR-27, BAT-25, BAT-26 and MONO-27, and the dinucleotide microsatellite gene loci includes one or more of D2S123, D17S250, D18S34 and D5S346.In the present invention, mononucleotide microsatellite locus and dinucleotide microsatellite site are combined and tested and analyzed to MSI, detection accuracy and sensitivity can be effectively improved, can be used for quickly detecting the parting of tumour with guiding treatment.
Description
Technical field
The invention belongs to field of biotechnology more particularly to a kind of compound expansions of microsatellite instability detection in Gene Mutation
Increasing system and kit.This application claims the Chinese patent submitted for 06th for 07 month for 2018, (number of patent application is
201810737101.5) equity, the full content by above-mentioned application is incorporated herein by reference herein.
Background technique
A large number of studies show that, tumour generation differs closely with mismatch repair system, especially in colorectal cancer, stomach in recent years
Cancer, carcinoma of endometrium etc..Due to the mutation and dysfunction of mispairing reparation (mismatch repair, MMR) gene, make gene
The copy error of group cannot repair in time, and constantly accumulation occurs for copy error frequency, so that cell genomic dna sequence be made to occur
Change, or cause certain tumor suppressor genes inactivate or oncogene activation and lead to the abnormal conversion and neoplasm of cell.Detection is wrong
Become one of the method for diagnosing this kind of tumour with repair system.
Being used clinically for the detection method of tumour mismatch repair system, there are mainly two types of:
The first is the abrupt climatic change of mismatch repair gene, and this method, which is predominantly detected, repairs highly relevant gene with mispairing
Such as hMSH2, hMLH1, hMLH6, PMS2 first extract DNA and carry out PCR amplification, then test PCR product, and this method needs
Several genes are detected, cumbersome, detection cycle is long, and testing cost is high.
Second is immunohistochemistry (Immunohistochemistry, IHC), and due to the change of MMR, albumen is produced
Object can occur accordingly to change, and the method detection MMR protein product of immunohistochemistry (IHC) can be used, predominantly detect at present
The isogenic protein product of hMSH2, hMLH1, hMLH6, PMS2.But this method is easy to ignore missense mutation and truncate mutation, and
And inhomogenous phenomenon is dyed due to existing in tumor tissues, sensitivity also will receive influence.
Both the above method is not widely used since there are many disadvantages.
Discovered in recent years, the mutation and dysfunction of mismatch repair gene, prevent the copy error of genome from obtaining and
Shi Xiufu, copy error frequency occur constantly accumulation, can cause tumour, while can also cause genome microsatellite DNA sequence
Column change.I.e. mismatch repair system failure cause it is tumorigenic simultaneously, also can cause shadow in the STR sequence of genome
It rings, therefore can be by detection STR sequence come the case where analyzing mismatch repair system, and then indicates the parting situation of tumour.
MMR is important DNA repair mechanism, accurately can identify and repair and generates in DNA replication dna or regrouping process
Base mispairing, small-scale base deletion or insertion, to Genome stability is maintained, the accuracy of hereditary offspring has important
Effect.DMMR refers to that MMR repair mechanism breaks down, and participates in the gene that MMR is repaired and is mutated, gene function is caused to lack
It falls into, the decline of MMR repair ability or missing.Lynch syndrome is caused since MMR repairs defect.MS, microsatellite, also known as simply
Repetitive sequence (SSR) is some short and duplicate DNA sequence dnas, is generally made of 1-6 nucleotide that tandem sequence repeats arrangement is common
Type is double alkali yl CA/GA or single base A/T etc., is present in the noncoding regions such as introne, and polymorphism is distributed in whole gene group,
Individual difference is big.Microsatellite instability (microsatellite instability, MSI) refers to due to inserting in DNA replication dna
Enter or deletion mutation caused by MS sequence length change the phenomenon that, often caused by MMR functional defect.MSI phenomenon was in quilt in 1993
Jacobs et al. has found for the first time in colorectal cancer, it has now been found that also has related to the generation of other tumours, can be used for tumor classification
Detection.
The frequency being detected according to MSI in tumour can be classified as three classes, and the first kind is stablized for microsatellite
(microsatellite stability, MSS), no apparent MSI occur；Second class is the unstable (low- of microsatellite minuent
Frequency MSI, MSI-L), the MSI frequency of occurrences is low, generally below 30-40%；Third class is that microsatellite is highly unstable
(high-frequency MSI, MSI-H), the MSI frequency of occurrences is high, generally greater than 30-40%.
MSI is often caused by MMR gene mutation and afunction.It therefore, both can be direct in detecting cancer cell when MSI
MSI sequence variation is detected, can also determine whether that MSI occurs by detection MMR gene delection.Generally, detection MMR gene lacks
It loses and uses immunohistochemistry, detection MSI sequence variation uses Molecular tools.It can be straight using immunohistochemistry detection MMR gene delection
The MMR dcc gene for identifying and MSI being caused to occur is connect, still, the MSI of about 5%-11% is not in MMR albumen
Missing, when furthermore the missense mutation of MMR gene causes MMR protein function to be lost, immunohistochemistry antibody test is positive.And it uses and divides
Sub- means detection MSI sequence variation accuracy is high.MSI detection discovery, amplification knot are carried out in the patient of all colorectal cancer histories
The MSI accuracy in detection of rectal cancer patient is high.Nearly 15% accidental colorectal cancer and Lynch syndrome colorectal cancer patients contains
MSI feature can accurately detect the MSI in colorectal cancer by MSI Molecular Detection, to the accuracy in detection of MSI-H patient
You Gao, accuracy is close to 100%, also, MSI detection is simple and direct, and benefit ratio is high.
Lynch syndrome is Familial Occurrence disease, and colorectal cancer patients have MSI feature, and offspring suffers from colorectal cancer year
Age is more early, and KRAS gene mutation can be often accumulated in cancer cell；MMR gene mutation is accumulated in Lynch syndrome patients' reproduction cell,
Including MLH1, MSH2 etc., it can be inherited to offspring.In sporadic MSI colorectal cancer, often it is rich in for MLH1 gene promoter region
DNA methylation inhibits it to express and cause MMR defect, leads to MSI.
PD-1 is immune t-cell surface self suppression receptor, passes through specifically binding partner albumen PD-L1 or PD-L2
Inhibit immune t-cell activity.Many cancer cells express PD-L1 by height, inhibit the anticancer activity of immune t-cell.PD-1 is mono-
Anti-immunity treatment specifically identifies the PD-1 receptor on immune t-cell by monoclonal antibody, hinders the knowledge of cancer cell PD-L1
Not, to activate immune t-cell active, the cancer cell for killing the heterologous antigen with there are many is specifically removed, is realized to cancer
Immunization therapy.It hinders PD-1 to be combined with PD-L1 identification using the monoclonal antibody of specificity, can be used for treating cancer advanced stage
Patient, including melanoma, non-small cell lung cancer and clear-cell carcinoma realize the immunization therapy of PD-1 monoclonal antibody.
DNA mismatch revision points and MSI and hereditary nonpolyposis colorectal cancer (HNPCC) and sporadic Colon and rectum
Cancer, gastric cancer, carcinoma of endometrium etc. occur closely related.The tumour of MMR defect is sensitive to PD-1 blocking treatment, and MSI inspection can be used
Examining system assesses mispairing in tumour cell and repairs state, further to assess whether to tumour cell using anti-PD-1 therapy.
U.S. Food and Drug Administration (FDA) ratifies in May, 2017 using MSI as the Medicine indication of anti-PD-1 therapy, and
The access of the drug pembrolizumab (Keytruda) for MSI-H or dMMR solid tumor clinical treatment has been examined in acceleration.
1997 American National institute of oncology (National Cancer Institute, NCI) hold meeting and promulgate
Bethesda guiding outlines, it is determined that for detecting the reference Panel (NCI panel) of MSI, recommend to apply 5 microsatellite positions
It puts to carry out the detection of colorectal cancer, i.e., mononucleotide repeats site BAT-25, BAT-26 and dinucleotides repeats site
D2S123, D5S346 and D17S250, and definition is made that MSI.Comparison of tumor and matched normal DNA have 2 in 5 sites
It is MSI-H that, which there is repetitive sequence length variation person in a or 2 or more sites, if only one site has length variation person for minuent
The variation of MSI-L, no any site are known as MSS.The research of Suraweera N et al. points out that for MSI-H patient, 5 quasi- single
State property mononucleotide repeats the Panel group (BAT-25, BAT-26, NR21, NR22 and NR24) in site than other microsatellite markers
It is more sensitive.
In NCI in 2002 in second of special meeting, the NCI Panel adopted before amplification carries out to detect MSI
It discusses, since NCI Panel has used 2 mononucleotides to repeat sites and 3 duplicate sites of dinucleotides, and dinucleotides
Duplicate site is in detection there are sensitivity when the tumour of mis-match repair deficient is lower, thus application NCI Panel is swollen to assess
It is had some limitations when tumor satellite unstable state.Nevertheless, dinucleotides site is still considered as its uniqueness
Place, this be also the intention of NCI Panel and be widely used in all the time MSI research in reason.Because of dinucleotides
Site can be less subject to the influence for influencing some Confounding Factors of mononucleotide microsatellite gene loci fidelity.Theoretically, base
Because the fidelity of mononucleotide and dinucleotides repetitive sequence is maintained by the mechanism of MMR and other biological process in group,
Length variation i.e. in simple repeated sequence may also be driven by certain biological processes other than MMR.And dinucleotides is micro-
Satellite gene loci is then less subject to the influence of these biological processes other than MMR, and MSI-L usually only shows dinucleotides position
The variation of point.
MSI detection architecture is established in the quasi- monomorphism site for being used to detect MSI for searching high sensitivity and specificity
It must work, it has also become the hot spot of Recent study MSI detection field, but the exploitation of this product is not easy to, at present
Until only Promega and other several companies there is the product for being able to detect mononucleotide microsatellite gene loci, exploitation is more
Multiple types site is imperative with the MSI detection system for improving sensitivity and accuracy.
Summary of the invention
In view of this, the present invention provides a kind of composite amplification system of microsatellite instability detection in Gene Mutation and examinations
The technical issues of agent box, accuracy and sensitivity for existing microsatellite instability detection in Gene Mutation are up for improving.
The specific technical solution of the present invention is as follows:
A kind of composite amplification system of microsatellite instability detection in Gene Mutation, composite amplification system is simultaneously to monokaryon glycosides
Sour microsatellite gene loci and dinucleotide microsatellite gene loci are expanded；
The mononucleotide microsatellite gene loci include NR-21, NR-22, NR-24, NR-27, BAT-25, BAT-26 and
MONO-27, the dinucleotide microsatellite gene loci include one in D2S123, D17S250, D18S34 and D5S346 or
It is multiple.
In the present invention, the composite amplification system of microsatellite instability detection in Gene Mutation is simultaneously to mononucleotide microsatellite
Gene loci and dinucleotide microsatellite gene loci are expanded, and mononucleotide microsatellite gene loci includes NR-21, NR-
22, NR-24, NR-27, BAT-25, BAT-26 and MONO-27, dinucleotide microsatellite gene loci include D2S123,
One or more of D17S250, D18S34 and D5S346, by mononucleotide microsatellite locus and dinucleotide microsatellite site
It combines and MSI is tested and analyzed, detection accuracy and sensitivity can be effectively improved, can quickly detect the parting of tumour
With guiding treatment.
Preferably, the composite amplification system is also simultaneously to tetranucleotide microsatellite gene loci and sex determining gene position
Point is expanded.
Preferably, the tetranucleotide microsatellite gene loci includes D13S317 and D7S820；
The sex determining gene site includes Amelogenin (AMEL).
In the present invention, composite amplification system is also simultaneously to tetranucleotide microsatellite gene loci D13S317, D7S820 and property
Not Jue Ding gene loci AMEL expanded, sample can be assisted to identify, prevent to malfunction when experimental implementation because obscuring sample
Accidentally result.
Preferably, the composite amplification system includes:
For expanding the primer pair of the specific region of following 7 mononucleotides microsatellite gene loci:
The sequence of NR-21 be the specific region of SEQ ID NO:1, NR-22 sequence be the given zone of SEQ ID NO:2
Domain, NR-24 sequence be the specific region of SEQ ID NO:3, NR-27 sequence be the specific region of SEQ ID NO:4, BAT-
25 sequence be the specific region of SEQ ID NO:5, BAT-26 sequence be specific region and the MONO-27 of SEQ ID NO:6
Sequence be SEQ ID NO:7 specific region；
For expanding the primer pair for being selected from the specific region of one or more of following dinucleotide microsatellite gene loci:
The sequence of D2S123 be the specific region of SEQ ID NO:8, D17S250 sequence be the specific of SEQ ID NO:9
Region, D18S34 the given zone that sequence is the specific region of SEQ ID NO:10 or the sequence of D5S346 is SEQ ID NO:11
Domain.
Preferably, the composite amplification system further include:
For expanding the primer pair of the specific region of following 2 tetranucleotides microsatellite gene loci: the sequence of D13S317
The specific region for being SEQ ID NO:13 for the specific region of SEQ ID NO:12 and the sequence of D7S820；
And the sequence for expanding sex determining gene site AMEL is the primer of the specific region of SEQ ID NO:14
It is right.
Preferably, a continuous bases of 18-26 for the position 1-123 that the primer pair for expanding NR-21 is selected from sequence SEQ ID NO:1
The primer of composition, and the continuous bases of 18-26 of complementary series of the position 140-200 selected from sequence SEQ ID NO:1 are constituted
Primer combination；
What 18-26 continuous bases of the position 1-130 that the primer pair for expanding NR-22 is selected from sequence SEQ ID NO:2 were constituted
Primer, and the position 150-250 selected from sequence SEQ ID NO:2 complementary series the primers that constitute of the continuous base of 18-26
Combination；
What 18-26 continuous bases of the position 1-120 that the primer pair for expanding NR-24 is selected from sequence SEQ ID NO:3 were constituted
Primer, and the position 140-250 selected from sequence SEQ ID NO:3 complementary series the primers that constitute of the continuous base of 18-26
Combination；
What 18-26 continuous bases of the position 1-138 that the primer pair for expanding NR-27 is selected from sequence SEQ ID NO:4 were constituted
Primer, and the position 158-260 selected from sequence SEQ ID NO:4 complementary series the primers that constitute of the continuous base of 18-25
Combination；
What 18-26 continuous bases of the position 1-132 that the primer pair for expanding BAT-25 is selected from sequence SEQ ID NO:5 were constituted
Primer, and the position 152-300 selected from sequence SEQ ID NO:5 complementary series the primers that constitute of the continuous base of 18-26
Combination；
What 18-26 continuous bases of the position 1-117 that the primer pair for expanding BAT-26 is selected from sequence SEQ ID NO:6 were constituted
Primer, and the position 145-270 selected from sequence SEQ ID NO:6 complementary series the primers that constitute of the continuous base of 18-26
Combination；
18-26 continuous bases of the position 1-116 that the primer pair for expanding MONO-27 is selected from sequence SEQ ID NO:7 are constituted
Primer, and the position 143-290 selected from sequence SEQ ID NO:7 complementary series drawing of constituting of the continuous bases of 18-26
The combination of object；
What 18-26 continuous bases of the position 1-132 that the primer pair for expanding D2S123 is selected from sequence SEQ ID NO:8 were constituted
Primer, and the position 174-310 selected from sequence SEQ ID NO:8 complementary series the primers that constitute of the continuous base of 18-26
Combination；
18-26 continuous bases of the position 1-104 that the primer pair for expanding D17S250 is selected from sequence SEQ ID NO:9 are constituted
Primer, and the position 141-280 selected from sequence SEQ ID NO:9 complementary series drawing of constituting of the continuous bases of 18-26
The combination of object；
18-26 continuous bases of the position 1-128 that the primer pair for expanding D18S34 is selected from sequence SEQ ID NO:10 are constituted
Primer, and the position 185-223 selected from sequence SEQ ID NO:10 complementary series drawing of constituting of the continuous bases of 18-26
The combination of object；
18-26 continuous bases of the position 1-154 that the primer pair for expanding D5S346 is selected from sequence SEQ ID NO:11 are constituted
Primer, and the position 193-300 selected from sequence SEQ ID NO:11 complementary series drawing of constituting of the continuous bases of 18-25
The combination of object；
18-26 continuous base structures of the position 45-228 that the primer pair for expanding D13S317 is selected from sequence SEQ ID NO:12
At primer, and the 18-26 continuous bases of complementary series of the position 268-480 selected from sequence SEQ ID NO:12 constitute
The combination of primer；
18-26 continuous bases of the position 45-216 that the primer pair for expanding D7S820 is selected from sequence SEQ ID NO:13 are constituted
Primer, and the position 69-460 selected from sequence SEQ ID NO:13 complementary series drawing of constituting of the continuous bases of 18-26
The combination of object；
What 18-26 continuous bases of the position 1-140 that the primer pair for expanding AMEL is selected from sequence SEQ ID NO:14 were constituted
Primer, and the position 145-310 selected from sequence SEQ ID NO:14 complementary series the primers that constitute of the continuous base of 18-25
Combination.
Preferably, in the primer pair for expanding NR-21, forward primer is selected from the primer that sequence is SEQ ID No.15-17, instead
The primer that sequence is SEQ ID No.18-20 is selected to primer；
In the primer pair for expanding NR-22, forward primer is selected from the primer that sequence is SEQ ID No.21-23, reverse primer
The primer for being SEQ ID No.24-26 selected from sequence；
In the primer pair for expanding NR-24, forward primer is selected from the primer that sequence is SEQ ID No.27-29, reverse primer
The primer for being SEQ ID No.30-32 selected from sequence；
In the primer pair for expanding NR-27, forward primer is selected from the primer that sequence is SEQ ID No.33-35, reverse primer
The primer for being SEQ ID No.36-38 selected from sequence；
In the primer pair for expanding BAT-25, forward primer is selected from the primer that sequence is SEQ ID No.39-41, reverse primer
The primer for being SEQ ID No.42-44 selected from sequence；
In the primer pair for expanding BAT-26, forward primer is selected from the primer that sequence is SEQ ID No.45-47, reverse primer
The primer for being SEQ ID No.48-50 selected from sequence；
In the primer pair for expanding MONO-27, forward primer is selected from the primer that sequence is SEQ ID No.51-53, reversely draws
Object is selected from the primer that sequence is SEQ ID No.54-56；
In the primer pair for expanding D2S123, forward primer is selected from the primer that sequence is SEQ ID No.57-59, reverse primer
The primer for being SEQ ID No.60-62 selected from sequence；
In the primer pair for expanding D17S250, forward primer is selected from the primer that sequence is SEQ ID No.63-65, reversely draws
Object is selected from the primer that sequence is SEQ ID No.66-68；
In the primer pair for expanding D18S34, forward primer is selected from the primer that sequence is SEQ ID No.69-71, reverse primer
The primer for being SEQ ID No.72-73 selected from sequence；
In the primer pair for expanding D5S346, forward primer is selected from the primer that sequence is SEQ ID No.74-76, reverse primer
The primer for being SEQ ID No.77-79 selected from sequence；
In the primer pair for expanding D13S317, forward primer is selected from the primer that sequence is SEQ ID No.80-81, reversely draws
Object is selected from the primer that sequence is SEQ ID No.82-83；
In the primer pair for expanding D7S820, forward primer is selected from the primer that sequence is SEQ ID No.84-85, reverse primer
The primer for being SEQ ID No.86-87 selected from sequence；
In the primer pair for expanding AMEL, forward primer is selected from the primer that sequence is SEQ ID No.88-89, reverse primer choosing
Author's preface is classified as the primer of SEQ ID No.90-91.
In the present invention, the primer pair for expanding 7 mononucleotide microsatellite gene locis can produce in the extension stage of PCR
The segment of raw uniform length, the primer pair for expanding dinucleotide microsatellite gene loci can generate in the extension stage of PCR
The segment of uniform length, for expanding the primer pair of tetranucleotide microsatellite gene loci and sex determining gene site in PCR
The extension stage can generate the segment of uniform length.
In the present invention, composite amplification system include for expand dinucleotide microsatellite gene loci D2S123,
The primer pair of one or more of D17S250, D18S34 and D5S346 expand the type of dinucleotide microsatellite gene loci
It is determined with quantity according to different tissues and different tumours, it should be noted that dinucleotide microsatellite gene loci is unlimited
In tetra- kinds of D2S123, D17S250, D18S34 and D5S346.
Preferably, in the primer pair, 5 ' ends of a primer have fluorescent marker.
Preferably, the primer pair is divided into three groups, is respectively provided with the fluorescent marker of three kinds of different colours: first group of primer pair
It is amplification NR-21, NR-24, D13S317, D5S346, D17S250；Second group of primer pair is amplification NR-27, BAT-25, MONO-
27,D18S34,D2S123；Third group primer pair is amplification BAT-26, NR-22, D7S820, AMEL.
In the present invention, fluorescent marker is selected from FAM, fluorescein, HEX, VIC, JOE, TMR or NED.
Preferably, the fluorescent marker of first group of primer pair is FAM or fluorescein, and the fluorescent marker of second group of primer pair is
HEX, VIC or JOE, the fluorescent marker of third group primer pair are TMR or NED.
The present invention also provides composite amplification systems described in above-mentioned technical proposal in tumour cell and the MSI base of tissue typing
Because of the application in abrupt climatic change.
Based on composite amplification system multiple fluorescence PCR technology of the present invention, it is applied to colorectal cancer, carcinoma of endometrium, stomach
The MSI detection in Gene Mutation of the tumour cells such as cancer and liver cancer and tissue typing, by mononucleotide microsatellite locus and dinucleotides
Microsatellite locus, which combines, tests and analyzes MSI, and testing result is more accurate, can effectively improve detection accuracy and spirit
Sensitivity；Sex Determination site AMEL is increased, experimental implementation person can be further prevented to obscure sample and obtain error result, such as
Patient is male, and the gender site result that detected is women, prompts to be likely to have taken wrong sample during operation experiments
Sheet or error flag；Increase by 2 tetranucleotide sites (D13S317, D7S820), is distinguished in combination with Sex Determination site AMEL
The sample of Different Individual further avoids obscuring between sample and obtaining error result.
The present invention also provides a kind of method of microsatellite instability detection, the by adopting the above technical scheme compound expansions
Increasing system carries out PCR amplification to measuring samples.
Preferably, further includes: Capillary Electrophoresis is carried out to the PCR product that PCR amplification obtains.
The present invention also provides a kind of kits for microsatellite instability detection in Gene Mutation, including above-mentioned technical side
Composite amplification system described in case.Kit of the present invention is easy to operate, can produce in batches, and use condition can be used with medelling
Process can be all using automation equipment, independent of the operating experience of people, when detection, and PCR amplification 2 hours, Capillary Electrophoresis
1.0~1.5 hours, data were analyzed about 1 hour, and required time is short, and entire detection time only needs 4~5 hours, convenient for extensive
It promotes.
In the present invention, the total volume of composite amplification system is 10 μ l, wherein 2 μ l of Multiplex PCR Mix (5x), primer
2 μ l of mixture (5x), 4 μ l of nuclease free deionized water, the template quantity (0.5ng/ μ l~5ng/ μ l) of addition are 2 μ l.It needs
Bright, the effect that the total volume of composite amplification system is 20 μ l and 10 μ l is suitable.
Primer pair concentration in primer mixture is respectively as follows:
| Primer pair | Concentration/μM |
| NR-21 | 0.04~0.2 |
| NR-22 | 0.04~0.2 |
| NR-24 | 0.04~0.2 |
| NR-27 | 0.04~0.2 |
| BAT-25 | 0.04~0.2 |
| BAT-26 | 0.04~0.2 |
| MONO-27 | 0.04~0.2 |
| D2S123 | 0.04~0.2 |
| D17S250 | 0.04~0.2 |
| D18S34 | 0.04~0.2 |
| D5S346 | 0.04~0.2 |
| D13S317 | 0.04~0.2 |
| D7S820 | 0.04~0.2 |
| AMEL | 0.04~0.2 |
In the present invention, the amplification condition of composite amplification system are as follows: 95 DEG C of denaturation, 1~10min；28~32 recycle 94 DEG C
10~30sec, 58 DEG C of 10~30sec, 72 DEG C of 20~60sec；Last 60 DEG C of 20~40min.
In the present invention, for the Tm value of each primer pair in the range of (60 ± 2) DEG C, amplification efficiency is similar and each primer pair
Amplified production size differs 15bp or more, does not generate non-specific product or dimer, and all under same amplification condition draws
Object bright and single purpose band can occur to equal.
Taq enzyme is the important component of amplification, and a variety of Taq enzymes can be applied to composite amplification system of the present invention.Present invention examination
Agent box uses the HsTaq thermal starting enzyme of our company.
In kit of the present invention, Mg2+Concentration be preferably 1.0mM~2.0mM, annealing temperature is preferably 58 DEG C~60 DEG C,
When 60 DEG C of extensions, extension of time 20min~60min can make Taq enzyme add A complete.
In conclusion the present invention provides a kind of composite amplification system of microsatellite instability detection in Gene Mutation, it is multiple
Amplification system is closed simultaneously to expand mononucleotide microsatellite gene loci and dinucleotide microsatellite gene loci；The list
Nucleotide microsatellite gene loci includes NR-21, NR-22, NR-24, NR-27, BAT-25, BAT-26 and MONO-27, and described two
Nucleotide microsatellite gene loci includes one or more of D2S123, D17S250, D18S34 and D5S346.In the present invention,
The composite amplification system of microsatellite instability detection in Gene Mutation is simultaneously to mononucleotide microsatellite gene loci and dinucleotide
Sour microsatellite gene loci is expanded, mononucleotide microsatellite gene loci include NR-21, NR-22, NR-24, NR-27,
BAT-25, BAT-26 and MONO-27, dinucleotide microsatellite gene loci include D2S123, D17S250, D18S34 and
Mononucleotide microsatellite locus and dinucleotide microsatellite site are combined and are carried out to MSI by one or more of D5S346
It tests and analyzes, detection sensitivity is 1~2ng, and accuracy can effectively improve detection accuracy and sensitivity close to 100%, can
For detecting the parting of tumour quickly with guiding treatment.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is to be obtained in the embodiment of the present invention 1 using composite amplification system of the present invention detection colon cancer tissue and normal tissue
The parting map arrived；
Fig. 2 is to be obtained in the embodiment of the present invention 2 using composite amplification system of the present invention detection colon cancer tissue and normal tissue
The parting map arrived；
Fig. 3 is the result figure for detecting cancer beside organism and cancerous tissue in the embodiment of the present invention 3 using immunohistochemistry；
Fig. 4 is to detect the parting map that cancer beside organism obtains using composite amplification system of the present invention in the embodiment of the present invention 3；
Fig. 5 is to detect the parting map that cancerous tissue obtains using composite amplification system of the present invention in the embodiment of the present invention 3；
Fig. 6 is the result figure for detecting cancer beside organism and cancerous tissue in the embodiment of the present invention 4 using immunohistochemistry；
Fig. 7 is to detect the parting map that cancer beside organism obtains using composite amplification system of the present invention in the embodiment of the present invention 4；
Fig. 8 is to detect the parting map that cancerous tissue obtains using composite amplification system of the present invention in the embodiment of the present invention 4.
Specific embodiment
The present invention provides the composite amplification systems and kit of a kind of microsatellite instability detection in Gene Mutation, are used for
The technical issues of accuracy and sensitivity of existing microsatellite instability detection in Gene Mutation are up for improving.
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Mode is only some embodiments of the invention, rather than whole embodiments.Based on the embodiment in the present invention, originally
Field those of ordinary skill every other embodiment obtained without making creative work, belongs to this hair
The range of bright protection.
Embodiment 1
The present embodiment carries out colon cancer tissue and normal tissue microsatellite instability using composite amplification system of the present invention
Detection, colon cancer tissue and normal tissue are first sample, and detecting step is as follows:
1.DNA is extracted
Using tissue DNA extracts kit (OMEGA) respectively to the cancerous tissue of patient and cancer beside organism's (normal tissue) into
Row extracting genome DNA, is carried out according to kit specification, fixed using ultraviolet specrophotometer after extracting genome DNA
Amount, and final concentration of 1ng/ μ l is diluted, the PCR reaction template for next step.
2.PCR
2.1 by Multiplex PCR Mix (5x) 2 site μ l, MSI primer mixture (5x) 2 μ l, nuclease free deionization
4 μ l of water,
The template quantity (0.5ng/ μ l~5ng/ μ l) of addition is 2 μ l, and total reaction volume is 10 μ l.
2.2PCR response procedures:
Step 1: 95 DEG C are denaturalized 10 minutes, step 2: 94 DEG C are denaturalized 30 seconds, step 3: 58 DEG C are annealed 30 seconds, step 4: 72 DEG C
Extend 40 seconds, repeat step 2 and recycled to step 4 32, step 5: 60 DEG C extend 30 seconds.Pcr amplification product is set after end of run
It is saved in 4 DEG C of refrigerator.
3. capillary electrophoresis detection
3.1 take pcr amplification product 1.0-2.0 μ l point sample obtained in the previous step on 1.5-2.0 Ago-Gel, and 140V is electric
Piezoelectricity is swum 20 minutes, and the observation under gel imager or ultraviolet lamp at 60-300bp as a result, if bright ladder strip band occur
Then be available on the machine detection.
3.2 internal standards (ROX500) provided using kit and formamide are mixed by 10:1000, and 9.5-9.0 μ l is taken to mix
Object is added in 96 orifice plates, then takes pcr amplification product 0.5-1.0 μ l, is mixed and is put into ABI 3100, ABI 3130 after of short duration centrifugation
Series, ABI 3500 are waited on testers, prepare detection.
3.3 open ABI 3100,3130 series of ABI, and ABI 3500 waits the data acquisition software of testers, machine on editor
The plate mark of detection imports detection program.
3.4 click operation, that is, start to detect.Different instrument detection times are different.
After 3.5 detections, by data copy to CD.
4. data are analyzed
4.1 importing initial data find sample in the File menu selection Add sample to project of homepage
File, filesselected folder, clicks add to list, clicks add, and sample file is shown in Project window.
4.2 selection analysis parameters define analysis method, panel and size standard.Browse sample electrophoresis
Initial data, name the same filename, under " sample " menu select " raw data ".Moving tracing line, makes cursor
Be parked at (before first red internal standard peak) on the right side of primer peak, the numerical value shown using in window lower left corner X-axis at this time as
Analysis mehtod analyzes the starting point in parameter.
4.3 click green analysis button, save project dialog box occur, save after name, software starts to process
Data, the lower left corner shows analysis completed after the completion of analysis.
4.4 data analyzed using GeneMapper software simultaneously generate map.
As a result referring to Fig. 1, in the embodiment of the present invention 1 using composite amplification system of the present invention detect colon cancer tissue with
The parting map that normal tissue obtains.The result shows that microsatellite instability detection is carried out using composite amplification system of the present invention,
Colon cancer carefully organizes the allele site occurred offset to be not present in normal tissue (shown in red arrow), shows colon
Cancerous tissue produces microsatellite instability (MSI).
Embodiment 2
The present embodiment carries out colon cancer tissue and normal tissue microsatellite instability using composite amplification system of the present invention
Detection, colon cancer tissue and normal tissue are second batch sample, and detecting step is the same as embodiment 1.
As a result referring to Fig. 2, in the embodiment of the present invention 2 using composite amplification system of the present invention detect colon cancer tissue with
The parting map that normal tissue obtains.The result shows that microsatellite instability detection is carried out using composite amplification system of the present invention,
Colon cancer carefully organizes have 2 allele site offset (shown in red arrow) occur in 8 sites, shows the colon cancer group
It knits and produces microsatellite instability (MSI), the offset in the two sites may miss in 5 site primers because can't detect
It examines, illustrates that composite amplification system of the present invention improves the sensitivity of microsatellite instability detection.
Embodiment 3
Immunohistochemistry is respectively adopted in the present embodiment and composite amplification system of the present invention examines cancer beside organism and cancerous tissue
It surveys, composite amplification system of the present invention carries out colon cancer tissue and the detection of normal tissue microsatellite instability, cancer beside organism and cancer
It organizes to be third lot sample sheet, in the present embodiment, using the detecting step of composite amplification system of the present invention with embodiment 1.
As a result please refer to Fig. 3 to Fig. 5, Fig. 3 shows to detect using immunohistochemistry, in cancer beside organism MLH1, PMS2, MSH2 and
MSH6 is the positive, and MLH1 and PMS2 is feminine gender in cancerous tissue, and MSH2 and MSH6 is the positive in cancerous tissue；Fig. 4 and Fig. 5 shows to use
Composite amplification system of the present invention carries out microsatellite instability detection, and cancer carefully organizes the allele site occurred offset (red
Shown in arrow) it is not present in cancer beside organism, show that colon cancer tissue produces microsatellite instability (MSI), the results showed that
It is consistent with the result using composite amplification system of the present invention detection using immunohistochemistry detection.
Embodiment 4
Immunohistochemistry is respectively adopted in the present embodiment and composite amplification system of the present invention examines cancer beside organism and cancerous tissue
It surveys, composite amplification system of the present invention carries out colon cancer tissue and the detection of normal tissue microsatellite instability, cancer beside organism and cancer
It organizes to be third lot sample sheet, in the present embodiment, using the detecting step of composite amplification system of the present invention with embodiment 1.
As a result please refer to Fig. 6 to Fig. 8, Fig. 6 shows to detect using immunohistochemistry, in cancer beside organism MLH1, PMS2, MSH2 and
MSH6 is the positive, and MLH1, PMS2, MSH2 and MSH6 are the positive in cancerous tissue；Fig. 7 and Fig. 8 shows using composite amplification of the present invention
System carries out microsatellite instability detection, and cancer carefully organizes no allele site to deviate, shows that cancerous tissue does not produce
Microsatellite instability (MSI), the results showed that the result for being detected using immunohistochemistry and being detected using composite amplification system of the present invention
Unanimously.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Guangzhou FulenGen Co., Ltd.
<120>a kind of composite amplification system and kit of microsatellite instability detection in Gene Mutation
<150> 201810737101.5
<151> 2018-07-06
<160> 91
<170> SIPOSequenceListing 1.0
<210> 1
<211> 200
<212> DNA
<213> Homo sapiens
<400> 1
aggaaccact gctactctct aaaaaaggca agcagataaa agagaacacg aaaaatattc 60
ctactccgca ttcacacttt ctggtcactc gcgtttacaa acaagaaaag tgttgctaaa 120
aaaaaaaaaa aaaaaaaaaa aggccagggg agacatacat ttaaatataa aaatagaact 180
gtgccagcga ctccggctgg 200
<210> 2
<211> 250
<212> DNA
<213> Homo sapiens
<400> 2
taaatattaa actgacatct ttatgttgca ggtaaaggac ctggataatc gaggcttgtc 60
aaggacataa atgtcacgtc cagctctgat atgcttcgca ctgagcacat cacatttagg 120
acgttgaaga tttttttttt tttttttttt taatatgcag tttgtaagaa caaaactgga 180
tggcatcaga attgtctgga agttttgtct tgggcagtat gggctgggcc aaatgaaatg 240
atttttataa 250
<210> 3
<211> 250
<212> DNA
<213> Homo sapiens
<400> 3
ctccctattt agtgaaaaat tatctgaata tttaaggtct gccttaacgt gatccccatt 60
gctgaatttt acctcctgac tccaaaaact cttctcttcc ctgggcccag tccttttttt 120
tttttttttt ttttttttgt gagacagagt ctcactctgt cacccaggtt ggaatgcaat 180
ggcacaatct ccgctcactg caagctccgc ctcccgggtt cacgccattc tcctgcctca 240
gcctcccgaa 250
<210> 4
<211> 260
<212> DNA
<213> Homo sapiens
<400> 4
cctttgaggg aaataattgt tggtaatgag atgtgatgtt tctcctgcca cctggaaaca 60
aagcattgaa gtctgcagtt gaaaagccca acgtctgtga gatccaggaa accatgcttg 120
caaaccactg gtaaaaaaaa aaaaaaaaaa aaaaaaaagc cacagtgact tgcttattgg 180
tcattgctag tattatcgac tcagaacctc tttactaatg gctagtaaat cataattgag 240
aaattctgaa ttttgacaag 260
<210> 5
<211> 300
<212> DNA
<213> Homo sapiens
<400> 5
gccatcatgg aggatgacga gttggcccta gacttagaag acttgctgag cttttcttac 60
caggtggcaa agggcatggc tttcctcgcc tccaagaatg taagtgggag tgattctcta 120
aagagttttg tgttttgttt ttttgatttt tttttttttt tttttttttt tgagaacaga 180
gcattttaga gccatagtta aaatgcagaa tgtcattttg aagtgtggta accaaaagca 240
gaggaaattt agtttcttca tgttccaact gctgtctctt tggaattcct gttctaattt 300
<210> 6
<211> 270
<212> DNA
<213> Homo sapiens
<400> 6
tttagaactc ttatcagatg attccaactt tggacagttt gaactgacta cttttgactt 60
cagccagtat atgaaattgg atattgcagc agtcagagcc cttaaccttt ttcaggtaaa 120
aaaaaaaaaa aaaaaaaaaa aaaagggtta aaaatgttga atggttaaaa aatgttttca 180
ttgacatata ctgaagaagc ttataaagga gctaaaatat tttgaaatat tattatactt 240
ggattagata actagcttta aatggctgta 270
<210> 7
<211> 290
<212> DNA
<213> Homo sapiens
<400> 7
agctactcgg gagactgagg caggagaatg gcatgaaccc gggaggcgga gcttgcagtg 60
agcagagatc gttccactgc actccagcct gagtgacaga gctagactct gtctcaaaaa 120
aaaaaaaaaa aaaaaaaaaa aagagtgaag cccaaaataa atctgaagcc atccaggatc 180
tgcaattcat aggtggttca cataacaatg aaattgaagc atacagctga tataaaaatt 240
tgttttaatt tctaatatgg taaataccaa tagctataaa ttatataaat 290
<210> 8
<211> 310
<212> DNA
<213> Homo sapiens
<400> 8
aacatcatac tcaataatga atgaccaaaa gcatttctct tatgataatg gacaaaaaca 60
ggatgcctgc ctttaacact gctattcaac attgctggaa gttctggcca gagaaattag 120
acacagtgat acacacacac acacacacac acacacacac acacacacac acatatttta 180
tagatagata gatggtatcc aagtcagaaa gggagaagta aaactatccc tattgtagat 240
gacacagtcc cataggtgga aagtcccctc caattcaaaa gagctcctaa ggctaaaaaa 300
tgaattcagc 310
<210> 9
<211> 280
<212> DNA
<213> Homo sapiens
<400> 9
aactcctgac ctcaggtgat ccacccacct cagcctccga aagtgctggg attacaggca 60
tgagccactc agccggccat atatatattt aaaccatttg aaagtgtgtg tgtgtgtgtg 120
tgtgtgtgtg tgtgtgtgtg tttgaaacca tttgaaagtt tatgtatgtg tatatatata 180
tataaacaca cacatatttt tattgtctat ttgattcttc ctttttatgc ttactttttt 240
cttctctctt ctttttgatc aattactttt catcacttca 280
<210> 10
<211> 223
<212> DNA
<213> Homo sapiens
<400> 10
agctcagtat gaaagagaag cagcaaaatt accatttcat caaatgcaaa aaaatcagtg 60
caattctttt aatgaaactc ccaagacttc agaaaattct ctctggctat ttttcattat 120
ttctttagac acacacacac acacacacac acacacacac acacacacac acacacacac 180
acacattctt gccaggaaca tgagtgaggg ttgaagaaga gct 223
<210> 11
<211> 300
<212> DNA
<213> Homo sapiens
<400> 11
aagaagacta aatcaacatg tttctccgct ttgaagataa aaccagaaat ggtttccatt 60
gtagcatctt gacaatagac aaatatgtaa agtttatagc agataagaca gtattactag 120
tttttcaggg aattgagagt tacaggttac tctgtgtgtg tgtgtgtgtg tgtgtgtgtg 180
tgtgtgtgtg tgtaaatttc ccgatttatc actagagtga gtaactaact aactaactgc 240
tttataaagc tatcctggta ttcatatgcc atatactacg gaaacaacca ggccaatctc 300
<210> 12
<211> 480
<212> DNA
<213> Homo sapiens
<400> 12
gtattgcaag cacttagtta catttctagc atataacaca tgatcaataa atattttgac 60
atgaacaaat ggtaattctg cctacagcca atgtgaatat tgggatgggt tgctggacat 120
ggtatcacag aagtctggga tgtggaggag agttcatttc tttagtgggc atccgtgact 180
ctctggactc tgacccatct aacgcctatc tgtatttaca aatacattat ctatctatct 240
atctatctat ctatctatct atctatctat caatcaatca tctatctatc tttctgtctg 300
tctttttggg ctgcctatgg ctcaacccaa gttgaaggag gagatttgac caacaattca 360
agctctctga atatgttttg aaaataatgt atattaatga atgtacaaat ttccccactt 420
gtactttcag actgttatct gtgagttaaa actcctccac tctttttcct acccaaataa 480
<210> 13
<211> 460
<212> DNA
<213> Homo sapiens
<400> 13
actgcaacct ccgcttcttg ggtcaagtgg ttctcctgcc ccagcctcct gagtagctgg 60
gactacaggc atgtgctact gcatccagct aatttttgta ttttttttag agacggggtt 120
tcaccatgtt ggtcaggctg actatggagt tattttaagg ttaatatata taaagggtat 180
gatagaacac ttgtcatagt ttagaacgaa ctaacgatag atagatagat agatagatag 240
atagatagat agatagatag atagatagac agattgatag ttttttttaa tctcactaaa 300
tagtctatag taaacattta attaccaata tttggtgcaa ttctgtcaat gaggataaat 360
gtggaatcgt tataattctt aagaatatat attccctctg agtttttgat acctcagatt 420
ttaagacctc acaattatct cataaggctt aaaatcaatc 460
<210> 14
<211> 310
<212> DNA
<213> Homo sapiens
<400> 14
gctaccacct catcctgggc accctggtta tatcaacttc agctatgagg taatttttct 60
ctttactaat tttgaccatt gtttgcgtta acaatgccct gggctctgta aagaatagtg 120
tgttgattct ttatcccaga tgtttctcaa gtggtcctga ttttacagtt cctaccacca 180
gcttcccagt ttaagctctg atggttggcc tcaagcctgt gtcgtcccag cagcctcccg 240
cctggccact ctgactcagt ctgtcctcct aaatatggcc gtaagcttac ccatcatgaa 300
ccactactca 310
<210> 15
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ctactccgca ttcacacttt ct 22
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gtcactcgcg tttacaaaca 20
<210> 17
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
cacgaaaaat attcctactc cgca 24
<210> 18
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
tgtcgctggc acagttctat t 21
<210> 19
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
ctatttttat atttaaatgt atgtct 26
<210> 20
<211> 0
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
<210> 21
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ggtaaaggac ctggataatc ga 22
<210> 22
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
atactgccca agacaaaact tcca 24
<210> 23
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
gttttgttct tacaaactgc atat 24
<210> 24
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
tgctctgata tgcttcgcac t 21
<210> 25
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
gagcacatca catttaggac gtt 23
<210> 26
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
catttcattt ggcccagccc ata 23
<210> 27
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
aattatctga atatttaagg tct 23
<210> 28
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
gtgccattgc attccaacct 20
<210> 29
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
gactccaaaa actcttctct tccctg 26
<210> 30
<211> 0
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
<210> 31
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
gggtgacaga gtgagactct gt 22
<210> 32
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
gcggagcttg cagtgagcgg ag 22
<210> 33
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
gtctgcagtt gaaaagccca acgt 24
<210> 34
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
gcaatgacca ataagcaagt ca 22
<210> 35
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
tccaggaaac catgcttgca aac 23
<210> 36
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
tgtgagatcc aggaaaccat gct 23
<210> 37
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
gaggttctga gtcgataata cta 23
<210> 38
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
ttatgattta ctagccatta gtaaa 25
<210> 39
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
agggcatggc tttcctcgcc tcc 23
<210> 40
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
acacttcaaa atgacattct gcat 24
<210> 41
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
ctatggctct aaaatgctct gtt 23
<210> 42
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
tggagtgatt ctctaaagag ttttgtgt 28
<210> 43
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
aactaaattt cctctgcttt tggt 24
<210> 44
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
caagaatgta agtgggagtg at 22
<210> 45
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
gatattgcag cagtcagagc 20
<210> 46
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
tgaaaacatt ttttaaccat tcaaca 26
<210> 47
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
aagcttcttc agtatatgtc aatga 25
<210> 48
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
gccagtatat gaaattggat attgca 26
<210> 49
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
gcagcagtca gagcccttaa cct 23
<210> 50
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
gcagtcagag cccttaacct t 21
<210> 51
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
gcggagcttg cagtgagcag a 21
<210> 52
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
tgcagagatc gttccactgc a 21
<210> 53
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
gttccactgc actccagcct 20
<210> 54
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
gtcattgtta tgtgaaccac ctatga 26
<210> 55
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
gcttcaattt cattgttatg tgaa 24
<210> 56
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
gttatgtgaa ccacctatga attgca 26
<210> 57
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
actgctattc aacattgctg gaa 23
<210> 58
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
ggactttcca cctatgggac 20
<210> 59
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
ttctggccag agaaattaga ca 22
<210> 60
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
aaacaggatg cctgccttta 20
<210> 61
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
gactgtgtca tctacaatag ggatagt 27
<210> 62
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
ccgcaacttt gctgaattca t 21
<210> 63
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
agccactcag ccggccatat atata 25
<210> 64
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
aaagtgctgg gattacaggc at 22
<210> 65
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
ccacccacct cagcctccga aa 22
<210> 66
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
ggaagaatca aatagacaat 20
<210> 67
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
atttagcttc caaactagta gag 23
<210> 68
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
cagccactaa aagatgagaa 20
<210> 69
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
ttaatgaaac tcccaagact tc 22
<210> 70
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
gctcttcttc aaccctcact ca 22
<210> 71
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
tcactcatgt tcctggcaag aa 22
<210> 72
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
agctcagtat gaaagagaag ca 22
<210> 73
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
cagtatgaaa gagaagcagc a 21
<210> 74
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
gtttccattg tagcatcttg aca 23
<210> 75
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
ccgctttgaa gataaaacca gaa 23
<210> 76
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
agcagttagt tagttagtta ct 22
<210> 77
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
actcactcta gtgataaatc g 21
<210> 78
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
cagggaattg agagttacag gtta 24
<210> 79
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 79
tggcatatga ataccaggat agct 24
<210> 80
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 80
atccgtgact ctctggactc tga 23
<210> 81
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 81
ggcagcccaa aaagacaga 19
<210> 82
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 82
tgagttcatt tctttagtgg gcat 24
<210> 83
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 83
gcccaaaaag acagacagaa aga 23
<210> 84
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 84
tataaagggt atgatagaac actt 24
<210> 85
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 85
gattccacat ttatcctcat tgac 24
<210> 86
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 86
ttgtcatagt ttagaacgaa ctaacga 27
<210> 87
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 87
attgacagaa ttgcaccaaa tatt 24
<210> 88
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 88
aagaatagtg tgttgattct tt 22
<210> 89
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 89
ggaagctggt ggtaggaact gta 23
<210> 90
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 90
ccctgggctc tgtaaagaa 19
<210> 91
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 91
ggccaaccat cagagcttaa act 23
Claims (10)
1. a kind of composite amplification system of microsatellite instability detection in Gene Mutation, which is characterized in that composite amplification system is same
When mononucleotide microsatellite gene loci and dinucleotide microsatellite gene loci are expanded；
The mononucleotide microsatellite gene loci include NR-21, NR-22, NR-24, NR-27, BAT-25, BAT-26 and
MONO-27, the dinucleotide microsatellite gene loci include one in D2S123, D17S250, D18S34 and D5S346 or
It is multiple.
2. composite amplification system according to claim 1, which is characterized in that the composite amplification system is also simultaneously to four cores
Thuja acid microsatellite gene loci and sex determining gene site are expanded.
3. composite amplification system according to claim 2, which is characterized in that the tetranucleotide microsatellite gene loci packet
Include D13S317 and D7S820；
The sex determining gene site includes AMEL.
4. composite amplification system according to claim 1, which is characterized in that the composite amplification system includes:
For expanding the primer pair of the specific region of following 7 mononucleotides microsatellite gene loci:
The sequence of NR-21 be the specific region of SEQ ID NO:1, NR-22 sequence be the specific region of SEQ ID NO:2, NR-
24 sequence be the specific region of SEQ ID NO:3, NR-27 sequence be the sequence of the specific region of SEQ ID NO:4, BAT-25
The sequence of the specific region, BAT-26 that are classified as SEQ ID NO:5 is the specific region of SEQ ID NO:6 and the sequence of MONO-27
For the specific region of SEQ ID NO:7；
For expanding the primer pair for being selected from the specific region of one or more of following dinucleotide microsatellite gene loci:
The sequence of D2S123 be the specific region of SEQ ID NO:8, D17S250 sequence be the specific region of SEQ ID NO:9,
The specific region that the sequence of D18S34 is the specific region of SEQ ID NO:10 or the sequence of D5S346 is SEQ ID NO:11.
5. composite amplification system according to claim 4, which is characterized in that the composite amplification system further include:
For expanding the primer pair of the specific region of following 2 tetranucleotides microsatellite gene loci: the sequence of D13S317 is
The specific region that the specific region of SEQ ID NO:12 and the sequence of D7S820 are SEQ ID NO:13,
And the sequence for expanding sex determining gene site AMEL is the primer pair of the specific region of SEQ ID NO:14.
6. composite amplification system according to claim 5, which is characterized in that the primer pair for expanding NR-21 is selected from sequence SEQ
The primer that the continuous base of 18-26 of the position 1-123 of ID NO:1 is constituted, and the 140-200 selected from sequence SEQ ID NO:1
The combination for the primer that the continuous base of 18-26 of the complementary series of position is constituted；
The primer that 18-26 continuous bases of the position 1-130 that the primer pair for expanding NR-22 is selected from sequence SEQ ID NO:2 are constituted,
And the group of the primer of 18-26 continuous bases compositions of the complementary series of the position 150-250 selected from sequence SEQ ID NO:2
It closes；
The primer that 18-26 continuous bases of the position 1-120 that the primer pair for expanding NR-24 is selected from sequence SEQ ID NO:3 are constituted,
And the group of the primer of 18-26 continuous bases compositions of the complementary series of the position 140-250 selected from sequence SEQ ID NO:3
It closes；
The primer that 18-26 continuous bases of the position 1-138 that the primer pair for expanding NR-27 is selected from sequence SEQ ID NO:4 are constituted,
And the group of the primer of 18-25 continuous bases compositions of the complementary series of the position 158-260 selected from sequence SEQ ID NO:4
It closes；
What the 18-26 continuous bases that the primer pair of amplification BAT-25 is selected from the position 1-132 of sequence SEQ ID NO:5 were constituted draws
Object, and the position 152-300 selected from sequence SEQ ID NO:5 complementary series the continuous bases of the 18-26 primers that constitute
Combination；
What the 18-26 continuous bases that the primer pair of amplification BAT-26 is selected from the position 1-117 of sequence SEQ ID NO:6 were constituted draws
Object, and the position 145-270 selected from sequence SEQ ID NO:6 complementary series the continuous bases of the 18-26 primers that constitute
Combination；
What the 18-26 continuous bases that the primer pair of amplification MONO-27 is selected from the position 1-116 of sequence SEQ ID NO:7 were constituted draws
Object, and the position 143-290 selected from sequence SEQ ID NO:7 complementary series the continuous bases of the 18-26 primers that constitute
Combination；
What the 18-26 continuous bases that the primer pair of amplification D2S123 is selected from the position 1-132 of sequence SEQ ID NO:8 were constituted draws
Object, and the position 174-310 selected from sequence SEQ ID NO:8 complementary series the continuous bases of the 18-26 primers that constitute
Combination；
What the 18-26 continuous bases that the primer pair of amplification D17S250 is selected from the position 1-104 of sequence SEQ ID NO:9 were constituted draws
Object, and the position 141-280 selected from sequence SEQ ID NO:9 complementary series the continuous bases of the 18-26 primers that constitute
Combination；
What the 18-26 continuous bases that the primer pair of amplification D18S34 is selected from the position 1-128 of sequence SEQ ID NO:10 were constituted draws
Object, and the position 185-223 selected from sequence SEQ ID NO:10 complementary series the continuous bases of the 18-26 primers that constitute
Combination；
What the 18-26 continuous bases that the primer pair of amplification D5S346 is selected from the position 1-154 of sequence SEQ ID NO:11 were constituted draws
Object, and the position 193-300 selected from sequence SEQ ID NO:11 complementary series the continuous bases of the 18-25 primers that constitute
Combination；
What 18-26 continuous bases of the position 45-228 that the primer pair for expanding D13S317 is selected from sequence SEQ ID NO:12 were constituted
Primer, and the position 268-480 selected from sequence SEQ ID NO:12 complementary series the primers that constitute of the continuous base of 18-26
Combination；
What the 18-26 continuous bases that the primer pair of amplification D7S820 is selected from the position 45-216 of sequence SEQ ID NO:13 were constituted draws
Object, and the position 69-460 selected from sequence SEQ ID NO:13 complementary series the continuous bases of the 18-26 primers that constitute
Combination；
The primer that 18-26 continuous bases of the position 1-140 that the primer pair for expanding AMEL is selected from sequence SEQ ID NO:14 are constituted,
And the group of the primer of 18-25 continuous bases compositions of the complementary series of the position 145-310 selected from sequence SEQ ID NO:14
It closes.
7. composite amplification system according to claim 6, which is characterized in that expand in the primer pair of NR-21, forward primer
The primer for being SEQ ID No.15-17 selected from sequence, reverse primer are selected from the primer that sequence is SEQ ID No.18-20；
In the primer pair for expanding NR-22, forward primer is selected from the primer that sequence is SEQ ID No.21-23, and reverse primer is selected from
Sequence is the primer of SEQ ID No.24-26；
In the primer pair for expanding NR-24, forward primer is selected from the primer that sequence is SEQ ID No.27-29, and reverse primer is selected from
Sequence is the primer of SEQ ID No.30-32；
In the primer pair for expanding NR-27, forward primer is selected from the primer that sequence is SEQ ID No.33-35, and reverse primer is selected from
Sequence is the primer of SEQ ID No.36-38；
In the primer pair for expanding BAT-25, forward primer is selected from the primer that sequence is SEQ ID No.39-41, and reverse primer is selected from
Sequence is the primer of SEQ ID No.42-44；
In the primer pair for expanding BAT-26, forward primer is selected from the primer that sequence is SEQ ID No.45-47, and reverse primer is selected from
Sequence is the primer of SEQ ID No.48-50；
In the primer pair for expanding MONO-27, forward primer is selected from the primer that sequence is SEQ ID No.51-53, reverse primer choosing
Author's preface is classified as the primer of SEQ ID No.54-56；
In the primer pair for expanding D2S123, forward primer is selected from the primer that sequence is SEQ ID No.57-59, and reverse primer is selected from
Sequence is the primer of SEQ ID No.60-62；
In the primer pair for expanding D17S250, forward primer is selected from the primer that sequence is SEQ ID No.63-65, reverse primer choosing
Author's preface is classified as the primer of SEQ ID No.66-68；
In the primer pair for expanding D18S34, forward primer is selected from the primer that sequence is SEQ ID No.69-71, and reverse primer is selected from
Sequence is the primer of SEQ ID No.72-73；
In the primer pair for expanding D5S346, forward primer is selected from the primer that sequence is SEQ ID No.74-76, and reverse primer is selected from
Sequence is the primer of SEQ ID No.77-79；
In the primer pair for expanding D13S317, forward primer is selected from the primer that sequence is SEQ ID No.80-81, reverse primer choosing
Author's preface is classified as the primer of SEQ ID No.82-83；
In the primer pair for expanding D7S820, forward primer is selected from the primer that sequence is SEQ ID No.84-85, and reverse primer is selected from
Sequence is the primer of SEQ ID No.86-87；
In the primer pair for expanding AMEL, forward primer is selected from the primer that sequence is SEQ ID No.88-89, and reverse primer is selected from sequence
It is classified as the primer of SEQ ID No.90-91.
8. composite amplification system according to claim 5, which is characterized in that in the primer pair, 5 ' ends of a primer
End has fluorescent marker.
9. composite amplification system according to claim 8, which is characterized in that the primer pair is divided into three groups, is respectively provided with
The fluorescent marker of three kinds of different colours: first group of primer pair is amplification NR-21, NR-24, D13S317, D5S346, D17S250；
Second group of primer pair is amplification NR-27, BAT-25, MONO-27, D18S34, D2S123；Third group primer pair is amplification BAT-
26、NR-22、D7S820、AMEL。
10. a kind of kit for microsatellite instability detection in Gene Mutation, which is characterized in that including claim 1 to
Composite amplification system described in claim 9 any one.
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