CN106367475A - MMR gene mutation detection kit - Google Patents
MMR gene mutation detection kit Download PDFInfo
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- CN106367475A CN106367475A CN201510437485.5A CN201510437485A CN106367475A CN 106367475 A CN106367475 A CN 106367475A CN 201510437485 A CN201510437485 A CN 201510437485A CN 106367475 A CN106367475 A CN 106367475A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention belongs to the field of molecular biology and medicine, and particularly relates to a DNA mismatch repair gene (MMR) mutation detection kit and uses thereof, more particularly to mutation detection of human MLH1 gene, human MSH2 gene, human MSH6 gene, human PMS1 gene and human PMS2 gene, and detection of the correlation with hereditary nonpolyposis colorectal cancer (HNPCC). According to the present invention, the kit can be used for detecting the genotype of the exon mutation of the individual MMR gene so as to provide the reference data for judging whether the individual has HNPCC and whether the suffering risk of family members is greater than the suffering risk of the general populations.
Description
Technical field
The present invention relates to molecular biology and medical domain are and in particular to a kind of mmr gene mutation detection kit.
Background technology
Colorectal cancer (colorectal cancer, crc) is the third-largest common cancer in the whole world.Research finds 35%
There is family's susceptibility in crc, most common of which is hereditary nonpolyposis colorectal cancer (hereditary nonpolyposis
Colorectal cancer, hnpcc), account for the 2%～5% of all cases.Dna mismatch repair gene (mismatch repair genes,
Mmr) there are hereditism's pathogenesis that germline mutation is lynch syndrome.Research finds at least 5 kinds mmr genes
The germline mutation of (msh1, mlh2, msh6, pms1, pms2) can lead to hnpcc, the wherein embryonal system of mlh1
Mutation accounts for 50%, msh2 and accounts for 40%, msh6 and accounts for 7%, and remaining gene accounts for 3%.In family, mmr gene germline
Carriers of mutation is that the high-risk group of hnpcc related neoplasms occurs, and detection mmr gene germ line mutation can be predicted well
The causing danger of hnpcc related neoplasms.For having been found that the patient that there is inherited mismatch repair gene mutation, can closely supervise
Survey Metachro nous Multiple Primary cancer and the generation of other hnpcc related neoplasms, and to other members in its family, particularly not yet go out
The younger members of existing tumor are accordingly detected, confirm mutant gene carrier, not only help and cancer susceptibility individual weight is clicked through
Early diagnosiss before row clinical monitoring and clinical symptoms appearance, alternatively genetic counselling and gene therapy provide foundation, and can make non-
Carriers of mutation exempts unnecessary spirit and financial burden, to mutation carriers, can carry out close follow-up, to accomplish
Early diagnosiss, early treatment.
Although the method being presently used for screening hnpcc is more, only detect the mmr genopathy of embryonal system (germline)
Rationality mutation just can be diagnosed as hnpcc patient.Direct gene sequencing is that mmr germline abrupt climatic change is the sensitiveest and
Special method, but it is sequenced time-consuming and expensive based on sanger method it is impossible to meet the detection demand of clinically large sample amount.
Compared with being sequenced with traditional sanger method, second filial generation high throughput sequencing technologies have that flux is high, speed is fast, the low advantage of expense,
In conjunction with specific nucleic acid sequence targeting beneficiation technologies, it is possible to achieve to disease, maximally related gene carries out deep sequencing.
Content of the invention
For the defect in the presence of prior art, it is an object of the invention to, provide and in a kind of detection sample, whether there is mmr
The reagent of genovariation.
Another object of the present invention is to, the purposes of the reagent that whether there is mmr genovariation in described detection sample is provided,
Test kit for preparation detection mmr genovariation.
It is another object of the present invention to provide a kind of test kit of detection mmr genovariation.
It is another object of the present invention to provide a kind of method obtaining amplified production.
The present invention is achieved by the following technical solutions:
A first aspect of the present invention, there is provided whether there is the reagent of mmr genovariation, described examination in a kind of detection sample
Agent 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 a second aspect of the present invention, the purposes of the reagent that whether there is mmr genovariation in described detection sample is provided, uses
Test kit in preparation detection mmr genovariation.
In a third aspect of the present invention, provide the test kit that whether there is mmr genovariation in a kind of detection sample, described
One or more primer pair of aforementioned 87 pairs of primer pairs is contained in test kit.
In another preference, in described test kit, contain aforementioned all 87 pairs of primer pairs.
In another preferred embodiment, in described test kit, containing 11 groups of multiple pcr primer pair combinations, 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
With 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
With 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
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 forms；
(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 forms；
(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
With 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 forms；
(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.
Use pcr based on the described test kit of the present invention to be detected, can also include some other in described test kit
Reagent, such as: total one or more of dna extraction agent, pcr reagent, pcr product purification reagent.Specifically need by
Which reagent is fitted into test kit, can configure according to actual needs.
Described total dna extraction agent can be using the reagent of various conventional extracting dna, the commercially available acquisition of these reagent.
Described pcr reagent can be using various conventional pcr reagent, the commercially available acquisition of this reagent.
Described pcr product purification reagent can be using various conventional pcr product purification reagent, the commercially available acquisition of this reagent.
Additionally, also including operation instructions in described test kit, being easy to those skilled in the art and using.
In a fourth aspect of the present invention, provide a kind of method of acquisition mmr gene amplification product, described method includes: with
Nucleic acid samples are template, carry out pcr amplification using selected from aforesaid primer pair or test kit, obtain amplified production.
For the template of pcr amplification, that is, nucleic acid samples (as genome dna) may also be employed the conventional method of this area and carry
Take acquisition.In another preference, using classical genome dna extracting method or the genome extracts reagent of commercialization
Box, extracts genome dna, as sample of nucleic acid from sample.
In another preference, carry out multiple pcr amplification by the use of aforementioned 11 groups of primer pairs combination respectively as primer, will obtain
11 groups of multiple pcr products merge, obtain final product amplified production.
Except the primer using the present invention, the present invention does not particularly limit to each components other in pcr reaction system and its final concentration
System, those skilled in the art can set up, according to routine, the general composition adopting during pcr system and its concentration sets up pcr reaction system.
In another preference, the multiple pcr reaction system 50 μ l of 1-8 group and 11 groups, comprising: 5x multiplex pcr
Master mix 10 μ l, template 50ng, 1.0 μm of 2.5-20 μ l of forward primer, 1.0 μm of reverse primer
2.5-20 μ l, ddh2O complements to 50 μ l.The multiple pcr reaction system 50 μ l:2x gc buffer i 25 μ l of 9-10 group,
Dntp 4ul, template 50ng, 1.0 μm of 2.0 μ l of forward primer, reverse 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 multiple pcr, its design difficulty can be double therewith.It can be said that design of primers be multiple pcr
Big obstacle.The present invention relates to a kind of multiple pcr targeting enrichment combine high-flux sequence detect mmr gene mutation test kit
And method, the present invention can expand 5 mmr genes (msh1, mlh2, msh6, pms1, pms2) by design
The primer of all exons, and the method that independent development multiple pcr targeting is enriched with mmr gene extron subsequence, realize mmr
Second filial generation high throughput sequencing technologies, the germline mutation of detection mmr gene is utilized after the amplification of gene extron.The present invention represents
Up-to-date secondary high throughput sequencing technologies are mutated in hereditary nonpolyposis colorectal cancer (hnpcc) mmr germline
The technological break-through of detection.It is enriched with mmr gene extron subsequence by multiple pcr targeting and combines second filial generation high-flux sequence skill
It is achieved that detect the germline mutation of 5 kinds of mmr genes simultaneously, the clinical diagnosises for hnpcc and research offer provide art
The technical method that flux is high, speed is fast, expense is low.The method of our inventions and test kit are surveyed with the miseq of illumina company
Sequence instrument is main platform, but this method and invention are equally applicable to other high-flux sequence platforms, including illumina company
Ion pgm/proton system of hiseq 2500/3000/4000, nextseq 500 and life company etc..
Brief description
Fig. 1: for the Technology Roadmap of the present 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 detecting.
Fig. 3: abscissa is clip size, i.e. bp number；Vertical coordinate is signal intensity, 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 detecting.
Fig. 5: abscissa is clip size, i.e. bp number；Vertical coordinate is signal intensity, i.e. nucleic acid content.
Fig. 6: it is the sanger sequence verification result sectional drawing 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, through extensively in-depth study, have found a class and is particularly suitable for expanding outside all 73 of 5 mmr genes
The primer of aobvious son, described primer designs, preferably obtains through rational, and when expanding for pcr, specificity is good, and expands
Efficiency high, the coverage for all 73 exons is up to 100%.The present inventor also optimizes pcr amplified reaction, for example,
Multiple pcr reaction system, further increases amplification efficiency.
For the shortcoming of current detection mmr gene mutation process very complicated, consuming time length, inventor have also been devised a kind of inspection
The method surveying 5 mmr gene mutation, methods described is simple, take short and low cost.Based on this, complete the present invention.
Mmr gene or mmr genetic mutation site can be detected using various technology, when detecting mmr genovariation, detection
Can be for cdna it is also possible to be directed to genome dna.Available existing prior art such as southern blotting, dna sequence
Analysis, pcr and in situ hybridization detection mutation.
It is conveniently to carry out pcr with the primer of mmr gene specific, mmr gene is expanded, amplified production is entered
Row sequencing, thus judge whether to morph.
The present inventor finds under study for action, and when detection mmr gene or mmr gene mutation, using general primer, pcr expands
Poor specificity during increasing, amplification efficiency is low.It is thus desirable to designing and screening the good primer of specificity.Through substantial amounts of test and
Relatively, the present inventor have found the primer corresponding respectively to all 73 exons of 5 mmr genes.Primer described in empirical tests
The amplified production obtaining both had included as complete as possible sequence on each exon of mmr, and specificity is very good, does not almost have
Non-specific amplification or the especially few situation of amplified production.It is particularly well-suited to the pcr amplification of complex system.The nucleotide of each primer
Sequence is as shown in table 2.
Multiple pcr has many good qualities relative to substance pcr, can be time-consuming, saves reagent, and reduction of expenditure is paid wages, and is
The prediction of disease and preventing and treating provide more more accurately diagnostic messages.Substance pcr detect, when the sequence that primer is directed to there occurs prominent
Then it is likely to occur false negative result during change, and be directed to the multiple pcr detection of cause of disease of the same race, because being directed to multiple genes, with
When the probability very little that morphs in multiple sites, the probability of false negative appearance can be substantially reduced.
Multiple pcr reaction system is more difficult to be set up it is desirable to guarantee will not interact between the primer in reaction, amplified production size
Close but can by electrophoresis separately.Multiple pcr amplification is very high to test operation requirement, and it is improper that any experiment condition controls,
The failure of amplification condition all will be easily lead to.In prior art, the foundation of the multiple pcr system of report, does not change pcr anti-
When answering condition, carry out double pcr and be easier to obtain expected resultss, and triple or more pcr is expanded relatively difficult.
Present inventor passes through to design multigroup primer combination, to multiple pcr system optimization, is successfully established multiple pcr detection system, energy
Enough disposably detect that several genes are mutated, not only can quickly, succinct, detection disease exactly, and detection can be greatlyd save
Cost.
Present invention also offers for detecting the test kit containing mmr gene or mmr genovariation in analyte, this reagent
Box includes being contained in the appropriate containers, primer for specific amplification mmr gene or mmr genovariation, and with described
The variation position related containing disease on mmr gene or mmr gene-correlation exon that primer amplification goes out.Described test kit
In containing selected from table 2 at least one pair of primer, for obtain correlation mmr gene or mmr gene extron on comprise become dystopy
The sequence of point.Optimal, contain all of primer pair in table 2 in described test kit, be so more beneficial for intactly analyzing mmr
Gene or the variation situation of each exon sequence of mmr gene.
Additionally, may also include in described test kit for extract dna, pcr amplification etc. needed for various reagents, including but not
It is limited to: extract, amplification liquid, hybridization solution, enzyme, washing liquid etc..These are all that those skilled in the art are understood.
Additionally, may also include operation instructions and/or Nucleotide Sequence Analysis Software etc. in described test kit, it is easy to those skilled in the art
Use and analyze.
The method obtaining mmr gene amplification product
The present inventor additionally provides a kind of method obtaining mmr gene amplification product from nucleic acid samples of optimization, described method
Including: with sample of nucleic acid as template, carry out pcr amplification using the primer pair selected from table 2, obtain amplified production.
Using described primer, ideal amplification can be obtained using conventional pcr amplification method.A kind of optional
In pcr amplification method, pcr thermal cycle step is as follows: 98 DEG C of degeneration are annealed 30 seconds, 72 DEG C and extended 60 seconds for 10 seconds, 55 DEG C, and three
Ten circulations.
Carrying out the template denaturation step before pcr thermal cycle can be according to the conventional method in this area.Preferably method is as follows: 94 DEG C pre-
Degeneration 1 minute.
Carrying out the extension step of the sequence after pcr thermal cycle can be according to the conventional method in this area.Preferably method is as follows: 72 DEG C are prolonged
Stretch 4 minutes.
Except the primer using the present invention, the present invention has no particular limits to each compositions other in pcr amplification system and its concentration,
Those skilled in the art can set up, according to routine, the general composition adopting during pcr system and its concentration sets up pcr amplification system.
Template (as genome dna) for pcr amplification can adopt the conventional method of this area to extract acquisition.
Using the acquisition mmr gene of the present invention or the method for mmr gene amplification product, amplification efficiency and all non-convention of specificity
Think, and be particularly suitable for the pcr amplification of complex system, such as using blood sample genome dna as the amplification of pcr template.
Present invention also offers a kind of determine the method with the presence or absence of mmr genovariation in testing sample, described method includes:
Mmr genetic fragment is expanded from determined nucleic acid sample using aforesaid method, obtains amplified production；And in analysing amplified product
The sequence of mmr genetic fragment, and be compared with corresponding sequence in wild type mmr gene；If there is difference, then table
There is mmr genovariation in bright determined nucleic acid sample.
By the situation of genovariation, the illness of experimenter's hereditary nonpolyposis colorectal cancer (hnpcc) can be learnt further
Risk, thus reach the purpose of early detection early prevention.
Main advantages of the present invention are:
(1) it is directed to 5 mmr genes first and all 73 exons of 5 mmr genes devise suitable primer, described
Primer specificity when expanding for pcr is good, and amplification efficiency is high.
(2) optimize the method obtaining mmr gene amplification product, methods described accurately and fast, stable, success rate high.
Before further describing the specific embodiment of the invention it should be appreciated that protection scope of the present invention be not limited to following specific
Specific embodiments；It is also understood that term is to describe specific specific embodiments used in the embodiment of the present invention,
Rather than in order to limit the scope of the invention.
When embodiment provides numerical range it should be appreciated that except non-invention is otherwise noted, two end points of each numerical range with
And any one numerical value all can be selected between two end points.Unless otherwise defined, used in the present invention, all technology and section are academic
The same meaning that language and those skilled in the art of the present technique are generally understood that.In addition to concrete grammar, equipment, material used in embodiment,
According to the record of the grasp to prior art for the those skilled in the art and the present invention, can also using and the embodiment of the present invention
Described in method, the similar or equivalent any method of prior art of equipment, material, equipment and material to be realizing the present invention.
Unless otherwise indicated, disclosed in this invention experimental technique, detection method, preparation method all normal using the art
The molecular biology of rule, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, restructuring dna technology and
The routine techniquess of association area.These technology have improved explanation in existing document, specifically can be found in 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 scheme route map is as shown in figure 1, mainly include following sport technique segment:
Sport technique segment one: carry out multiple pcr targeting enrichment mmr gene extron using the primer pair and combinations thereof of the present invention.
Sport technique segment two: 5 genes (msh1, mlh2, msh6, pms1, pms2) that sport technique segment one is produced are outward
Show sub- amplified production and carry out sequencing and identification using secondary high throughput sequencing technologies.
Embodiment 1 design of primers and synthesis
The present invention is to 73 exons of 5 genes (msh1, mlh2, msh6, pms1, pms2) altogether 11419
Individual base devises 87 couples of primer, and pcr amplified production length is 257-528bp, and this combination probe primer can cover 5
All exon regions of mmr gene (being shown in Table 1).Primer synthesis is synthesized by Shanghai Sheng Gong biological engineering company limited.Primer
Sequence information refers to 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 embodiment 2 primer and multiple pcr targeting enrichment method
First, experiment material
(1), main agents
1st, fluorescence quantitative detection kit: qubittmDsdna hs assay kit (500assays), life technologies company；
2nd, immue quantitative detection reagent box: agilent dna 7500and 12000kit, agilent company, this test kit comprises；
3rd, multiplex pcr test kit: mixmultiplex pcr 5 × master mix, neb company；
4th, sequencing library jointing test kit: nextflextmDna barcode-48, bioo scientific company；
5th, sequencing library builds test kit: nextflextmRapid dna-seq kit, bioo scientific company；
6th, sequencing kit: miseq reagent kit v3 (600-cycles), illumina company
7th, purification kit: agencourt ampure xp beads, beckman coulter (agencourt) company
(2), key instrument
1st, 0.5ul～2ml pipettor, eppendof, Germany；
2nd, micro centrifuge, Hangzhou Ao Sheng Instrument Ltd.；
3rd, high speed centrifuge, sigma, Germany；
4th, turbula shaker, scientific industries, the U.S.；
5th, dry-type thermostat, techne, Germany；
6th, tc-412 type pcr instrument, techne, Germany；
7th, agilent 2100 biological analyser, agilent, Germany；
8th, qubit fluorescent quantitation instrument, invitrogen, the U.S.；
9th, miseq sequencer sequenator, illumina, the U.S.；
2nd, single tube pcr amplification
1., using the gdna of commercialization as template, it is respectively adopted as shown in table 2 87 pair primer pair as primer, carries out single tube
Pcr expands.When being expanded using the independent pcr of every group of primer pair of 87 centerings, reaction system used and amplification condition are as follows, used
Pcr kit be takara ex
Reaction system:
Reaction condition:
2. pair each pair independent to primer amplification obtained by single tube pcr amplified production carry out purification, concrete grammar with the present embodiment three,
Method in 2.
3. by obtain 87 groups of pcr products respectively after purification, mixed in equal amounts, and using the detection of agilent 2100 biological analyser,
Result is as shown in Figures 2 and 3.Wherein, as shown in Fig. 2 the top band (green) and nethermost band (purple)
It is dna ladder, be equivalent to marker；Middle a plurality of black stripe is the sample strip detecting.As Fig. 3, abscissa is
Clip size, i.e. bp number；Vertical coordinate is signal intensity, i.e. nucleic acid content.87 pairs of primers designed by the present invention are to pin 5
The specific primer of all exon regions of individual mmr gene, according to Fig. 2's and Fig. 3 as a result, it is possible to find out using this 87 couple
When primer pair carries out single tube pcr, can expand out and obtain corresponding pcr amplified production, absolutely prove this 87 pairs of primer pairs
Specificity and effectiveness.
3rd, multiple pcr amplification
1. further, the 87 pairs of primer pairs (as shown in table 2) embodiment 1 designed and optimizes are combined, and obtain as table 3
11 groups of shown primer pair combinations.Then using the gdna of commercialization as template, drawn using this 11 groups of primer pair combination conducts
Thing carries out multiple pcr amplification respectively.The dna template amount of every group of multiple pcr is 10-200 nanogram, wherein 1-8 and 11
Group carries out multiple pcr amplification, the 9th and 10 group of use using neb mixmultiplex pcr 5 × master mix test kit
Takara la tag with gc buffer test kit carries out multiple pcr amplification, and amplification condition is: 95 DEG C of denaturations 1 minute；
95 DEG C of degeneration are annealed 60 seconds, 68 DEG C and are extended 30 seconds for 20 seconds, 63 DEG C, 30 circulations；68 DEG C extend 5 minutes.
Table 3:11 group primer pair combines
1-8 group and 11 groups of multiple pcr 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 groups of multiple pcr reaction systems:
| 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. purification will be carried out respectively using the multiple pcr product obtained by the combination of every group of primer pair:
(1) after pcr amplified reaction terminates, take out 96 hole pcr Sptting plates, tear sealed membrane, every hole adds 48ul ampure
Xp beads, upper and lower mix homogeneously；
(2) room temperature places 5min；
(3) centrifuge tube is positioned on 96 orifice plate magnetic frames, places 5min so that magnetic bead adsorbs completely on tube wall；
(4) with pipettor, the liquid in centrifuge tube is carefully drawn and abandon；
(5) add freshly prepared 80% ethanol of 200ul in 96 hole pcr plates on magnetic frame, inhaled with pipettor after placing 30s
Take supernatant and abandon；
(6) repeat step 1.3.6.3.5 it is ensured that in centrifuge tube remaining ethanol be all sucked out；
(7) 96 orifice plates are removed from magnetic frame, spontaneously dry 2min so that the magnetic bead on wall is completely dried；
(8) add 21ul resuspension buffer, carefully mix it is ensured that the magnetic bead on tube wall is completely dissolved in buffer；
(9) 96 orifice plates are reapposed on magnetic frame, place 5min so that magnetic bead is adsorbed onto on tube wall completely；
(10) careful 20ul eluent of drawing is to new 1.5ml centrifuge tube.
3., after adopting 11 groups of primer pair combinations individually to expand pcr product purification, obtain 11 groups of multiple pcr after purification and produce
Thing, then by after this 11 groups multiple pcr product mixed in equal amounts after purification, is detected using agilent 2100 biological analyser,
Result is as shown in Figure 4 and Figure 5.Wherein, as shown in figure 4, the top band (green) and nethermost band (purple)
It is dna ladder, be equivalent to marker；Middle a plurality of black stripe is the sample strip detecting.As Fig. 5, abscissa is
Clip size, i.e. bp number；Vertical coordinate is signal intensity, i.e. nucleic acid content.The result of Fig. 4, Fig. 5 and Fig. 2, Fig. 3 are entered
Row compares, and carries out multiple pcr respectively using 11 groups of primer pair combinations and expands the g dna of commercialization and using 87 groups of primer pairs
Carry out the g dna that substance pcr expands commercialization respectively, the result obtaining is consistent, absolutely proves, using 11 groups of primers
The g dna that multiple pcr expands commercialization is carried out respectively to combination, is feasible.
4th, using second filial generation high throughput sequencing technologies, sequencing and identification are carried out to mmr gene extron amplified production
The multiple pcr product that above-mentioned steps are produced, after qualitative and detection by quantitative, for the accuracy of the present invention can be investigated,
Carry out carrying out sequencing to mmr gene extron amplified production using second filial generation high throughput sequencing technologies simultaneously, to judge described
The accuracy of primer amplification.The mainly preparation of sequencing library and sequencing.Library preparation includes pcr product end-filling, 5` end
Phosphorylation, 3` end add a, connect illumina sequence measuring joints, connection product pcr enrichment and purification, then using miseq sequenator
Carry out Sequencing and Characterization.
Specifically, (1) sequencing library builds
Using nextflextmRapid dna-seq kit carries out the structure of sequencing library, comprises the following steps that
1) dna end-filling and 3 ' ends plus a
1.1st, template uses gdna multiple pcr product, and building storehouse initial amount is 200ng, and reaction cumulative volume is 50ul, prepares enzyme anti-
Answer mixed liquor.
Reaction system:
1.2nd, it is placed in pcr instrument after mixed liquor being mixed and carry out enzyme reaction.
Enzyme reaction condition:
2) sequence measuring joints adapter are connected
2.1st, prepare coupled reaction mixed liquor
Reaction system:
2.2nd, it is placed in pcr instrument after mixing night being mixed and carries out enzyme reaction, reaction condition is: 25 DEG C of incubation 30min.
3) connection product purification
3.1st, connection product is transferred in new 1.5ml centrifuge tube, is subsequently adding 48ul ampure xp beads, mix homogeneously；
3.2nd, room temperature places 5min；
3.3rd, centrifuge tube is positioned on magnetic frame, places 5min so that magnetic bead adsorbs completely on tube wall；
3.4th, with pipettor, the liquid in centrifuge tube is carefully drawn and abandon；
3.5th, add freshly prepared 80% ethanol of 200ul in the centrifuge tube on magnetic frame, after placing 30s, draw supernatant with pipettor
Liquid simultaneously abandons；
3.6th, repeat step 3.5 it is ensured that in centrifuge tube remaining ethanol be all sucked out；
3.7th, centrifuge tube is transferred on common centrifuge tube shelf from magnetic frame, spontaneously dry 2min so that the magnetic bead on centrifugation tube wall is complete
White drying；
3.8th, add 21ul resuspension buffer, carefully mix it is ensured that the magnetic bead on tube wall is completely dissolved in buffer；
3.9th, centrifuge tube is reapposed on magnetic frame, place 5min so that magnetic bead is adsorbed onto on tube wall completely；
3.10th, careful 20ul eluent of drawing is to new 1.5ml centrifuge tube.
4) pcr enrichment
4.1st, prepare pcr reaction system, and reaction mixture is added in pcr reaction tube；
Reaction condition:
4.2nd, pcr reaction tube is placed on pcr instrument and carries out pcr amplification；
Reaction condition
4.3rd, pcr product is transferred in new 1.5ml centrifuge tube, is subsequently adding 44ul ampure xp beads, mix homogeneously is simultaneously
Room temperature places 5min；
4.4th, centrifuge tube is positioned on magnetic frame, places 5min so that magnetic bead adsorbs completely on tube wall；
4.5th, with pipettor, the liquid in centrifuge tube is carefully drawn and abandon；
4.6th, add freshly prepared 80% ethanol of 200ul in the centrifuge tube on magnetic frame, after placing 30s, draw supernatant with pipettor
Liquid simultaneously abandons；
4.7th, repeat step 1.3.6.3.5 it is ensured that in centrifuge tube remaining ethanol be all sucked out；
4.8th, centrifuge tube is transferred on common centrifuge tube shelf from magnetic frame, spontaneously dry 2min so that the magnetic bead on centrifugation tube wall is complete
White drying；
4.9th, add 21ul resuspension buffer, carefully mix it is ensured that the magnetic bead on tube wall is completely dissolved in buffer；
4.10th, centrifuge tube is reapposed on magnetic frame, place 5min so that magnetic bead is adsorbed onto on tube wall completely；
4.11st, careful 20ul eluent of drawing is to new 1.5ml centrifuge tube.
(2) sequencing library quality inspection
1) using agilent 2100bioanalyzer and agilent dna 7500and 12000assay kit, library quality inspection is examined
Survey, specifically comprise the following steps that
1.1st, prepare gel-dye mix (glue, dyestuff mixed liquor): by agilent dna 7500and 12000assay kit room temperature solution
Freeze 30min, vibration mixes in dna dye, plus 25ul dye to dna gel glue, and vibration mixes and is transferred to containing centrifugal column
Centrifuge tube in, 1500g is centrifuged 10min, and the gel-dye solution after filtration keeps in Dark Place；
1.2nd, encapsulating: prepare a new dna chip and be placed on chip shelf, plus 9ul gel-dye mixed liquor is to being labeled as g's
Then syringe is adjusted to 1ml position and covers on chip by Kong Zhong, pushes syringe to screens, after standing 30s adjusts syringe
It is back to 1ml position, in the hole that two other is labeled as g position, then add 9ul gel-dye mixed liquor；
1.3rd, add 5ul marker (Green Marker) in chip well and ladder hole；
1.4th, 1ul library dna and 1ul ladder is taken to be separately added in chip well and ladder hole, 2400rpm vibrates 1min
To mix sample, then chip is placed on agilent 2100 biological analyser and is detected.
2) using qubit spectrophotometer, quantitation is carried out to library
2.1st, by qubittmDsdna hs assay kit (500assays) takes out reagent room temperature from 4 DEG C of refrigerators and places 30min, then presses
Prepare mixed liquor according to following system in 0.5ml centrifuge tube:
Quantitative mixture system:
2.2nd, lucifuge standing 2min after vibration mixes；
2.3rd, spectrophotometer, examination criteria sample standard 1 and 2 are calibrated；
2.4th, detect sample sample, record testing result.
(3) high-flux sequence
Using miseq sequenator, sample library is sequenced, sequencing agents useful for same is miseq reagent kit v3
(600-cycles).
(4) data results
Sequencing data analysis is compared with human genome reference sequences (hg19) using bowtie2 software, uses sam instrument
Software carries out Mutation Screening analysis (screening conditions: 1, compare quality and be more than 20 to comparison result；2nd, sequencing depth is more than 100),
With annovar software, functional annotation is carried out to the mutation of screening.Analysis acquired results sanger method sequence verification.
By above-mentioned experiment and analytical procedure, analysis result, 5 mmr gene always amplification rate up to 61.89%～88.17%, outward
Show sub- coverage be 100%, also can 5 mmr genes of amplification gene all exons.
Data above absolutely proves, the primer pair combination 1～11 using the present invention carries out pcr amplification to 5 mmr gene extrons,
Accuracy height, high specificity, sensitivity are high.
3 17 colorectal cancer cases of embodiment and 14 normal person's mmr detection in Gene Mutation
First, experiment material, with embodiment 2.
2nd, pattern detection
(1), multiple pcr targeting enrichment
Employ the test kit of qiagen, the method in illustrating according to test kit, to 31 samples (17 colorectal cancer cases and 14
Example normal person) extract genome dna.Using the base to 31 samples for the 11 groups of primer pair combinations as shown in embodiment 2 table 3
Because group dna carries out multiple pcr amplification, concrete grammar is with embodiment 2.
(2), multiple pcr product purification, concrete grammar is with embodiment 2.
(3), sequencing library builds, and concrete grammar is with embodiment 2.
(4), sequencing library quality inspection, concrete grammar is with embodiment 2.
3rd, high-flux sequence, concrete grammar is with embodiment 2.
4th, data results
Sequencing data analysis is compared with human genome reference sequences (hg19) using bowtie2 software, uses sam instrument
Software carries out Mutation Screening analysis (screening conditions: 1, compare quality and be more than 20 to comparison result；2nd, sequencing depth is more than 100),
With annovar software, functional annotation is carried out to the mutation of screening.Analysis acquired results sanger method sequence verification, part is tied
Fruit is as shown in Figure 6.Again, confirmed the accuracy of the inventive method.
5 mmr genes and high-flux sequence are enriched with by multiple pcr targeting, 31 samples are obtained 2.7gigabases (gb)
Data, averagely each sample 86mb, average reads number is 287048.As shown in table 4, five sieves of wherein 30 samples
Looking into gene (mlh1, msh2, msh6, pms1, pms2) exon sequencing coverage is 100%, and 1 is 96.8%,
Mean coverage is 99.9%, and exon region is averagely sequenced depth for 2284.Through analysis of biological information, 31 sequencing samples
Find 13 kinds of non-synonymous single nucleotide variations (single nucleotide variation, snv), 2 kinds of synonymous snv altogether, non-same
In adopted snv, positioned at mlh1 gene have 3: c.a655g:p.i219v, c.t1151a:p.v384d,
c.c2101a:p.q701k；Positioned at msh2 gene have 3: c.c1168t:p.l390f, c.a1690g:p.t564a,
c.g2425a:p.e809k；Positioned at msh6 gene have 3: c.g116a:p.g39e, c.g3205c:p.g1069r,
c.a3488t:p.e1163v；Positioned at pms2 gene have 4: c.g59a:p.r20q, c.a1621g:p.k541e,
c.c1408t:p.p470s、c.c1454a:p.t485k.2 synonymous snv are msh6 gene c.t3306a and pms2 gene
c.c780g；Gene pms1 is not detected by snv.These snv and data base dbsnp and insight enters line retrieval, finds
14 kinds is that the wherein c.g3205c:p.g1069r of msh6 is not reported it has been reported that crossing.
As shown in table 5, by above-mentioned experiment and analytical procedure, in 17 colorectal cancer cases and 14 normal person's samples altogether
Find 13 kinds of non-synonymous single nucleotide variations of exon region, 2 kinds of synonymous single nucleotide variations.One of them causes a disease or may cause
The variation of disease, i.e. c.c2101a:p.q701k；Two pathogenic uncertain variations, that is, 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 any to the present invention in form and substantial limit it should
Point out, for those skilled in the art, on the premise of without departing from the inventive method, also can make some
Improve and supplement, these improve and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, not
In the case of departing from the spirit and scope of the present invention, a little change of making when available disclosed above technology contents, repair
Decorations and the equivalent variations developing, are the Equivalent embodiments of the present invention；Meanwhile, all substantial technological according to the present invention are to above-mentioned reality
Apply the change of any equivalent variations, modification and the differentiation that example is made, all still fall within the range of technical scheme.
Claims (9)
1. whether there is the reagent of mmr genovariation in a kind of detection sample it is characterised in that described reagent is primer pair, choosing
Any one or more from the following group 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.
2. whether there is the purposes of the reagent of mmr genovariation in detection sample according to claim 1, for preparation detection
The test kit of mmr genovariation.
3. whether there is the test kit of mmr genovariation in a kind of detection sample, contain in described test kit as in claim 1
One or more primer pair of 87 pairs of primer pairs in described reagent.
4. test kit according to claim 3 is it is characterised in that contain as described in claim 1 examination in described test kit
Whole 87 pairs of primer pairs in agent.
5. test kit according to claim 4 is it is characterised in that in described test kit, containing 11 groups of multiple pcr primer pairs
Combination, 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
With 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
With 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
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 forms；
(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 forms；
(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
With 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 forms；
(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.
6. test kit according to claim 3 is it is characterised in that in described test kit, also contain total dna extraction agent,
One or more of pcr reagent, pcr product purification reagent or operation instructions.
7. a kind of method of acquisition mmr gene amplification product, described method includes: with nucleic acid samples as template, using as right
Require the test kit described in 3～7 any claim to carry out pcr amplification, obtain amplified production.
8. method according to claim 8 is it is characterised in that drawn using 11 groups in test kit as claimed in claim 6
Thing carries out multiple pcr amplification to combination respectively as primer, obtain 11 groups of multiple pcr products is merged, obtains final product expansion
Volume increase thing.
9. method according to claim 9 is it is characterised in that the multiple pcr reaction system 50 μ l of 1-8 group and 11 groups,
Including: 5x multiplex pcr master mix 10 μ l, template 50ng, 1.0 μm of 2.5-20 μ l of forward primer,
1.0 μm of 2.5-20 μ l of reverse primer, ddh2O complements to 50 μ l；The multiple pcr reaction system 50 μ l of 9-10 group:
2x gc buffer i 25 μ l, dntp 4ul, template 50ng, 1.0 μm of 2.0 μ l of forward primer, reverse
1.0 μm of 2.0 μ l of primer, ddh2O complements to 50 μ l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510437485.5A CN106367475B (en) | 2015-07-23 | 2015-07-23 | A kind of MMR gene mutation detection kit |
Applications Claiming Priority (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192971A (en) * | 2018-02-02 | 2018-06-22 | 厦门基源医疗科技有限公司 | A kind of detection method of Jessica Lynch's syndrome related genes variants |
| CN112513292A (en) * | 2018-08-27 | 2021-03-16 | 深圳华大生命科学研究院 | Method and device for detecting homologous sequence based on high-throughput sequencing |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192971A (en) * | 2018-02-02 | 2018-06-22 | 厦门基源医疗科技有限公司 | A kind of detection method of Jessica Lynch's syndrome related genes variants |
| CN108192971B (en) * | 2018-02-02 | 2020-10-16 | 厦门基源医疗科技有限公司 | Method for detecting gene variation related to forest syndrome |
| CN112513292A (en) * | 2018-08-27 | 2021-03-16 | 深圳华大生命科学研究院 | Method and device for detecting homologous sequence based on high-throughput sequencing |
| CN112513292B (en) * | 2018-08-27 | 2023-12-26 | 深圳华大生命科学研究院 | Method and device for detecting homologous sequences based on high-throughput sequencing |
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