CN106367475B - A kind of MMR gene mutation detection kit - Google Patents
A kind of MMR gene mutation detection kit Download PDFInfo
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Abstract
The invention belongs to molecular biology and medical domain, more particularly to human DNA mismatch repair gene (mismatch repair genes, MMR mutation detection kit) and application thereof, more particularly to human MutL protein homolog 1., the abrupt climatic change of MSH2, MSH6, PMS1, PMS2 gene and its detection with hereditary nonpolyposis colorectal cancer (HNPCC) correlation.Kit of the invention can be used for detecting the genotype of the MMR gene extron mutation of individual, to judge whether the individual suffers from HNPCC and whether family member's risk is greater than general population and provides reference data.
Description
Technical field
The present invention relates to molecular biology and medical domain, and in particular to a kind of MMR gene mutation detection kit.
Background technique
Colorectal cancer (Colorectal Cancer, CRC) is the third-largest common cancer in the whole world.Research is found
For 35% CRC there are family's neurological susceptibility, one of the most common is hereditary nonpolyposis colorectal cancer (hereditary
Nonpolyposis colorectal cancer, HNPCC), the 2%~5% of Zhan Suoyou case.DNA mismatch revision points
The science of heredity pathogenesis that germline mutation is Lynch syndrome occurs for (mismatch repair genes, MMR).Research is found
The germline mutation of at least five kinds of MMR genes (MSH1, MLH2, MSH6, PMS1, PMS2) will lead to HNPCC, wherein the embryonal system of MLH1
Mutation, which accounts for 50%, MSH2 and accounts for 40%, MSH6, accounts for 7%, and remaining gene accounts for 3%.In family, MMR gene germ line mutation is carried
Person is the people at highest risk that HNPCC related neoplasms occur, and detection MMR gene germ line mutation can predict HNPCC related neoplasms well
Cause danger.For having found there are the patient of inherited mismatch repair gene mutation, Metachro nous Multiple Primary can be monitored closely
The generation of cancer and other HNPCC related neoplasms, and to other members in its family, especially not yet occur tumour it is young at
Member accordingly detected, confirm mutated gene carrier, not only help to cancer susceptibility individual weight point carry out clinical monitoring with
Early diagnosis before clinical symptoms appearance, can also provide foundation for genetic counselling and gene therapy, and can make not mutated carrier
Close follow-up can be carried out to mutation carriers by exempting unnecessary spirit and financial burden, to accomplish to early diagnose,
Early treatment.
Currently, the method for screening HNPCC only detects the MMR gene of embryonal system (germline) although more
Pathologic mutation can just be diagnosed as HNPCC patient.Direct gene PCR sequencing PCR is that MMR germline abrupt climatic change is most sensitive and most special
Different method, but it is time-consuming and expensive based on the sequencing of Sanger method, it is unable to satisfy the detection demand of clinically large sample size.With
Traditional sanger method sequencing is compared, and second generation high throughput sequencing technologies have the advantages that flux is high, speed is fast, expense is low etc., knot
It closes specific nucleic acid sequence and targets beneficiation technologies, may be implemented to carry out deep sequencing to the maximally related gene of disease.
Summary of the invention
For the defect in the presence of the prior art, whether the object of the present invention is to provide deposit in a kind of test sample
In the reagent of MMR genetic mutation.
Another object of the present invention is to provide the use for the reagent that whether there is MMR genetic mutation in the test sample
On the way, it is used to prepare the kit of detection MMR genetic mutation.
It is another object of the present invention to provide a kind of kits for detecting MMR genetic mutation.
It is another object of the present invention to provide a kind of methods for obtaining amplified production.
The present invention is achieved by the following technical solutions:
The first aspect of the present invention provides the reagent that whether there is MMR genetic mutation in a kind of test sample, described
Reagent is primer pair, is selected from the group any one or more in primer pair:
(1) SEQ ID NO.1 and SEQ ID NO.2；(2) SEQ ID NO.3 and SEQ ID NO.4；
(3) SEQ ID NO.5 and SEQ ID NO.6；(4) SEQ ID NO.7 and SEQ ID NO.8；
(5) SEQ ID NO.9 and SEQ ID NO.10；(6) SEQ ID NO.11 and SEQ ID NO.12；
(7) SEQ ID NO.13 and SEQ ID NO.14；(8) SEQ ID NO.15 and SEQ ID NO.16；
(9) SEQ ID NO.17 and SEQ ID NO.18；(10) SEQ ID NO.19 and SEQ ID NO.20；
(11) SEQ ID NO.21 and SEQ ID NO.22；(12) SEQ ID NO.23 and SEQ ID NO.24；
(13) SEQ ID NO.25 and SEQ ID NO.26；(14) SEQ ID NO.27 and SEQ ID NO.28；
(15) SEQ ID NO.29 and SEQ ID NO.30；(16) SEQ ID NO.31 and SEQ ID NO.32；
(17) SEQ ID NO.33 and SEQ ID NO.34；(18) SEQ ID NO.35 and SEQ ID NO.36；
(19) SEQ ID NO.37 and SEQ ID NO.38；(20) SEQ ID NO.39 and SEQ ID NO.40；
(21) SEQ ID NO.41 and SEQ ID NO.42；(22) SEQ ID NO.43 and SEQ ID NO.44；
(23) SEQ ID NO.45 and SEQ ID NO.46；(24) SEQ ID NO.47 and SEQ ID NO.48；
(25) SEQ ID NO.49 and SEQ ID NO.50；(26) SEQ ID NO.51 and SEQ ID NO.52；
(27) SEQ ID NO.53 and SEQ ID NO.54；(28) SEQ ID NO.55 and SEQ ID NO.56；
(29) SEQ ID NO.57 and SEQ ID NO.58；(30) SEQ ID NO.59 and SEQ ID NO.60；
(31) SEQ ID NO.61 and SEQ ID NO.62；(32) SEQ ID NO.63 and SEQ ID NO.64；
(33) SEQ ID NO.65 and SEQ ID NO.66；(34) SEQ ID NO.67 and SEQ ID NO.68；
(35) SEQ ID NO.69 and SEQ ID NO.70；(36) SEQ ID NO.71 and SEQ ID NO.72；
(37) SEQ ID NO.73 and SEQ ID NO.74；(38) SEQ ID NO.75 and SEQ ID NO.76；
(39) SEQ ID NO.77 and SEQ ID NO.78；(40) SEQ ID NO.79 and SEQ ID NO.80；
(41) SEQ ID NO.81 and SEQ ID NO.82；(42) SEQ ID NO.83 and SEQ ID NO.84；
(43) SEQ ID NO.85 and SEQ ID NO.86；(44) SEQ ID NO.87 and SEQ ID NO.88；
(45) SEQ ID NO.89 and SEQ ID NO.90；(46) SEQ ID NO.91 and SEQ ID NO.92；
(47) SEQ ID NO.93 and SEQ ID NO.94；(48) SEQ ID NO.95 and SEQ ID NO.96；
(49) SEQ ID NO.97 and SEQ ID NO.98；(50) SEQ ID NO.99 and SEQ ID NO.100；
(51) SEQ ID NO.101 and SEQ ID NO.102；(52) SEQ ID NO.103 and SEQ ID NO.104；
(53) SEQ ID NO.105 and SEQ ID NO.106；(54) SEQ ID NO.107 and SEQ ID NO.108；
(55) SEQ ID NO.109 and SEQ ID NO.110；(56) SEQ ID NO.111 and SEQ ID NO.112；
(57) SEQ ID NO.113 and SEQ ID NO.114；(58) SEQ ID NO.115 and SEQ ID NO.116；
(59) SEQ ID NO.117 and SEQ ID NO.118；(60) SEQ ID NO.119 and SEQ ID NO.120；
(61) SEQ ID NO.121 and SEQ ID NO.122；(62) SEQ ID NO.123 and SEQ ID NO.124；
(63) SEQ ID NO.125 and SEQ ID NO.126；(64) SEQ ID NO.127 and SEQ ID NO.128；
(65) SEQ ID NO.129 and SEQ ID NO.130；(66) SEQ ID NO.131 and SEQ ID NO.132；
(67) SEQ ID NO.133 and SEQ ID NO.134；(68) SEQ ID NO.135 and SEQ ID NO.136；
(69) SEQ ID NO.137 and SEQ ID NO.138；(70) SEQ ID NO.139 and SEQ ID NO.140；
(71) SEQ ID NO.141 and SEQ ID NO.142；(72) SEQ ID NO.143 and SEQ ID NO.144；
(73) SEQ ID NO.145 and SEQ ID NO.146；(74) SEQ ID NO.147 and SEQ ID NO.148；
(75) SEQ ID NO.149 and SEQ ID NO.150；(76) SEQ ID NO.151 and SEQ ID NO.152；
(77) SEQ ID NO.153 and SEQ ID NO.154；(78) SEQ ID NO.155 and SEQ ID NO.156；
(79) SEQ ID NO.157 and SEQ ID NO.158；(80) SEQ ID NO.159 and SEQ ID NO.160；
(81) SEQ ID NO.161 and SEQ ID NO.162；(82) SEQ ID NO.163 and SEQ ID NO.164；
(83) SEQ ID NO.165 and SEQ ID NO.166；(84) SEQ ID NO.167 and SEQ ID NO.168；
(85) SEQ ID NO.169 and SEQ ID NO.170；(86) SEQ ID NO.171 and SEQ ID NO.172；
(87) SEQ ID NO.173 and SEQ ID NO.174.
In the second aspect of the present invention, the purposes that whether there is the reagent of MMR genetic mutation in the test sample is provided,
It is used to prepare the kit of detection MMR genetic mutation.
In the third aspect of the present invention, the kit that whether there is MMR genetic mutation in a kind of test sample is provided, it is described
Kit in contain aforementioned 87 pairs of primer pairs one or more primer pairs.
In another preferred example, aforementioned all 87 pairs of primer pairs are contained in the kit.
In another preferred embodiment, in the kit, containing 11 groups of multiple PCR primers to combination, it is respectively as follows:
(1) primer pair combination 1: by SEQ ID NO.7 and SEQ ID NO.8；SEQ ID NO.23 and SEQ ID NO.24；
SEQ ID NO.27 and SEQ ID NO.28；SEQ ID NO.63 and SEQ ID NO.64；SEQ ID NO.97 and SEQ ID
NO.98；SEQ ID NO.111 and SEQ ID NO.112；SEQ ID NO.127 and SEQ ID NO.128；SEQ ID NO.149
It is formed with SEQ ID NO.150；
(2) primer pair combination 2: by SEQ ID NO.3 and SEQ ID NO.4；SEQ ID NO.33 and SEQ ID NO.34；
SEQ ID NO.39 and SEQ ID NO.40；SEQ ID NO.67 and SEQ ID NO.68；SEQ ID NO.77 and SEQ ID
NO.78；SEQ ID NO.81 and SEQ ID NO.82；SEQ ID NO.117 and SEQ ID NO.118；SEQ ID NO.137 and
SEQ ID NO.138；SEQ ID NO.153 and SEQ ID NO.154；SEQ ID NO.173 and SEQ ID NO.174 composition；
(3) primer pair combination 3: by SEQ ID NO.17 and SEQ ID NO.18；SEQ ID NO.25 and SEQ ID
NO.26；SEQ ID NO.37 and SEQ ID NO.38；SEQ ID NO.59 and SEQ ID NO.60；SEQ ID NO.79 and SEQ
ID NO.80；SEQ ID NO.101 and SEQ ID NO.102；SEQ ID NO.113 and SEQ ID NO.114；SEQ ID
NO.123 and SEQ ID NO.124；SEQ ID NO.141 and SEQ ID NO.142；SEQ ID NO.163 and SEQ ID
NO.164 composition；
(4) primer combination 4: by SEQ ID NO.21 and SEQ ID NO.22；SEQ ID NO.35 and SEQ ID NO.36；
SEQ ID NO.61 and SEQ ID NO.62；SEQ ID NO.87 and SEQ ID NO.88；SEQ ID NO.99 and SEQ ID
NO.100；SEQ ID NO.119 and SEQ ID NO.120；SEQ ID NO.133 and SEQ ID NO.134；SEQ ID
NO.147 and SEQ ID NO.148；SEQ ID NO.155 and SEQ ID NO.156；SEQ ID NO.159 and SEQ ID
NO.160 composition；
(5) primer combination 5: by SEQ ID NO.5 and SEQ ID NO.6；SEQ ID NO.19 and SEQ ID NO.20；
SEQ ID NO.41 and SEQ ID NO.42；SEQ ID NO.47 and SEQ ID NO.48；SEQ ID NO.55 and SEQ ID
NO.56；SEQ ID NO.89 and SEQ ID NO.90；SEQ ID NO.95 and SEQ ID NO.96；SEQ ID NO.115 and
SEQ ID NO.116 composition；
(6) primer combination 6: by SEQ ID NO.11 and SEQ ID NO.12；SEQ ID NO.31 and SEQ ID NO.32；
SEQ ID NO.53 and SEQ ID NO.54；SEQ ID NO.65 and SEQ ID NO.66；SEQ ID NO.75 and SEQ ID
NO.76；SEQ ID NO.91 and SEQ ID NO.92；SEQ ID NO.103 and SEQ ID NO.104；SEQ ID NO.109 and
SEQ ID NO.110；SEQ ID NO.125 and SEQ ID NO.126；SEQ ID NO.161 and SEQ ID NO.162 composition；
(7) primer combination 7: by SEQ ID NO.1 and SEQ ID NO.2；SEQ ID NO.15 and SEQ ID NO.16；
SEQ ID NO.57 and SEQ ID NO.58；SEQ ID NO.85 and SEQ ID NO.86；SEQ ID NO.93 and SEQ ID
NO.94；SEQ ID NO.129 and SEQ ID NO.130；SEQ ID NO.145 and SEQ ID NO.146；SEQ ID NO.9 and
SEQ ID NO.10 composition；
(8) primer combination 8: by SEQ ID NO.13 and SEQ ID NO.14；SEQ ID NO.29 and SEQ ID NO.30；
SEQ ID NO.49 and SEQ ID NO.50；SEQ ID NO.69 and SEQ ID NO.70；SEQ ID NO.83 and SEQ ID
NO.84；SEQ ID NO.121 and SEQ ID NO.122；SEQ ID NO.131 and SEQ ID NO.132；SEQ ID NO.135
With SEQ ID NO.136；SEQ ID NO.43 and SEQ ID NO.44 composition；
(9) primer combination 9: by SEQ ID NO.105 and SEQ ID NO.106；SEQ ID NO.73 and SEQ ID
NO.74；
(10) primer combination 10: by SEQ ID NO.107 and SEQ ID NO.108；SEQ ID NO.71 and SEQ ID
NO.72；SEQ ID NO.171 and SEQ ID NO.172 composition；
(11) primer combination 11: by SEQ ID NO.139 and SEQ ID NO.140；SEQ ID NO.151 and SEQ ID
NO.152；SEQ ID NO.165 and SEQ ID NO.166；SEQ ID NO.167 and SEQ ID NO.168；SEQ ID
NO.169 and SEQ ID NO.170；SEQ ID NO.51 and SEQ ID NO.52；SEQ ID NO.143 and SEQ ID
NO.144；SEQ ID NO.157 and SEQ ID NO.158；SEQ ID NO.45 and SEQ ID NO.46 composition.
It is detected based on the kit of the invention using PCR, can also include it in the kit
His some reagents, such as: one of total DNA extraction agent, PCR reagent, PCR product purified reagent are a variety of.Specifically need by
Which reagent is fitted into kit, can configure according to actual needs.
The reagent of various conventional extracting DNA, the commercially available acquisition of these reagents can be used in the total DNA extraction agent.
Various conventional PCR reagents, the commercially available acquisition of this reagent can be used in the PCR reagent.
Various conventional PCR product purified reagents can be used in the PCR product purified reagent, this reagent is commercially available to be obtained
?.
In addition, further including operation instructions in the kit, used convenient for those skilled in the art.
In the fourth aspect of the present invention, a kind of method for obtaining MMR gene amplification product is provided, the method includes:
Using nucleic acid samples as template, PCR amplification is carried out using selected from primer pair above-mentioned or kit, obtains amplified production.
For the template of PCR amplification, that is, nucleic acid samples (such as genomic DNA) can also be used the conventional method of this field and mention
Take acquisition.In another preferred example, reagent is extracted using classical genome DNA extracting method or the genome of commercialization
Box extracts genomic DNA from sample, as sample of nucleic acid.
In another preferred example, multiplexed PCR amplification is carried out respectively as primer using the combination of aforementioned 11 groups of primer pairs, will
The 11 groups of multiple PCR products arrived merge to get amplified production.
In addition to using primer of the invention, the present invention is to other each components in PCR reaction system and its final concentration without spy
Other limitation, the general ingredient and its concentration that those skilled in the art use when can establish PCR system according to routine react to establish PCR
System.
In another preferred example, 1-8 group and 11 groups of 50 μ l of multi-PRC reaction system, comprising: 5X Multiplex
1.0 μM of 2.5-20 μ l, Reverse Primer 1.0 of PCR Master Mix 10 μ l, template 50ng, Forward Primer
μM 2.5-20 μ l, ddH2O complements to 50 μ l.50 μ l:2X GC Buffer I of multi-PRC reaction system, the 25 μ l of 9-10 group,
1.0 μM of 2.0 μ l, Reverse Primer of dNTP 4ul, template 50ng, Forward Primer 1.0 μM of 2.0 μ l, ddH2O
Complement to 50 μ l.
Compared with prior art, beneficial effects of the present invention are as follows:
From general PCR to multiplex PCR, design difficulty can be double therewith.It can be said that design of primers be multiplex PCR most
Big obstacle.The present invention relates to a kind of multiplex PCR targeting enrichment combine high-flux sequence detection MMR gene mutation kit and
Method, the present invention can expand drawing for 5 MMR gene (MSH1, MLH2, MSH6, PMS1, PMS2) all exons by design
Object, and the method for independent development multiplex PCR targeting enrichment MMR gene extron subsequence, after the amplification for realizing MMR gene extron
Using second generation high throughput sequencing technologies, the germline mutation of MMR gene is detected.Present invention represents the measurements of newest two generations high pass
Technological break-through of the sequence technology in hereditary nonpolyposis colorectal cancer (HNPCC) MMR germline abrupt climatic change.It passes through more
Weight PCR targeting enrichment MMR gene extron subsequence combines second generation high throughput sequencing technologies, realizes while detecting 5 kinds of MMR bases
The germline mutation of cause provides for the clinical diagnosis and research of HNPCC and provides the technical method that flux is high, speed is fast, expense is low.
The method and kit that we invent are using the Miseq sequenator of illumina company as main platform, but this method and invention
Other high-flux sequence platforms are equally applicable to, Hiseq 2500/3000/4000, NextSeq including illumina company
Ion PGM/Proton system of 500 and Life company etc..
Detailed description of the invention
Fig. 1: for Technology Roadmap of the invention.
Fig. 2: the top band (green) and nethermost band (purple) are dna ladder, are equivalent to marker；In
Between a plurality of black stripe be the sample strip detected.
Fig. 3: abscissa is clip size, i.e. bp number；Ordinate is signal strength, i.e. nucleic acid content.
Fig. 4: the top band (green) and nethermost band (purple) are dna ladder, are equivalent to marker；In
Between a plurality of black stripe be the sample strip detected.
Fig. 5: abscissa is clip size, i.e. bp number；Ordinate is signal strength, i.e. nucleic acid content.
Fig. 6: being the sanger sequence verification result screenshot of the embodiment of the present invention 3, A) MLH1:exon18:c.C2101A；B)
MSH2:exon14:c.G2425A；C)MSH6:exon5:c.G3205C.
Specific embodiment
The present inventor after extensive and in-depth study, has found a kind of particularly suitable for expanding 5 MMR genes all 73
The primer of a exon, the primer is by reasonably designing, preferably obtaining, and specificity is good when for PCR amplification, and expansion
Increasing Efficiency is high, is up to 100% for the coverage of all 73 exons.The present inventor also optimizes pcr amplification reaction, example
Such as, multi-PRC reaction system further improves amplification efficiency.
For current detection MMR gene mutation process very complicated, consuming time is long the shortcomings that, inventor has also been devised one
The method of kind 5 MMR gene mutations of detection, the method are simple, time-consuming short and at low cost.Based on this, the present invention is completed.
Various technologies can be used to detect MMR gene or MMR genetic mutation site, when detecting MMR genetic mutation, detect
It can be directed to cDNA, genomic DNA can also be directed to.The existing prior art such as Southern blotting, DNA sequence dna point can be used
Analysis, PCR and in situ hybridization detection mutation.
Be conveniently to carry out PCR with the primer of MMR gene specific, MMR gene expanded, to amplified production into
Row sequencing, to judge whether to morph.
The present inventor has found under study for action, and when detecting MMR gene or MMR gene mutation, using general primer, PCR expands
Poor specificity when increasing, amplification efficiency are low.Therefore it needs to design and screen the good primer of specificity.By largely testing and
Compare, the present inventor has found the primer for corresponding respectively to all 73 exons of 5 MMR genes.The verified primer obtains
The amplified production obtained had both included sequence as complete as possible on each exon of MMR, and specificity is very good, almost without non-spy
Specific amplification or the especially few situation of amplified production.PCR amplification especially suitable for complex system.The nucleotide sequence of each primer
As shown in table 2.
Multiplex PCR has many good qualities with respect to substance PCR, can save the time, saves reagent, and reduction of expenditure spending is disease
The prediction and prevention and treatment of disease provide more more accurate diagnostic messages.Substance PCR detection, when the sequence that primer is directed to is mutated
Shi Ze is likely to occur false negative result, and detects for the multiplex PCR of cause of disease of the same race, because being directed to multiple genes, simultaneously
Very little a possibility that multiple sites are morphed can substantially reduce the probability of false negative appearance.
The more difficult foundation of multi-PRC reaction system, it is desirable that will not interact, expand between the primer in ensuring to react
Primer size is close but can be separated by electrophoresis.Multiplexed PCR amplification requires very high, any experiment condition to test operation
It is improper to control, and will all easily lead to the failure of amplification condition.The foundation for the multiplex PCR system reported in the prior art, does not change
When becoming PCR reaction condition, carries out double PCR and be easier to obtain expected results, and it is relatively more tired to triple or above PCR amplifications
It is difficult.Present inventor, to multiplex PCR system optimization, is successfully established multiplex PCR detection architecture, energy by design multiple groups primer combination
It is enough disposably to detect that several genes are mutated, not only can quickly, it is succinct, accurately detect disease, and detection can be greatlyd save
Cost.
The present invention also provides for detecting the kit containing MMR gene or MMR genetic mutation, the examination in analyte
Agent box include it is in the appropriate containers, for specific amplification MMR gene or the primer of MMR genetic mutation, and described in using
What primer amplification went out contains the relevant variation position of disease on MMR gene or MMR gene-correlation exon.In the kit
Containing at least pair of primers for being selected from table 2, for obtaining on relevant MMR gene or MMR gene extron comprising variant sites
Sequence.It is optimal, containing primer pair all in table 2 in the kit, it is more advantageous to completely analysis MMR base in this way
The variation situation of cause or each exon sequence of MMR gene.
In addition, may also include in the kit for various reagents needed for extracting DNA, PCR amplification etc., including but
It is not limited to: extract, amplification liquid, hybridization solution, enzyme, washing lotion etc..These are all that those skilled in the art are understood.
In addition, may also include operation instructions and/or Nucleotide Sequence Analysis Software etc. in the kit, it is convenient for ability
Domain personnel use and analyze.
The method for obtaining MMR gene amplification product
The present inventor additionally provides the method that MMR gene amplification product is obtained in the slave nucleic acid samples of optimization a kind of, described
Method include: using sample of nucleic acid as template, utilize selected from table 2 primer pair carry out PCR amplification, obtain amplified production.
Using the primer, ideal amplification can be obtained using conventional PCR amplification method.One kind can
In the PCR amplification method of choosing, steps are as follows for PCR thermal cycle: 98 DEG C be denaturalized 10 seconds, 55 DEG C anneal 30 seconds, 72 DEG C extend 60 seconds, three
Ten circulations.
Template denaturation step before carrying out PCR thermal cycle can be according to the method for this field routine.Preferable method is as follows: 94
DEG C initial denaturation 1 minute.
Sequence after carrying out PCR thermal cycle extends step can be according to the method for this field routine.Preferable method is as follows: 72
DEG C extend 4 minutes.
In addition to using primer of the invention, the present invention is not special to each ingredients other in PCR amplification system and its concentration
Limitation, those skilled in the art can according to it is conventional establish PCR system when the general ingredient that uses and its concentration expand to establish PCR
Increasing system.The conventional method that this field can be used in template (such as genomic DNA) for PCR amplification, which is extracted, to be obtained.
Using the method for obtaining MMR gene or MMR gene amplification product of the invention, amplification efficiency and specificity are very
Ideal, and the PCR amplification particularly suitable for complex system, such as using blood sample genomic DNA as the amplification of pcr template.
The present invention also provides a kind of method that whether there is MMR genetic mutation in determining sample to be tested, the methods
Include: that MMR genetic fragment is expanded from determined nucleic acid sample using method above-mentioned, obtains amplified production；And analysis amplification
The sequence of MMR genetic fragment in product, and be compared with corresponding sequence in wild type MMR gene；If there is difference, then
Show that there are MMR genetic mutations in determined nucleic acid sample.
The case where passing through genetic mutation can further learn subject's hereditary nonpolyposis colorectal cancer (HNPCC)
Risk, to achieve the purpose that early detection early prevention.
Main advantages of the present invention are:
(1) suitable primer, institute are devised for all 73 exons of 5 MMR genes and 5 MMR genes for the first time
It is good to state primer specificity when for PCR amplification, amplification efficiency height.
(2) method for obtaining MMR gene amplification product is optimized, the method is accurately and fast, stable, success rate is high.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Technical solution of the present invention route map is as shown in Figure 1, mainly include following sport technique segment:
Sport technique segment one: multiplex PCR targeting enrichment MMR gene extron is carried out using primer pair of the invention and combinations thereof
Son.
Sport technique segment two: 5 gene (MSH1, MLH2, MSH6, PMS1, PMS2) exons that sport technique segment one is generated
Amplified production carries out sequencing and identification using two generation high throughput sequencing technologies.
1 design of primers of embodiment and synthesis
The present invention is to 73 exons of 5 genes (MSH1, MLH2, MSH6, PMS1, PMS2), 11419 bases in total
87 couples of Primer are devised, pcr amplification product length is 257-528bp, and combination probe primer can cover 5 MMR genes
All exon regions of (being shown in Table 1).Primer synthesis is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.Primer sequence information is detailed
It is shown in Table 2.
Table 1: design of primers reference sequences information table
Table 2: for 87 pairs of primers of 5 MMR genes
The combination of 2 primer of embodiment and multiplex PCR target enrichment method
One, experimental material
(1), main agents
1, fluorescence quantitative detection kit: QubitTM dsDNA HS Assay Kit(500assays),Life
Technologies company；
2, immue quantitative detection reagent box: Agilent DNA 7500and 12000Kit, Agilent company, the kit packet
Contain；
3, Multiplex PCR kit: MixMultiplex 5 × Master of PCR Mix, NEB company；
4, sequencing library jointing kit: NEXTflexTMDNA Barcode-48, BIOO SCIENTIFIC are public
Department；
5, sequencing library constructs kit: NEXTflexTMRapid DNA-Seq Kit, BIOO SCIENTIFIC company；
6, sequencing kit: MiSeq Reagent Kit v3 (600-cycles), Illumina company
7, purification kit: Agencourt AMPure XP beads, Beckman Coulter (Agencourt) company
(2), key instrument
1,0.5ul~2ml pipettor, Eppendof, Germany；
2, micro centrifuge, Hangzhou Ao Sheng Instrument Ltd.；
3, supercentrifuge, sigma, Germany；
4, turbula shaker, Scientific Industries, the U.S.；
5, dry-type thermostat, TECHNE, Germany；
6, TC-412 type PCR instrument, TECHNE, Germany；
7,2100 biological analyser of Agilent, Agilent, Germany；
8, Qubit fluorescent quantitation instrument, Invitrogen, the U.S.；
9, Miseq sequencer sequenator, Illumina, the U.S.；
Two, one tube PCR amplification
1. 87 pairs of primer pairs as shown in table 2 are respectively adopted as primer, carry out using the gDNA of commercialization as template
One tube PCR amplification.Reaction system and amplification condition used are as follows when PCR amplification independent using every group of primer pair of 87 centerings,
PCR kit used is TaKaRa Ex
Reaction system:
Reaction condition:
2. a pair each pair of pair of primer individually expands obtained one tube PCR amplification product and purify, specific method is the same as this reality
Apply the method in example three, 2.
3. respectively after purification by obtain 87 groups of PCR products, mixed in equal amounts, and use 2100 biological analyser of Agilent
Detection, as a result as shown in Figures 2 and 3.Wherein, as shown in Fig. 2, the top band (green) and nethermost band (purple) are
Dna ladder, is equivalent to marker；Intermediate a plurality of black stripe is the sample strip detected.Such as Fig. 3, abscissa is piece
Duan great little, i.e. bp number；Ordinate is signal strength, i.e. nucleic acid content.87 pairs of primers designed by the present invention are to 5 MMR of needle
The specific primer of all exon regions of gene, according to fig. 2 with the result of Fig. 3, it can be seen that using this 87 pairs of primer pairs into
When row single tube PCR, can amplify to obtain corresponding pcr amplification product, absolutely proved this 87 pairs of primer pairs specificity and
Validity.
Three, multiplexed PCR amplification
1. further, 87 pairs of primer pairs (as shown in table 2) that embodiment 1 is designed and optimized are combined, obtain as
11 groups of primer pair combinations shown in table 3.Then it using the gDNA of commercialization as template, is used as and is drawn using this 11 groups of primer pair combinations
Object carries out multiplexed PCR amplification respectively.The DNA profiling amount of every group of multiplex PCR is 10-200 nanogram, wherein the 1-8 and 11 group of use
NEB MixMultiplex PCR 5 × Master Mix kit carries out multiplexed PCR amplification, and the 9th and 10 group uses TaKaRa
LA Tag with GC Buffer kit carry out multiplexed PCR amplification, amplification condition are as follows: 95 DEG C initial denaturation 1 minute；95 DEG C of changes
Property 20 seconds, 63 DEG C anneal 60 seconds, 68 DEG C extend 30 seconds, 30 circulation；68 DEG C extend 5 minutes.
The combination of table 3:11 group primer pair
1-8 group and 11 groups of multi-PRC reaction systems:
| Component | 50μl reaction | Final Conc. |
| Multiplex PCR 5X Master Mix | 10μl | 1X |
| 1μM Primer Stock | variable | 0.15μM(0.05-0.4μM) |
| Template DNA | variable | 50ng |
| Nuclease-free water | to 50μl |
9th, 10 group of multi-PRC reaction system:
| Component | 50μl reaction |
| 2X GC Buffer I | 25μl |
| dNTP | 4ul |
| Template DNA | 50ng |
| Primer mix | 2ul |
| La Tag | 0.5ul |
| Nuclease-free water | to 50μl |
2. obtained multiple PCR products will be combined using every group of primer pair to purify respectively:
(1) after to pcr amplification reaction, 96 hole PCR reaction plates are taken out, tear sealed membrane, 48ul is added in every hole
AMPure XP Beads is uniformly mixed up and down；
(2) it is placed at room temperature for 5min；
(3) centrifuge tube is placed on 96 orifice plate magnetic frames, 5min is placed, so that magnetic bead is adsorbed on tube wall completely；
(4) liquid in centrifuge tube is carefully drawn and is abandoned with pipettor；
(5) 80% ethyl alcohol of 200ul Fresh is added in the 96 hole PCR plates on magnetic frame, uses liquid relief after placing 30s
Device Aspirate supernatant simultaneously abandons；
(6) step 1.3.6.3.5 is repeated, it is ensured that remaining ethyl alcohol is all sucked out in centrifuge tube；
(7) 96 orifice plates are removed from magnetic frame, 2min is spontaneously dried, so that the magnetic bead on wall is completely dried；
(8) 21ul Resuspension Buffer is added, it is careful to mix, it is ensured that the magnetic bead on tube wall is completely dissolved in
In Buffer；
(9) 96 orifice plates are reapposed on magnetic frame, 5min is placed, so that magnetic bead is adsorbed on tube wall completely；
(10) 20ul eluent is carefully drawn into new 1.5ml centrifuge tube.
3. using 11 groups of primer pair combinations, individually amplification PCR product after purification, obtains 11 groups of multiplex PCRs after purification
Product is examined then by after this 11 groups multiple PCR products mixed in equal amounts after purification using 2100 biological analyser of Agilent
It surveys, as a result as shown in Figure 4 and Figure 5.Wherein, as shown in figure 4, the top band (green) and nethermost band (purple) are
Dna ladder, is equivalent to marker；Intermediate a plurality of black stripe is the sample strip detected.Such as Fig. 5, abscissa is piece
Duan great little, i.e. bp number；Ordinate is signal strength, i.e. nucleic acid content.The result of Fig. 4, Fig. 5 are compared with Fig. 2, Fig. 3, adopted
The g DNA of multiplexed PCR amplification commercialization is carried out respectively with 11 groups of primer pair combinations and carries out substance respectively using 87 groups of primer pairs
The g DNA of PCR amplification commercialization, obtained result are consistent, absolutely prove, are carried out respectively using 11 groups of primer pair combinations more
The g DNA of weight PCR amplification commercialization, is feasible.
Four, sequencing and identification are carried out using second generation high throughput sequencing technologies to MMR gene extron amplified production
For the multiple PCR products that above-mentioned steps generate, after qualitative and quantitative detection, for standard of the invention can be investigated
True property, while carrying out carrying out sequencing to MMR gene extron amplified production using second generation high throughput sequencing technologies, to sentence
The accuracy of the fixed primer amplification.The mainly preparation and sequencing of sequencing library.Library preparation includes that PCR product end is mended
The flat, end 5` phosphorylation, the end 3` add A, connection illumina sequence measuring joints, connection product PCR enrichment and purifying, then use
Miseq sequenator carries out Sequencing and Characterization.
Specifically, (1) sequencing library constructs
Use NEXTflexTMRapid DNA-Seq Kit carries out the building of sequencing library, and specific step is as follows
1) DNA end-filling and 3 ' ends plus A
1.1, template uses gDNA multiple PCR products, and building library initial amount is 200ng, and reaction total volume is 50ul, prepares enzyme
React mixed liquor.
Reaction system:
1.2, mixed liquor mixing is placed on PCR instrument and carries out enzyme reaction.
Enzyme reaction condition:
2) sequence measuring joints Adapter is connected
2.1, connection reaction mixed liquor is prepared
Reaction system:
2.2, mixing night is mixed and is placed on PCR instrument progress enzyme reaction, reaction condition is: 25 DEG C of incubation 30min.
3) connection product purifies
3.1, connection product is transferred in new 1.5ml centrifuge tube, 48ul AMPure XP Beads is then added, mixed
It closes uniform；
3.2, it is placed at room temperature for 5min；
3.3, centrifuge tube is placed on magnetic frame, 5min is placed, so that magnetic bead is adsorbed on tube wall completely；
3.4, the liquid in centrifuge tube is carefully drawn and is abandoned with pipettor；
3.5,80% ethyl alcohol of 200ul Fresh is added in the centrifuge tube on magnetic frame, uses pipettor after placing 30s
Aspirate supernatant simultaneously abandons；
3.6, step 3.5 is repeated, it is ensured that remaining ethyl alcohol is all sucked out in centrifuge tube；
3.7, centrifuge tube is transferred on common centrifuge tube shelf from magnetic frame, spontaneously dries 2min, so that centrifugation tube wall
On magnetic bead be completely dried；
3.8,21ul Resuspension Buffer is added, it is careful to mix, it is ensured that the magnetic bead on tube wall is completely dissolved in
In Buffer；
3.9, centrifuge tube is reapposed on magnetic frame, 5min is placed, so that magnetic bead is adsorbed on tube wall completely；
3.10,20ul eluent is carefully drawn into new 1.5ml centrifuge tube.
4) PCR is enriched with
4.1, PCR reaction system is prepared, and reaction mixture is added in PCR reaction tube；
Reaction condition:
4.2, PCR reaction tube is placed into PCR instrument and carries out PCR amplification；
Reaction condition
4.3, PCR product is transferred in new 1.5ml centrifuge tube, 44ul AMPure XP Beads is then added, mixed
It closes uniformly and is placed at room temperature for 5min；
4.4, centrifuge tube is placed on magnetic frame, 5min is placed, so that magnetic bead is adsorbed on tube wall completely；
4.5, the liquid in centrifuge tube is carefully drawn and is abandoned with pipettor；
4.6,80% ethyl alcohol of 200ul Fresh is added in the centrifuge tube on magnetic frame, uses pipettor after placing 30s
Aspirate supernatant simultaneously abandons；
4.7, step 1.3.6.3.5 is repeated, it is ensured that remaining ethyl alcohol is all sucked out in centrifuge tube；
4.8, centrifuge tube is transferred on common centrifuge tube shelf from magnetic frame, spontaneously dries 2min, so that centrifugation tube wall
On magnetic bead be completely dried；
4.9,21ul Resuspension Buffer is added, it is careful to mix, it is ensured that the magnetic bead on tube wall is completely dissolved in
In Buffer；
4.10, centrifuge tube is reapposed on magnetic frame, 5min is placed, so that magnetic bead is adsorbed on tube wall completely；
4.11,20ul eluent is carefully drawn into new 1.5ml centrifuge tube.
(2) sequencing library quality inspection
1) using Agilent 2100bioanalyzer and Agilent DNA 7500and 12000Assay Kit to text
Library quality inspection is detected, the specific steps are as follows:
1.1, prepare gel-dye Mix (glue, dyestuff mixed liquor): by Agilent DNA 7500and 12000Assay
Kit thaw at RT 30min, oscillation mix DNA dye, add in 25ul dye to DNA gel glue, oscillation mix and be transferred to containing
In the centrifuge tube of centrifugal column, 1500g is centrifuged 10min, and filtered gel-dye solution is kept in dark place；
1.2, encapsulating: prepare a new DNA chip and be placed on chip shelf, add 9ul gel-dye mixed liquor to mark
It is denoted as in the hole of G, syringe is then adjusted to the position 1ml and is covered on chip, push syringe to detent, will be infused after standing 30s
Emitter is recalled to the position 1ml, and 9ul gel-dye mixed liquor is then added in other two hole labeled as the position G；
1.3,5ul marker (Green Marker) is added in chip well and the hole ladder；
1.4,1ul library DNA and 1ul Ladder is taken to be separately added into chip well and the hole ladder, 2400rpm vibration
1min is swung to mix sample, and then chip is placed on 2100 biological analyser of Agilent and is detected.
2) library is quantified using Qubit spectrophotometer
2.1, by QubitTMDsDNA HS Assay Kit (500assays) takes out reagent from 4 DEG C of refrigerators and is placed at room temperature for
Then 30min prepares mixed liquor in 0.5ml centrifuge tube according to following system:
Quantitative mixture system:
2.2, oscillation is protected from light after mixing stands 2min；
2.3, spectrophotometer, examination criteria sample Standard 1 and 2 are calibrated；
2.4, sample Sample is detected, testing result is recorded.
(3) high-flux sequence
Sample library is sequenced using Miseq sequenator, sequencing agents useful for same is MiSeq Reagent Kit v3
(600-cycles)。
(4) data analysis result
Sequencing data analysis is compared using Bowtie2 software with human genome reference sequences (hg19), with SAM work
Have software comparison result progress Mutation Screening analysis (screening conditions: 1, is compared quality and is greater than 20；2, sequencing depth is greater than
100), functional annotation is carried out with mutation of the ANNOVAR software to screening.Analyze acquired results sanger method sequence verification.
By above-mentioned experiment and analytical procedure, analysis as a result, 5 MMR genes always amplification rate be up to 61.89%~
88.17%, exon coverage be 100%, also can 5 MMR genes of amplification gene all exons.
Above data absolutely proves, combines 1~11 pair of 5 MMR gene extron using primer pair of the invention and carries out PCR
Amplification, accuracy height, high specificity, high sensitivity.
3 17 colorectal cancer cases of embodiment and 14 normal person's MMR detection in Gene Mutation
One, experimental material, with embodiment 2.
Two, pattern detection
(1), multiplex PCR targeting enrichment
Using the kit of Qiagen, according to kit illustrate in method, to 31 samples (17 Colon and rectum carninomatosis
Example and 14 normal persons) extract genomic DNA.Using 11 groups of primer pair combinations as shown in 2 table 3 of embodiment to 31 samples
Genomic DNA carries out multiplexed PCR amplification, and specific method is the same as embodiment 2.
(2), multiple PCR products purify, and specific method is the same as embodiment 2.
(3), sequencing library constructs, and specific method is the same as embodiment 2.
(4), sequencing library quality inspection, specific method is the same as embodiment 2.
Three, high-flux sequence, specific method is the same as embodiment 2.
Four, data analysis result
Sequencing data analysis is compared using Bowtie2 software with human genome reference sequences (hg19), with SAM work
Have software comparison result progress Mutation Screening analysis (screening conditions: 1, is compared quality and is greater than 20；2, sequencing depth is greater than
100), functional annotation is carried out with mutation of the ANNOVAR software to screening.Analyze acquired results sanger method sequence verification, portion
Divide result as shown in Figure 6.Again, the accuracy of the method for the present invention has been confirmed.
5 MMR genes of enrichment are targeted by multiplex PCR and 2.7gigabases is obtained in high-flux sequence, 31 samples
(Gb) data, average each sample 86Mb, average reads number are 287048.As shown in table 4, wherein five of 30 samples are sieved
Looking into gene (MLH1, MSH2, MSH6, PMS1, PMS2) exon sequencing coverage is 100%, and 1 is 96.8%, average to cover
Degree is 99.9%, and exon region average sequencing depth is 2284.By analysis of biological information, 31 sequencing samples find 13 altogether
Kind single nucleotide variations (single nucleotide variation, SNV) non-synonymous, 2 kinds of synonymous SNV, in SNV non-synonymous
In, there are 3 positioned at MLH1 gene: c.A655G:p.I219V, c.T1151A:p.V384D, c.C2101A:p.Q701K；It is located at
MSH2 gene has 3: c.C1168T:p.L390F, c.A1690G:p.T564A, c.G2425A:p.E809K；Positioned at MSH6 base
Cause has 3: c.G116A:p.G39E, c.G3205C:p.G1069R, c.A3488T:p.E1163V；Positioned at having for PMS2 gene
4: c.G59A:p.R20Q, c.A1621G:p.K541E, c.C1408T:p.P470S, c.C1454A:p.T485K.2 synonymous
SNV is MSH6 gene c.T3306A and PMS2 gene c.C780G；SNV is not detected in gene PMS1.These SNV and database
DbSNP and InSight are retrieved, and 14 kinds of discovery is it has been reported that crossing, wherein the c.G3205C:p.G1069R of MSH6 has no
Report.
As shown in table 5, by above-mentioned experiment and analytical procedure, in 17 colorectal cancer cases and 14 normal person's samples
13 kinds of exon region of discovery single nucleotide variations non-synonymous, 2 kinds of synonymous single nucleotide variations altogether.One of them causes a disease or may
Pathogenic variation, i.e. c.C2101A:p.Q701K；Two pathogenic uncertain variations, i.e. c.G2425A:p.E809K,
C.G3205C:p.G1069R, remaining is tolerable variation.
Table 4
Table 5:MMR gene mutation for screening result
The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation,
It should be pointed out that under the premise of not departing from the method for the present invention, can also be made for those skilled in the art
Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more
Dynamic, modification and the equivalent variations developed, are equivalent embodiment of the invention；Meanwhile all substantial technologicals pair according to the present invention
The variation, modification and evolution of any equivalent variations made by above-described embodiment, still fall within the range of technical solution of the present invention
It is interior.
Claims (5)
1. whether there is the kit of MMR genetic mutation in a kind of test sample, which is characterized in that in the kit, contain
11 groups of multiple PCR primers are respectively as follows: combination
(1) primer pair combination 1: by SEQ ID NO.7 and SEQ ID NO.8；SEQ ID NO.23 and SEQ ID NO.24；SEQ
ID NO.27 and SEQ ID NO.28；SEQ ID NO.63 and SEQ ID NO.64；SEQ ID NO.97 and SEQ ID NO.98；
SEQ ID NO.111 and SEQ ID NO.112；SEQ ID NO.127 and SEQ ID NO.128；SEQ ID NO.149 and SEQ
ID NO.150 composition；
(2) primer pair combination 2: by SEQ ID NO.3 and SEQ ID NO.4；SEQ ID NO.33 and SEQ ID NO.34；SEQ
ID NO.39 and SEQ ID NO.40；SEQ ID NO.67 and SEQ ID NO.68；SEQ ID NO.77 and SEQ ID NO.78；
SEQ ID NO.81 and SEQ ID NO.82；SEQ ID NO.117 and SEQ ID NO.118；SEQ ID NO.137 and SEQ ID
NO.138；SEQ ID NO.153 and SEQ ID NO.154；SEQ ID NO.173 and SEQ ID NO.174 composition；
(3) primer pair combination 3: by SEQ ID NO.17 and SEQ ID NO.18；SEQ ID NO.25 and SEQ ID NO.26；
SEQ ID NO.37 and SEQ ID NO.38；SEQ ID NO.59 and SEQ ID NO.60；SEQ ID NO.79 and SEQ ID
NO.80；SEQ ID NO.101 and SEQ ID NO.102；SEQ ID NO.113 and SEQ ID NO.114；SEQ ID NO.123
With SEQ ID NO.124；SEQ ID NO.141 and SEQ ID NO.142；SEQ ID NO.163 and SEQ ID NO.164 group
At；
(4) primer combination 4: by SEQ ID NO.21 and SEQ ID NO.22；SEQ ID NO.35 and SEQ ID NO.36；SEQ
ID NO.61 and SEQ ID NO.62；SEQ ID NO.87 and SEQ ID NO.88；SEQ ID NO.99 and SEQ ID
NO.100；SEQ ID NO.119 and SEQ ID NO.120；SEQ ID NO.133 and SEQ ID NO.134；SEQ ID
NO.147 and SEQ ID NO.148；SEQ ID NO.155 and SEQ ID NO.156；SEQ ID NO.159 and SEQ ID
NO.160 composition；
(5) primer combination 5: by SEQ ID NO.5 and SEQ ID NO.6；SEQ ID NO.19 and SEQ ID NO.20；SEQ ID
NO.41 and SEQ ID NO.42；SEQ ID NO.47 and SEQ ID NO.48；SEQ ID NO.55 and SEQ ID NO.56；SEQ
ID NO.89 and SEQ ID NO.90；SEQ ID NO.95 and SEQ ID NO.96；SEQ ID NO.115 and SEQ ID
NO.116 composition；
(6) primer combination 6: by SEQ ID NO.11 and SEQ ID NO.12；SEQ ID NO.31 and SEQ ID NO.32；SEQ
ID NO.53 and SEQ ID NO.54；SEQ ID NO.65 and SEQ ID NO.66；SEQ ID NO.75 and SEQ ID NO.76；
SEQ ID NO.91 and SEQ ID NO.92；SEQ ID NO.103 and SEQ ID NO.104；SEQ ID NO.109 and SEQ ID
NO.110；SEQ ID NO.125 and SEQ ID NO.126；SEQ ID NO.161 and SEQ ID NO.162 composition；
(7) primer combination 7: by SEQ ID NO.1 and SEQ ID NO.2；SEQ ID NO.15 and SEQ ID NO.16；SEQ ID
NO.57 and SEQ ID NO.58；SEQ ID NO.85 and SEQ ID NO.86；SEQ ID NO.93 and SEQ ID NO.94；SEQ
ID NO.129 and SEQ ID NO.130；SEQ ID NO.145 and SEQ ID NO.146；SEQ ID NO.9 and SEQ ID
NO.10 composition；
(8) primer combination 8: by SEQ ID NO.13 and SEQ ID NO.14；SEQ ID NO.29 and SEQ ID NO.30；SEQ
ID NO.49 and SEQ ID NO.50；SEQ ID NO.69 and SEQ ID NO.70；SEQ ID NO.83 and SEQ ID NO.84；
SEQ ID NO.121 and SEQ ID NO.122；SEQ ID NO.131 and SEQ ID NO.132；SEQ ID NO.135 and SEQ
ID NO.136；SEQ ID NO.43 and SEQ ID NO.44 composition；
(9) primer combination 9: by SEQ ID NO.105 and SEQ ID NO.106；SEQ ID NO.73 and SEQ ID NO.74 group
At；
(10) primer combination 10: by SEQ ID NO.107 and SEQ ID NO.108；SEQ ID NO.71 and SEQ ID NO.72；
SEQ ID NO.171 and SEQ ID NO.172 composition；
(11) primer combination 11: by SEQ ID NO.139 and SEQ ID NO.140；SEQ ID NO.151 and SEQ ID
NO.152；SEQ ID NO.165 and SEQ ID NO.166；SEQ ID NO.167 and SEQ ID NO.168；SEQ ID
NO.169 and SEQ ID NO.170；SEQ ID NO.51 and SEQ ID NO.52；SEQ ID NO.143 and SEQ ID
NO.144；SEQ ID NO.157 and SEQ ID NO.158；SEQ ID NO.45 and SEQ ID NO.46 composition.
2. kit according to claim 1, which is characterized in that in the kit, also containing total DNA extraction agent,
One of PCR reagent, PCR product purified reagent or operation instructions are a variety of.
3. a kind of method for obtaining MMR gene amplification product, the method is non-disease diagnostic purpose, the method packet
It includes: using nucleic acid samples as template, carrying out PCR amplification using the kit as described in claim 1~2 any claim, obtain
Obtain amplified production.
4. according to the method described in claim 3, it is characterized in that, using in kit as stated in claim 1 or 2
11 groups of primer pair combinations carry out multiplexed PCR amplification as primer respectively, and obtain 11 groups of multiple PCR products are merged to get expansion
Increase production object.
5. according to the method described in claim 4, it is characterized in that, 1-8 group and 11 groups of 50 μ l of multi-PRC reaction system,
It include: 10 μ l of 5X Multiplex PCR Master Mix, 1.0 μM of 2.5-20 μ l of template 50ng, Forward Primer,
Reverse Primer 1.0 μM of 2.5-20 μ l, ddH2O complements to 50 μ l；The 50 μ l of multi-PRC reaction system of 9-10 group:
2X GC Buffer I25 μ l, dNTP 4ul, template 50ng, Forward Primer 1.0 μM of 2.0 μ l, Reverse
Primer 1.0 μM of 2.0 μ l, ddH2O complements to 50 μ l.
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