CN106399479A - SNP typing kit used for detecting susceptibility genes of type-II diabetes - Google Patents
SNP typing kit used for detecting susceptibility genes of type-II diabetes Download PDFInfo
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Abstract
The invention discloses an SNP typing kit used for detecting susceptibility genes of type-II diabetes. The kit comprises primers of 7 SNP loci of 6 susceptibility genes. A method for performing detection by adopting the SNP typing kit comprises the steps of extracting genomic DNA; preparing a PCR reaction solution, and amplifying the 7 SNP loci including RS10886471, RS13062383, RS780094, RS7903146, RS3817588, RS2241766 and RS7403531; and performing fluorescent gel electrophoresis detection and genotyping on amplified products. The SNP typing kit is high in specificity, high in sensitivity, simple in operation and low in cost, can realize batch detection, can be used for assessing the risk of suffering from the type-II diabetes by to-be-detected people, and is used for auxiliary diagnosis and prevention of the type-II diabetes.
Description
Technical field
The invention belongs to biological technical field is and in particular to a kind of SNP for the detection of type ii diabetes tumor susceptibility gene divides
Type test kit and its using method.
Background technology
Noninfectiouss (non-communicable diseases, NCD) are that on our times, topmost death is former
Cause, mainly includes diabetes (diabetes mellitus, DM), tumor, cardiovascular disease etc..What the whole world was occurred every year is dead
In dying, 63% is had to be led to by NCD.Diabetes are one of most important NCD of current threat global human health, be with
Hyperglycemia is principal character, with one group of chronic incretion metabolism disease of fat, protein metabolism disorder etc., has heredity easily
Perception.
Over nearly 30 years, China's diabetes prevalence dramatically increases.At present, China's diabeticss up to 1.14 hundred million, account for complete
The 1/3 of ball is it is meant that just have 1 to be derived from China in every 3 diabeticss in the whole world.Diabetes are not single diseases, are by losing
Pass and environmental factorss are in the interior coefficient result of many factors.Type ii diabetes (T2DM) are Adult Onset's patients with type Ⅰ DMs,
Many sequela at 35～40 years old, account for diabeticss more than 90%.Type ii diabetes have higher heritability and environmental factorss,
And in significantly heterogeneous.It is now recognized that pathogenic factor is insulin resistant (is mainly shown as hyperinsulinemia；Glucose profit
Reduced with rate) and hypoinsulinism merging exist, its performance is inhomogenous, have based on insulin resistant with
Hypoinsulinism, have is then with insulin resistant with hypoinsulinism.Recent studies indicate that, hereditary because
Element plays an important role in the generation of type ii diabetes, and its pathogenesis is in familial aggregation, and about 60% patient has family to lose
Pass history.But the heredity of diabetes-susceptible gene is very complicated, it does not simply follow the dominant and stealthy law of heredity of Mendel, and
It is in complex inheritance pattern, i.e. the impact of multiple genovariations and environmental factorss together decides on the susceptibility of disease.At present, Quan Ji
Because group association study (genome-wide association study, GWAS) has identified the susceptible of 69 type ii diabetes
Gene (Nature Genetics, 2014), these genes maintain in beta Cell of islet growth, growth and function respectively, and outward
The aspects such as all insulin resistants are played an important role.
With the fast development of single nucleotide polymorphism (single nucleotide polymorphism, SNP) technology,
Current research tendency is become using the positioning that SNP is used for diseases predisposing gene as genetic map.The principle of ARMS technology be
Two primers are designed, mutational site is located at 3 ' ends of primer, one corresponds to normal equipotential base at the mutation of DNA sequence one end
Because of aligning primer, one corresponds to mutant gene sequence primer, then designs a general primer in the DNA other end, thus using PCR
Mutant gene is detected.The method being presently used for the detection of type ii diabetes tumor susceptibility gene focuses primarily upon sonde method, fluorescence
Quantitative PCR method, high-resolution solubility curve (HRM) and sequencing etc., although said method can complete to a certain extent right
The detection of SNP, but come with some shortcomings, it is mainly shown as that detection process is loaded down with trivial details, detection time is long, expensive reagents etc., from
And it is unfavorable for popularization and application aborning.For example, Chinese Patent Application No. " 201510428933.5 ", " can be used for detection and II
The related test kit of PTPN1 gene promoter zone methylation degree of patients with type Ⅰ DM and its application " is and in particular to pyrosequencing
Technology, though the method only needs a PCR amplification, sequencing steps are loaded down with trivial details, time-consuming, are unfavorable for batch detection.ARMS-PCR is
A kind of new method growing up on the basis of PCR for detecting various point mutation in DNA.ARMS-PCR and fluorescence gel electrophoresis
The combination of technology has many detection advantages, for example：Detection site quantity is many, sensitivity height, economical and efficient, time-consuming short and logical
Amount is high.
Content of the invention
For above-mentioned technical problem, it is an object of the invention to provide a kind of for the detection of type ii diabetes tumor susceptibility gene
SNP parting kit, this test kit has that high specificity, sensitivity are high, simple to operate, low cost, flux are high can achieve batch
Many advantages, such as detection, this test kit not only can quick detection go out person under test type ii diabetes susceptible genotype, simultaneously
Testing result can be also used for auxiliary diagnosis and the prevention of type ii diabetes.
Technical scheme is as follows：
A kind of SNP parting kit for the detection of type ii diabetes tumor susceptibility gene, including 7 SNP of 6 tumor susceptibility genes
The primer in site；7 SNP site of described 6 tumor susceptibility genes are respectively：
The RS7903146 site of TCF7L2 gene；
The RS13062383 site of SLC6A20 gene；
The RS780094 site of GCKR gene and RS3817588 site；
Observe the RS2241766 site of apm 1 gene；
The RS10886471 site of GRK5 gene；
The RS7403531 site of RASGRP1 gene.
Preferably, described primer is divided into two groups and is marked.
Preferably, the primer sequence of 7 SNP site of described 6 tumor susceptibility genes is respectively：
RS10886471, forward primer SEQ ID NO.1-2, reverse general primer, SEQ ID NO.3；
RS13062383, forward primer SEQ ID NO.4-5, reverse general primer, SEQ ID NO.6；
RS780094, forward primer SEQ ID NO.7-8, reverse general primer, SEQ ID NO.9；
RS7903146, forward primer SEQ ID NO.10-11, reverse general primer, SEQ ID NO.12；
RS3817588, forward primer SEQ ID NO.13-14, reverse general primer, SEQ ID NO.15；
RS2241766, forward primer SEQ ID NO.16-17, reverse general primer, SEQ ID NO.18；
RS7403531, forward primer SEQ ID NO.19-20, reverse general primer, SEQ ID NO.21.
Preferably, described reverse general primer all has fluorescent labeling；The fluorescent labeling packet bag of described reverse general primer
Include two groups, respectively first group RS10886471, RS13062383, RS780094, RS7903146；Second group of RS3817588,
RS2241766 and RS7403531.
Preferably, the fluorescent labeling of described reverse general primer include FAM color marker, HEX color marker, ROX color marker,
In TAMRA color marker any two kinds；And the fluorescent labeling between every group is different.
Preferably, final concentration in amplification system for the described primer is respectively：
A kind of using method of the SNP parting kit for the detection of type ii diabetes tumor susceptibility gene, comprises the steps：
Step A, extracts genomic DNA from biological material, and extracting method includes paramagnetic particle method, Chelex-100 method, extracts
DNA solution afterwards dilutes after quantitation；
Step B, the PCR amplification of SNP site：Prepare PCR reactant liquor first, sequentially add Reaction Mix, thermal starting
Taq polymerase, composite primer, water and DNA profiling, then enter performing PCR amplification using three step TRAP to it；
Step C, the capillary electrophoresis detection of amplified production, the genotype of each SNP site is determined according to testing result.
The configuration processor of described PCR amplification includes：A, denaturation, 95 DEG C of 3min；B, thermal cycle, 94 DEG C of 15s, 60 DEG C
30s；Totally 30 circulations；C, eventually extension, 60 DEG C of 15min；D, maintenance, 4 DEG C.
Described biological material includes blood, hair, saliva, exfoliative cyte.
Described SNP parting kit can be used for the risk that auxiliary evaluation personnel to be detected suffer from type ii diabetes, and is used for II
The auxiliary diagnosis of patients with type Ⅰ DM and prevention.
The invention has the beneficial effects as follows：The present invention establishes a kind of new utilization fluorescent dye primer specific amplification knot
Close the classifying method of the type ii diabetes tumor susceptibility gene detection of capillary electrophoresis technique, can be detected by once experiment simultaneously
RS10886471, RS13062383, RS780094, RS7903146, RS3817588, RS2241766 and RS7403531 etc. 7
The related SNP site of type ii diabetes, and obtain genotyping result simultaneously, and the invention of other correlations or product can only detect it
In one or two site；The method is simple to operate, accuracy is strong, sensitivity is high, can be by non-specific amplification product, primer two
Aggressiveness separates with specific amplification products, at utmost reduces false positive；The II type to person under test for the test kit of the application present invention
Diabetes-susceptible gene is detected, can draw experimental result the soonest in two hours, also can carry out batch detection, be to be measured
The risk that crowd suffers from type ii diabetes is estimated, and the auxiliary diagnosis for type ii diabetes and prevention；The present invention first will
ARMS-PCR technology and fluorescence gel electrophoretic techniquess combine in the detection applying to type ii diabetes tumor susceptibility gene, and can single tube
Detect the focus susceptibility loci of multiple type ii diabetes, testing result is the genotyping result of tumor susceptibility gene simultaneously.
Brief description
Fig. 1 is the electrophoretogram of the genotype standard substance of the present invention；
Fig. 2 is that the pleomorphism site of the present invention corresponds to design of primers schematic diagram；
Fig. 3 a is the electrophoretogram before RS780094 site-tag primer change of the present invention；
Fig. 3 b is the electrophoretogram of RS780094 site-tag primer deletion experiments of the present invention；
Fig. 3 c is the electrophoretogram after RS780094 site-tag primer change of the present invention；
Fig. 4 a is the electrophoretogram of the testing result of patient 1 of the present invention and patient 2；
Fig. 4 b is the electrophoretogram of the testing result of patient 3 of the present invention and patient 4；
Fig. 4 c is the electrophoretogram of the testing result of patient 5 of the present invention.
Specific embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to description literary composition
Word can be implemented according to this.
As illustrated, the present invention discloses a kind of SNP parting kit for the detection of type ii diabetes tumor susceptibility gene, including
The primer of 7 SNP site of 6 tumor susceptibility genes；7 SNP site of described 6 tumor susceptibility genes are respectively：
The RS7903146 site of TCF7L2 gene；
The RS13062383 site of SLC6A20 gene；
The RS780094 site of GCKR gene and RS3817588 site；
Observe the RS2241766 site of apm 1 gene；
The RS10886471 site of GRK5 gene；
The RS7403531 site of RASGRP1 gene.
Preferably, described primer is divided into two groups and is marked.
Preferably, the primer sequence of 7 SNP site of described 6 tumor susceptibility genes is respectively：
RS10886471, forward primer SEQ ID NO.1-2, reverse general primer, SEQ ID NO.3；
RS13062383, forward primer SEQ ID NO.4-5, reverse general primer, SEQ ID NO.6；
RS780094, forward primer SEQ ID NO.7-8, reverse general primer, SEQ ID NO.9；
RS7903146, forward primer SEQ ID NO.10-11, reverse general primer, SEQ ID NO.12；
RS3817588, forward primer SEQ ID NO.13-14, reverse general primer, SEQ ID NO.15；
RS2241766, forward primer SEQ ID NO.16-17, reverse general primer, SEQ ID NO.18；
RS7403531, forward primer SEQ ID NO.19-20, reverse general primer, SEQ ID NO.21.
Preferably, described reverse general primer all has fluorescent labeling；The fluorescent labeling packet bag of described reverse general primer
Include two groups, respectively first group RS10886471, RS13062383, RS780094, RS7903146；Second group of RS3817588,
RS2241766 and RS7403531.
Preferably, the fluorescent labeling of described reverse general primer include FAM color marker, HEX color marker, ROX color marker,
In TAMRA color marker any two kinds；And the fluorescent labeling between every group is different.
Preferably, final concentration in amplification system for the described primer is respectively：
A kind of using method of the SNP parting kit for the detection of type ii diabetes tumor susceptibility gene, comprises the steps：
Step A, extracts genomic DNA from biological material, and extracting method includes paramagnetic particle method, Chelex-100 method, extracts
DNA solution afterwards dilutes after quantitation；
Step B, the PCR amplification of SNP site：Prepare PCR reactant liquor first, sequentially add Reaction Mix, thermal starting
Taq polymerase, composite primer, water and DNA profiling, then enter performing PCR amplification using three step TRAP to it；
Step C, the capillary electrophoresis detection of amplified production, the genotype of each SNP site is determined according to testing result.
The configuration processor of described PCR amplification includes：A, denaturation, 95 DEG C of 3min；B, thermal cycle, 94 DEG C of 15s, 60 DEG C
30s；Totally 30 circulations；C, eventually extension, 60 DEG C of 15min；D, maintenance, 4 DEG C.
Described biological material includes blood, hair, saliva, exfoliative cyte.
Described SNP parting kit can be used for the risk that auxiliary evaluation personnel to be detected suffer from type ii diabetes, and is used for II
The auxiliary diagnosis of patients with type Ⅰ DM and prevention.
Embodiment 1：
The screening of SNP site of the present invention, the foundation of the design of primer, the optimization of concentration and reaction system
1st, the design of primer and screening
The download of 1.1SNP sequence
The gene order of 7 SNP site of the present invention is all downloaded from down NCBI genebank, specific sequence information
See table (table 1).The allele C in wherein RS10886471 site is wild type, and allele T is saltant type (susceptible type)；
The allele G in RS13062382 site is wild type, and allele A is saltant type (susceptible type)；RS780094 site etc.
Position Gene A is wild type, and allele G is saltant type (susceptible type)；The allele C in RS7903146 site is wild type, etc.
Position gene T is saltant type (susceptible type)；The allele A in RS3817588 site is wild type, and G is saltant type (susceptible type)；
The allele A in RS2241766 site is wild type, and allele C is saltant type (susceptible type)；RS7403531 site etc.
Position gene C is wild type, and allele T is saltant type (susceptible type).
Table 1 SNP sequence information
1.2 design of primers
The present invention carries out design of primers using OLIGO6.0 software, and each SNP site designs three primers, PCR primer altogether
Between 100-210bp, 5 ' ends of a wherein general primer are marked size with corresponding fluorescein, and two other is nonstandard
3 ' ends of note primer identify to mutating alkali yl sequence specific, and wherein one cold 5 ' end Jia 3 again～5 nucleotide (lead to
It is often att or tata, the lower case at primer sequence 5 ' end in table 2) it is used for indicating allele.Because two non-marked are drawn
Thing length difference leads to amplified production length different, and electrophoretic mobility is different, is finally reached the purpose of detection.
The principle of design of primers of the present invention as shown in Fig. 2 in addition to following the rule of general primer design, for increasing by two
The specificity of bar non-marked primer, is artificially induced base mismatch (in primer sequence holding apart from primer 3 ' at 2～5 bases
The band underscore lower case at nearly 3 ' ends), the principle introducing mispairing is：If 3 ' hold as weak mispairing (A-C, G-T), then introduce wrong by force
Join (A-G, C-T)；If 3 ' hold as middle mispairing (A-A, G-G, C-C, T-T), then mispairing in being introduced into；If 3 ' hold as strong mispairing, draw
Enter weak mispairing.
The primer of the present invention is all designed by this rule, but the present invention gropes to find through test of many times repeatedly：When
3 ' hold and can cannot get amplification because primer specificity is too strong for during weak mispairing, being artificially introduced strong base mismatch, in addition, artificially
Introduce base mismatch distance 3 ' end position different primers specificity also different, so be artificially introduced base mismatch type and
Position will regard concrete condition, verify through test of many times.Finally, for the optimization of follow-up primer concentration, here design
Primer Tm is all between 58～62 DEG C.
1.3 primer screening
All primers of the present invention are synthesized by Sangon Biotech (Shanghai) Co., Ltd., the template used by screening primer
It is through the medical test portion sequencing success of son deep pool judicial expertise institute and determines the individuality of genotype.The concrete grammar of primer screening is such as
Under：
(1) the single locus amplification of primer
Synthetic each pair primer (labeled primer and two non-marked primers) is diluted to 1 μM and carries out amplification examination
Test, observe and have or not non-specific amplification peak, and amplification efficiency is how.If there being non-specific amplification peak, carry out disappearance investigation real
Test, find out the primer producing non-specific amplification peak and change.
(2) the monochromatic composite amplification of primer
The single locus primer composition composite primer of the same color marker determining is carried out amplification experiment, observing has nothing but
Behind specific amplification peak, and primer mixing, amplification efficiency how.If there being non-specific amplification peak, carry out disappearance investigation experiment,
Find out the primer producing non-specific amplification peak and change.
(3) primer mixture amplification
All primer composition primer mixtures of the different color markers determining are carried out amplification experiment, observes and have spy nothing but
Specific amplification peak, and the amplification efficiency of primer mixture is how.If there being non-specific amplification peak, carry out disappearance investigation experiment,
Find out the primer producing non-specific amplification peak and change, determine final primer sequence.
Preferably, no non-specific amplification peak occurs for the primer single locus amplification of the present invention and monochromatic amplification assay result.
And find when testing primer mixture：A non-specific amplification peak occurs in FAM color, size is 108bp, result
See Fig. 3 a.The present invention carries out deletion experiments successively to FAM color primer：When all primers of HEX color all add, and FAM color disappearance
During the labeled primer of RS780094, the non-specific amplification peak of amplified production disappears, and result is shown in Fig. 3 b；Then use RS780094 again
The non-marked primer of labeled primer and HEX color carry out deletion experiments successively, amplification show when the open country that lack RS3817588
During raw type non-marked primer, disappearance result in non-specific amplification peak is shown in Fig. 3 c.Therefore this experiment is ensureing the expansion of RS3817588 primer
Increasing Efficiency and specific in the case of, change RS780094 labeled primer, before change RS780094 site primer amplification produce
Thing size is 173bp (susceptible type) and 177bp (wild type), and after change, the primer extension product size in RS780094 site is
150bp (susceptible type) and 154bp (wild type).
The present invention also finds when screening primer：Carry out allele-specific primerses in strict accordance with introducing mispairing principle
During design it may appear that the specificity of primer too strong and expand not appearance or amplification efficiency high situations such as.For example：
The second that the mutant primers of RS13062383 are held in primer 3 ' is introduced into when strong base mismatch " T " and middle base mismatch " C " all
The situation that expand not appearance occurs；And when introducing weak base mismatch " A ", then the not high situation of amplification efficiency occurs, constantly carry
High primer concentration still cannot preferably expanding effect；Therefore its base mismatch is introduced the 4th and by strong mispairing alkali by the present invention
When base " G " makes middle base mismatch " A " into, expanding effect is preferable.As can be seen here, being artificially introduced during mispairing can not be in strict accordance with mispairing
Principle is carried out, and be determined on a case-by-case basis, and the situation of the primer introducing mispairing of different base compositions can be variant.The present invention is to this
7 groups of primers have carried out multiple change and have repeatedly tested, and finally determine in the case of the specificity ensureing primer and amplification efficiency
The primer sequence of each SNP site, specific primer information is shown in Table 2.
The each SNP site primer sequence of table 2
In table 2, " F1 " numbering refers to the wild type in concrete site during allele-specific primerses amplification, and " F2 " numbering refers to
The saltant type (susceptible type) in concrete site during the gene-specific primer amplification of position, " R " numbering refers to being total to of 5 ' Terminal fluorescent element labellings
Use primer.In above-mentioned primer, each SNP site all has two allele-specific non-marked primers, is respectively used for amplifying
Wild type and saltant type (susceptible type) base sequence, and the general primer in each site, for nonstandard with allele-specific
Note primer forms pairing, so that detecting target base sequence is expanded.
The present invention on the basis of determining dichromatism fluorescence assembled scheme, by lot of experiments it is determined that above-mentioned 7 SNP positions
The compound mode of point and fluorescent labeling type.Fluorescent labeling used can be optional two kinds in FAM, HEX, TAMRA and ROX, examines
Consider cost and fluorescence efficiency, this programme is preferably with FAM, HEX dye marker general primer.The primer of 7 SNP site is divided into two
Group, first group includes RS10886471, RS13062383, RS780094, RS7903146 totally four sites, the sharing of each site
Primer is marked with FAM, and second group includes tri- sites of RS3817588, RS2241766 and RS7403531, being total to of each site
It is marked with HEX with primer, molecular weight internal standard uses orange fluorescent dye LIZ500 (Applied biosystems)
It is marked.
The optimization of 1.4 primer concentrations
Because the amplification efficiency of every primer is different, cause every primer will reach during same amplification peak value in system
Dosage different.Therefore, the present invention is after determining primer sequence, and the concentration of primer will be optimized.
After primer synthesis, first the concentration of each primer is done from 0.02 μM to 0.2 μM with Concentraton gradient experiment, spacing gradient is
0.02μM；Then the annealing temperature of primer is done with thermograde experiment, spacing gradient is 2 DEG C, does primer to human gene group DNA
Single expansion is tested, the impact to amplification efficiency of the different primer concentration of comparative observation and different annealing temperatures；Finally, according to detection
As a result, select primer concentration between 2000～5000RFU for the peak height and annealing temperature and annealing temperature, prepare initial primer
Mixture, expands to human gene group DNA, according to testing result, each primer concentration is finely adjusted, and makes composite primer each
The amplification peak height in site is basically identical.
In the present invention, two non-marked primers of each SNP site occur suppression and the situation of competition in amplification, because
This dosage when preparing primer mixture should be more than labeled primer.Because two non-marked primers are artificially introduced the position of base mismatch
Put difference and then amplification efficiency is different, so need to ceaselessly be finely adjusted to its dosage when carrying out primer concentration optimization, so that
Article two, the peak height when expanding heterozygous mutant for the non-marked primer is basically identical.
The present invention, through the multiple fine setting experiment of primer concentration, finally determines each SNP site primer in amplification system
Optimum concentration.Specific concentration see table：
Final concentration in amplification system for each SNP site primer of table 3
1.5 amplifications and product detection
Amplification system
Complete the preparation of the PCR reaction system outside removing template first, be eventually adding amplification template；When extracting genomic DNA,
First use ultraviolet scene photometer measurement concentration, then dilute and make its final concentration in 0.05～1ng/ μ L, in practical operation, one
As take the DNA profiling of 1～2 μ L to be expanded.In system, the sample-adding amount of each component is as shown in table 4.
Table 4 test kit amplification system
Amplification program
Above-mentioned system uses hand held centrifuge after preparing immediately, after be placed on thermal cycler.Select the journey of table 5
Sequence is expanded, and the product after amplification should carry out fluorescence gel electrophoresis detection immediately or keep in Dark Place.
Because the size of the primer extension product of 7 SNP site in the present invention is all in below 210bp, in order to save test kit
Detection time, in the case of determining primer concentration, the present invention will be optimized to the amplification program of PCR.In denaturation
In the case that time is constant, the annealing time in change thermocycling program and whole extension of time, filter out proliferation time short and expand
The high program of Increasing Efficiency.Optimization process is as follows：(1) in denaturation 3 minutes, degeneration 15 seconds, 30 periods and 20 points of extension eventually
In the case of clock, annealing time 60 seconds and the annealing time expanding effect no significant difference of 30 seconds, electrophoresis detection result is all preferable.
This 30 seconds amplification program of optimized choice annealing time；(2) because the amplified production fragment of all sites of the present invention is shorter, required
Extension of time is short compared with other standard PCR amplification programs, therefore this suboptimization does gradient experiment to whole extension of time, and spacing gradient is 5
Minute.In the case of denaturation 3 minutes, degeneration 15 seconds, annealing 30 seconds, 30 periods, extend eventually and be respectively set to 20 points
Clock, 15 minutes, 10 minutes and 5 minutes, amplified production electrophoresis result shows：Extend 20 minutes and extend eventually the result of 15 minutes eventually
No significant difference, the electrophoresis result extending 10 minutes eventually occurs the unbalanced situation of each SNP site peak height, and extends 5 eventually
The electrophoresis result of minute occurs the too low situation of indivedual SNP site peak heights.The present invention finally determines PCR's through test of many times
Amplification program, particular situation is shown in Table 5.
Table 5 test kit amplification program
Amplified production fluoroscopic examination on genetic analyzer
It is made up of deionized formamide and test kit middle-molecular-weihydroxyethyl internal standard LIZ-500 (Applied biosystems)
Sample mixture (9.5: 0.5 mixing by volume).10 μ L loading mixture are mixed with 1 μ L amplified production, centrifugation, it is to avoid produce
Bubble.95 DEG C of degeneration 3 minutes, ice bath 3 minutes, with genetic analyzer 3130 (Applied biosystems) detection and analysis.
The preparation of allelic ladder
Choose through the medical test portion sequencing success of son deep pool judicial expertise institute and determine the patient of genotype, choosing in the present embodiment
Go out the individuality of each SNP site different genotype, respectively use RS10886471, RS13062383, RS780094, RS7903146,
The single locus primer in each site of RS3817588, RS2241766 and RS7403531 is expanded, wherein, the individuality of wild type
With the primer of wild type, the individuality of saltant type is expanded with the primer of saltant type.Primer sequence is shown in Table 2.
(1) amplification system
Complete the preparation of the PCR reaction system outside removing template first, be eventually adding amplification template；When extracting genomic DNA,
First use ultraviolet scene photometer measurement concentration, then dilute and make its final concentration in 0.05～1ng/ μ L, in practical operation, one
As take the DNA profiling of 1～2 μ L to be expanded.In system, the sample-adding amount of each component is as shown in table 4.
(2) amplification program
Use hand held centrifuge immediately after above-mentioned system configurations are good, after PCR pipe is placed on thermal cycler, select table 5
Program expanded, the product after amplification should carry out fluorescence gel electrophoresis detection immediately or keep in Dark Place.
(3) amplified production fluoroscopic examination on genetic analyzer
It is made up of deionized formamide and test kit middle-molecular-weihydroxyethyl internal standard LIZ-500 (Applied biosystems)
Sample mixture (9.5: 0.5 mixing by volume).10 μ L loading mixture are mixed with 1 μ L amplified production, centrifugation, it is to avoid produce
Bubble.95 DEG C of degeneration 3 minutes, ice bath 3 minutes, with genetic analyzer 3130 (Applied biosystems) detection and analysis.
(4) phenotypic analysis
The data collected with genetic analyzer detection in fragment analysis software GeneMapperID-X analytical procedure (3).
(5) the assembling of allelic ladder
According to the amplified allele peak height ratio of each SNP site, the few of peak height adds, and low the adding in peak, each equipotential base
Gene-amplification product mixes, and forms allelic ladder.Electrophoresis detection again, detection figure such as accompanying drawing 1.
The Accuracy Verification of test kit of the present invention
The present embodiment is chosen and has been diagnosed as type ii diabetes patient 5, and the genotype of this 5 patients is judicial by son deep pool
Medical test portion of identification institute is successfully sequenced, and then with the test kit of the present invention, each SNP site of above-mentioned 5 patients is examined
Survey.The numbering of 5 patients is respectively C1, C2, C3, C4 and C5.Testing result is shown in Table 6, and specific detection method is as follows：
Amplification system
Complete the preparation of the PCR reaction system outside removing template first, be eventually adding amplification template；When extracting genomic DNA,
First use ultraviolet scene photometer measurement concentration, then dilute and make its final concentration in 0.05～1ng/ μ L, in practical operation, one
As take the DNA profiling of 1～2 μ L to be expanded.In system, the sample-adding amount of each component is as shown in table 4.
Table 4 test kit amplification system
Amplification program
Above-mentioned system uses hand held centrifuge after preparing immediately, after be placed on thermal cycler, select table 5 journey
Sequence is expanded, and the product after amplification should carry out fluorescence gel electrophoresis detection immediately or keep in Dark Place.
Table 5 test kit amplification program
Amplified production fluoroscopic examination on genetic analyzer
It is made up of deionized formamide and test kit middle-molecular-weihydroxyethyl internal standard LIZ-500 (Applied biosystems)
Sample mixture (9.5: 0.5 mixing by volume).10 μ L loading mixture are mixed with 1 μ L amplified production, centrifugation, it is to avoid produce
Bubble.95 DEG C of degeneration 3 minutes, ice bath 3 minutes, with genetic analyzer 3130 (Applied biosystems) detection and analysis.
Test kit using the present invention see table to the testing result of above-mentioned 5 patients.By relative analyses, utilize
The survey to the testing result of type ii diabetes Patient genotype and son deep pool medical test portion of judicial expertise institute for the test kit of the present invention
Sequence result is completely the same.
The genotype that 6 test kits of table detect to 5 patients
By above-described embodiment, we can see that the test kit testing result of the present invention accurately and reliably, operational approach letter
Singly, only need to once test the susceptible SNP site that can detect 7 type ii diabetes, and obtain the genotype of corresponding site.Detection
Result not only can be used for assessing risk and the prevention that healthy population suffers from type ii diabetes, is also used as type ii diabetes patient
Auxiliary diagnosis and treatment.
The present invention establishes a kind of new utilization fluorescent dye primer specific amplification and combines capillary electrophoresis technique
Type ii diabetes tumor susceptibility gene detection classifying method, by once experiment can detect simultaneously RS10886471,
7 type ii diabetes phases such as RS13062383, RS780094, RS7903146, RS3817588, RS2241766 and RS7403531
The SNP site closed, and obtain genotyping result simultaneously, and the invention of other correlations or product can only detect wherein one or two
Site；The method is simple to operate, accuracy is strong, sensitivity is high, can be by non-specific amplification product, primer dimer and specificity
Amplified production separates, and at utmost reduces false positive；The easy sensillary base of type ii diabetes to person under test for the test kit of the application present invention
Because being detected, experimental result can be drawn the soonest in two hours, also can carry out batch detection, be that crowd to be measured suffers from II type sugar
The risk of urine disease is estimated, and the auxiliary diagnosis for type ii diabetes and prevention；The present invention is first by ARMS-PCR technology
Combine in the detection applying to type ii diabetes tumor susceptibility gene with fluorescence gel electrophoretic techniquess, and can single tube detect simultaneously multiple
The focus susceptibility loci of type ii diabetes, testing result is the genotyping result of tumor susceptibility gene.
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in description and embodiment
With, it can be applied to various suitable the field of the invention completely, for those skilled in the art, can be easily
Realize other modification, therefore under the general concept being limited without departing substantially from claim and equivalency range, the present invention does not limit
In specific details with shown here as the legend with description.
Claims (9)
1. a kind of for type ii diabetes tumor susceptibility gene detection SNP parting kit it is characterised in that：Including 6 easy sensillary bases
The primer of 7 SNP site of cause；7 SNP site of described 6 tumor susceptibility genes are respectively：
The RS7903146 site of TCF7L2 gene；
The RS13062383 site of SLC6A20 gene；
The RS780094 site of GCKR gene and RS3817588 site；
Observe the RS2241766 site of apm 1 gene；
The RS10886471 site of GRK5 gene；
The RS7403531 site of RASGRP1 gene.
2. the SNP parting kit for the detection of type ii diabetes tumor susceptibility gene according to claim 1, its feature exists
In：Described primer is divided into two groups and is marked.
3. the SNP parting kit for the detection of type ii diabetes tumor susceptibility gene according to claim 1, its feature exists
In：The primer sequence of 7 SNP site of described 6 tumor susceptibility genes is respectively：
RS10886471, forward primer SEQ ID NO.1-2, reverse general primer, SEQ ID NO.3；
RS13062383, forward primer SEQ ID NO.4-5, reverse general primer, SEQ ID NO.6；
RS780094, forward primer SEQ ID NO.7-8, reverse general primer, SEQ ID NO.9；
RS7903146, forward primer SEQ ID NO.10-11, reverse general primer, SEQ ID NO.12；
RS3817588, forward primer SEQ ID NO.13-14, reverse general primer, SEQ ID NO.15；
RS2241766, forward primer SEQ ID NO.16-17, reverse general primer, SEQ ID NO.18；
RS7403531, forward primer SEQ ID NO.19-20, reverse general primer, SEQ ID NO.21.
4. the SNP parting kit for the detection of type ii diabetes tumor susceptibility gene according to claim 3, its feature exists
In：Described reverse general primer all has fluorescent labeling；The fluorescent labeling packet of described reverse general primer includes two groups, respectively
First group of RS10886471, RS13062383, RS780094, RS7903146；Second group of RS3817588, RS2241766 and
RS7403531.
5. the SNP parting kit for the detection of type ii diabetes tumor susceptibility gene according to claim 4, its feature exists
In：The fluorescent labeling of described reverse general primer include FAM color marker, HEX color marker, ROX color marker, in TAMRA color marker
Any two kinds；And the fluorescent labeling between every group is different.
6. the SNP parting kit for the detection of type ii diabetes tumor susceptibility gene according to claim 1, its feature exists
In：Final concentration in amplification system for the described primer is respectively：
7. the SNP parting kit for the detection of type ii diabetes tumor susceptibility gene according to any one of claim 1～6
Using method, comprise the steps：
Step A, extracts genomic DNA, extracting method includes paramagnetic particle method, Chelex-100 method, after extraction from biological material
DNA solution dilutes after quantitation；
Step B, the PCR amplification of SNP site：Prepare PCR reactant liquor first, sequentially add Reaction Mix, thermal starting Taq and gather
Synthase, composite primer, water and DNA profiling, then enter performing PCR amplification using three step TRAP to it；
Step C, the capillary electrophoresis detection of amplified production, the genotype of each SNP site is determined according to testing result.
8. the using method of the SNP parting kit for the detection of type ii diabetes tumor susceptibility gene according to claim 7,
It is characterized in that：The configuration processor of described PCR amplification includes：A, denaturation, 95 DEG C of 3min；B, thermal cycle, 94 DEG C of 15s, 60 DEG C
30s；Totally 30 circulations；C, eventually extension, 60 DEG C of 15min；D, maintenance, 4 DEG C.
9. the using method of the SNP parting kit for the detection of type ii diabetes tumor susceptibility gene according to claim 7,
It is characterized in that：Described biological material includes blood, hair, saliva, exfoliative cyte.
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| CN114574575A (en) * | 2022-05-06 | 2022-06-03 | 深圳会众生物技术有限公司 | Detection primer and detection kit for deep venous thrombosis SNP locus |
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| CN107083429A (en) * | 2017-04-17 | 2017-08-22 | 江苏苏博生物医学科技南京有限公司 | A kind of SNP parting kits detected for atrial fibrillation tumor susceptibility gene and its application |
| CN107083429B (en) * | 2017-04-17 | 2019-06-14 | 江苏苏博生物医学科技南京有限公司 | A kind of SNP parting kit and its application for the detection of atrial fibrillation tumor susceptibility gene |
| CN107252098A (en) * | 2017-06-27 | 2017-10-17 | 上海中优精准医疗科技股份有限公司 | A kind of diabetic individualization intervenes formula food and its preparation system |
| CN107955834A (en) * | 2017-12-05 | 2018-04-24 | 北京市计算中心 | Mutational site for detecting diabetes B tumor susceptibility gene is combined and its detection primer and application |
| CN109295210A (en) * | 2018-10-30 | 2019-02-01 | 深圳市万众基因转化医学研究院 | A kind of combination primer of mankind's diabetes B associated gene mutation screening and application |
| CN114574575A (en) * | 2022-05-06 | 2022-06-03 | 深圳会众生物技术有限公司 | Detection primer and detection kit for deep venous thrombosis SNP locus |
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