CN106868136A - A kind of kit of detection JAG2 gene SNP site rs741859 genotype and preparation method thereof - Google Patents
A kind of kit of detection JAG2 gene SNP site rs741859 genotype and preparation method thereof Download PDFInfo
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Abstract
The present invention discloses a kind of kit of detection JAG2 gene SNP site rs741859 genotype and preparation method thereof, and the kit includes：Standard items, primer, the qPCR amplifing reagents containing fluorescent dye；The standard items are comprising JAG2 rs741859 (c*1053G>A) the wild-type DNA-sequence in site and comprising JAG2 rs741859 (c*1053G>A) the homozygous mutant DNA sequence dna in site；The primer is used to expand standard items and detected sample.Kit provided by the present invention has sensitivity high, and detection speed is fast, simple to operate, expends low advantage.
Description
Technical field
The present invention relates to genetic engineering field, and in particular to the detection examination of JAG2 gene SNP site rs741859 genotype
Agent box and preparation method thereof.
Background technology
JAG2 genes are one of parts of NOTCH signal paths, its Main Function be take part in secondary palate formation and
Growth course, it is considered to be one of candidate gene of harelip.JAG2 gene rs741859 (c*1053G>A) site is located at the base
Near 5 ' ends of cause, site AG, GG genotypic expression goes out under study for action has conspicuousness correlation with non-syndromic cleft lip with cleft palate.
Used as common inborn defect and Craniofacial growth defect, harelip is a kind of complicated different substantiality disease, in the world
It is classified as syndromic (syndromic cleft lip or palate, SCLP) and non-syndromic cleft lip with cleft palate (non-
Syndromic cleft lip or palate, NSCLP).The incidence of global harelip is 3-22/10000, and China
Incidence is about 1.82/1000.The pathogenic factor of NSCLP has a lot, and the influence of wherein h and E occupies Main Function.
Research finds that many signal paths take part in the development of lip palate, and coloured differently body is interval to have one to be set in harelip morbidity
With.The NSCLP area of liability being currently known has Iq32,2p, 2q, 3q27-28,4q, 6p23-p25,8q24,9q21,12p11,
14q21-24,16q24 and 19q13, possible tumor susceptibility gene have IRF6, SATB2, TP63, FQXE1, PVRL1, MSX1,
GABRB3, CRISPLD2, MAFB and ABCA4 etc..
The analysis principle of high-resolution melting curve (high resolution melting, HRM) is in typical polymerization enzyme
Saturated fluorescence dye Tu is combined with double-stranded DNA during chain reaction (PCR), glimmering when making DNA double chain dissociate by heat temperature raising
Photoinitiator dye discharges from the local DNA molecular for unwinding, and due to being mismatched between mutation, SNP site double-strand base double-stranded DNA can be made to exist
This site is untied first, by the fluorescence intensity in real-time monitoring temperature-rise period, just may be used from fluorescence intensity and time axial curve
Judge whether mutation or SNP, and different loci, heterozygote whether, G/C content, amplicon length etc. can all influence to melt
The peak shape of curve.So, HRM analyses can effectively distinguish different mutational sites, SNP site and possess the amplification of different G/C contents
Fragment.
The content of the invention
In order to overcome the deficiencies in the prior art, first purpose of the invention is to provide a kind of detection JAG2 gene SNPs
The kit of site rs741859 genotype.
Second object of the present invention is to provide for a kind of preparation method of mentioned reagent box.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：
A kind of kit of detection JAG2 gene SNP site rs741859 genotype, including：Standard items, primer, containing glimmering
The qPCR amplifing reagents of photoinitiator dye；The standard items are comprising JAG2rs741859 (c*1053G>A) the wild type DNA sequences in site
Arrange and comprising JAG2rs741859 (c*1053G>A) the homozygous mutant DNA sequence dna in site；The primer is used to expand standard items
And detected sample.
Preferably, the wild-type DNA-sequence is sequence W；The homozygous mutant DNA sequence dna is sequence M.
Preferably, the standard items are the plasmid for being connected with sequence W and the plasmid for being connected with sequence M.
Preferably, the primer is JAG2-F and JAG2-R.
Preferably, the qPCR amplifing reagents containing fluorescent dye are made a living work bioengineering (Shanghai) limited company
The Green-2-Go qPCR Mastermix kits containing EvaGreen for being produced.
The present invention also provides the preparation side of the kit of described detection JAG2 gene SNP site rs741859 genotype
Method, comprises the following steps：
1) design of primers and synthesis
According to JAG2rs741859 gene orders, using the softwares of Primer express 3, the softwares of PrimerPremier 5
And the softwares of Oligo 7, design one group of primer；
2) standard items are prepared
A, synthesis contain JAG2rs741859 (c*1053G>A) wild-type DNA-sequence in site and homozygous mutant DNA sequences
Row；
B, wild-type DNA-sequence and homozygous mutant DNA sequence dna are connected with plasmid respectively；Plasmid after connection is imported
In bacterium；Cultivate bacterium and obtain positive colony；
The positive colony that C, Amplification Culture step B are obtained, extracts plasmid and obtains standard items；
3) choose or prepare the qPCR amplifing reagents containing fluorescent dye.
Preferably, the plasmid described in step B is pMD18-T plasmids.
Preferably, the wild-type DNA-sequence is sequence W；The homozygous mutant DNA sequence dna is sequence M.
Preferably, sequence W or sequence M are connected with pMD18-T plasmids respectively in step B, the linked system for being used for：
SolutionI is from TaKaRa companies, the product of numbering D6020A.
The reaction time of connection is 10-16 hours, and reaction temperature is 16 DEG C.
Preferably, the concrete operations of step C are that positive colony is inoculated in the LB fluid nutrient mediums containing ammonia benzyl resistance,
37 DEG C of 220rpm cultivate 14-16h；Plasmid is extracted from thalline again and obtains standard items.
Sequence W of the present invention is：
TATATTTGATTGAATAATGCCACCATTCCGGCCTCCCTTGTTCTTTCGGTGCTGTCCCTTTTGTATTGAGAGTGAGG
TTGGGGGAGAGCCACGCCGGCAGAGAGGCTTGGGGCAGTGGGGCGCGTGCTGGGTATTGGCCCACGTGGCTGTGGTG
GCTGTAGAGGGCGAGACGGTTCTGTTGAGTCGGGGCCTGCCAGGGCCTCGAATGCGTTGGCATGCCAAGGTGGTGG；
The base being wherein underlined is gene mutation site；
Sequence M of the present invention is：
TATATTTGATTGAATAATGCCACCATTCCGGCCTCCCTTGTTCTTTCGGTGCTGTCCCTTTTGTATTGAGAGTGAGG
TTGGGGGAGAGCCACGCCGGCAGAGAGGCTTGGGGCAGTGGGGCACGTGCTGGGTATTGGCCCACGTGGCTGTGGTG
GCTGTAGAGGGCGAGACGGTTCTGTTGAGTCGGGGCCTGCCAGGGCCTCGAATGCGTTGGCATGCCAAGGTGGTGG；
The base being wherein underlined is gene mutation site.
The sequence of JAG2-F of the present invention and JAG2-R is specially：
JAG2-F：5'-CAGAGAGGCTTGGGGCAGT-3'
JAG2-R：5'-AACAGAACCGTCTCGCCCTC-3'
Compared to existing technology, the beneficial effects of the present invention are：
The present invention is prepared for a kind of kit, makes HRM methods for identifying JAG2 gene SNP site rs741859 genes
Type, the kit has sensitivity high, low cost, simple to operate, the fireballing advantage of identification.The present invention also provides the kit
Preparation method, make kit sensitivity of the invention higher by controlling the parameter of preparation process.
Brief description of the drawings
The sensitivity test result figure of Fig. 1, kit of the present invention
The testing result figure of Fig. 2, the present invention to genotype
Fig. 3, Sanger sequencing result figure
Specific embodiment
Below, with reference to accompanying drawing and specific embodiment, the present invention is described further：
Embodiment 1：
A kind of kit of detection JAG2 gene SNP site rs741859 genotype, including：Standard items, primer, containing glimmering
The qPCR amplifing reagents of photoinitiator dye；
The standard items are the pMD18-T plasmids for being connected with sequence W and the pMD18-T plasmids for being connected with sequence M.
The primer is JAG2-F and JAG2-R.
The qPCR amplifing reagents containing fluorescent dye work bioengineering (Shanghai) limited company of making a living is produced
Green-2-Go qPCR Mastermix kits containing EvaGreen.
Embodiment 2
Structure and the conversion of restructuring wild type and homozygous mutant plasmid pMD18-T-rs741859
1st, composition sequence W and sequence M.
2nd, coupled reaction：The sequence W and sequence M of above-mentioned synthesis are attached with pMD18-T respectively, using following connection
System is prepared：
16 DEG C are placed in after the completion of preparation carries out overnight coupled reaction, the plasmid for being connected with sequence W for obtaining and is connected with sequence M
Plasmid.
3rd, the conversion of pMD18-T-rs741859 plasmids and PCR are identified
(1) the DH5 α competent cells for freezing are taken out from -80 DEG C of ultra low temperature freezer, being placed on ice chest makes it thaw.
(2) the DH5 α that connection product (plasmid for being connected with sequence W and the plasmid for being connected with sequence M) 10 μ L add 50 μ L are taken
In competent cell, rearmounted ice bath is gently shaken up 30 minutes.
(3) 42 DEG C of water-bath heat shocks 90 seconds, put cooled on ice 2min immediately after heat shock.
(4) after adding the piping and druming of the LB fluid nutrient mediums without resistance of the 1ml of precooling to mix in 1.5ml EP pipes, 37 DEG C
120 turns of jog culture 90min.
(5) the of short duration centrifugation of above-mentioned nutrient solution is sucked and take remaining 100ul after 900ul and coat the LB containing Amp antibiotic
On flat board, face up placement 30min, after bacterium solution is cultured base absorption completely, is inverted 37 DEG C of insulating box cultures of culture dish
Night.
(6) from 1.5ml EP pipe of the picking monoclonal bacterium colony on flat board in 500 μ L Amp resistance LB fluid nutrient mediums, 37
DEG C 220rpm shaken cultivations 5-6 hours.
(7) take 1 μ L carries out bacterium solution PCR identifications as template.The bacterium solution that the positive will be accredited as is added to the LB liquid of 20ml
Carry out expanding the positive colony for shaking and obtaining in culture medium.
(8) above-mentioned dilution bacterium solution is expanded with primer JAG2-F and JAG2-R, PCR primer uses 2% agarose gel electrophoresis,
By detecting that PCR primer identifies positive transformant.
Primer sequence is that HRM analyzes the primer, and sequence is as follows：
JAG2-F：5'-CAGAGAGGCTTGGGGCAGT-3'
JAG2-R：5'-AACAGAACCGTCTCGCCCTC-3'
PCR system is as follows：
PCR buffer are the Power Taq DNA polymerases from the Tyke Bioisystech Co., Ltd of Beijing hundred, article No.
PR1001。
Amplification program/reaction condition：94 DEG C of predegeneration 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30sec, 35 circulations；
72℃10min。
4th, negative and positive plasmid is recombinated to extract：The Plasmid Preparation kit produced using the Imtech of Beijing hundred extracts weight
Group plasmid, determines concentration and purity, is sequenced while drawing a part of plasmid purification and delivering to Shanghai bioengineering Co., Ltd,
Determine that the gene order of Insert Fragment is consistent with aim sequence.
Embodiment 3
The acquisition of standard items and quantitative
(1) the positive colony 100ul containing recombinant plasmid that what Example 2 was obtained freeze is inoculated in 15ml ammonia benzyl resistances
LB fluid nutrient mediums in, 37 DEG C of 220rpm cultivate 14-16h；
(2) thalline for obtaining, the Plasmid Preparation kit produced using the Tyke bio tech ltd of Beijing hundred extracts weight
Group plasmid；
(3) the ultramicron ultraviolet-uisible spectrophotometer (ND5000) using the Tyke bio tech ltd of Beijing hundred is right
The plasmid of extraction determines concentration, and the purity of plasmid is judged according to A260/A280.
Embodiment 4
The foundation of HRM-PCR detection JAG2rs741859 methods and sensitivity technique
First, the preparation of sample to be tested
30 poba gene group DNA of sample are extracted, the template as the amplification of JAG2 gene PCRs.Poba gene group DNA's
Extraction step is as follows：
(1) 1ml whole bloods are taken, 3ml TE are added, 10min is stood after reverse mixing, 8000rpm is centrifuged 5 minutes, abandons supernatant
Liquid；
(2) 2-3 times is repeated the above steps to being precipitated as white；
(3) 900ul 10%SDS and 10ul 10mg/ml Proteinase Ks, 55 DEG C of water-bath 1h are added；
(4) centrifuge tube is cooled to room temperature, adds isometric phenol/chloroform/isoamyl alcohol (25:24:1) mix,
12000rpm is centrifuged 10min；
(5) isometric phenol/chloroform/isoamyl alcohol (25 is added after careful absorption supernatant:24:1) mix, 12000rpm centrifugations
10min；
(6) supernatant is taken, adds the absolute ethyl alcohol of two volumes, mixing of turning upside down, 12000rpm centrifugation 10min to abandon
Clearly；
(7) add the ethanol washing precipitation of 500ul 70%, supernatant is abandoned after of short duration centrifugation, uncap to dry to ethanol and wave completely
Hair；
(8) 50ul distilled waters or TE dissolving DNAs, -20 DEG C of preservations are added.
2nd, the design of specific primer and synthesis
The present invention is retrieved by JAG2rs741859 gene orders in ncbi database, is chosen and is adapted to design primer
Fragment sequence be target, it is soft using the softwares of Primer express 3, the softwares of Primer Premier 5 and Oligo 7
Part, devises one group of HRM-PCR primer.The primer sequence is as follows：
JAG2-F：5'-CAGAGAGGCTTGGGGCAGT-3'
JAG2-R：5'-AACAGAACCGTCTCGCCCTC-3'
3rd, HRM-PCR reaction systems and reaction condition
With DNA as template, with above-mentioned primer JAG2-F and JAG2-R as amplimer, using such as following systems and reaction bar
Part enters performing PCR amplification.
PCR system：
Wherein primer uses raw work biology (Shanghai) Green-2-Go qPCR using JAG2-F and JAG2-R, HRM-PCR
Mastermix kits.HRM analyzers are hundred source Gene A SA-9600 real-time fluorescence quantitative PCR instrument.
PCR amplification programs：
94 DEG C of predegeneration 3min；94 DEG C of denaturation 10s；60 DEG C of annealing/extension 20s.
HRM is analyzed：
94℃ 90s
60℃ 30sec
83-94 DEG C of collection data, temperature rate-of-rise is 0.06 DEG C/s.
4th, HRM sensitivity experiments
Positive plasmid will be recombinated and the negative plasmid of restructuring is diluted to 50ng/ μ l, then the two mixes to recombinate positive matter
Grain accounting example is followed successively by 50%, 25%, 12.5%, 5%, 2% and 1% carries out gradient dilution, according to above-mentioned HRM-PCR reactants
System and program are expanded, analysis mutation inspection range, i.e. sensitivity.
Reaction system is as follows：
PCR amplification programs：
94 DEG C of predegeneration 3min；94 DEG C of denaturation 10s；60 DEG C of annealing/extension 20s
HRM is analyzed：
94℃ 90s
60℃ 30sec
83-94 DEG C of collection data, temperature rate-of-rise is 0.06 DEG C/s.
As a result reference picture 1, experimental data shows, when positive plasmid is respectively 1 with negative plasmid volume ratio:1、1:3、1:7、
1:19、1:49 and 1:When 99, positive plasmid can be detected, so the sensitivity of HRM detection methods used by this patent is 1%.
Embodiment 5
Sample JAG2rs741859 genotype is detected using detection method of the invention
With the testing conditions in step 2, HRM analyses are carried out to 30 samples, with restructuring positive plasmid and the negative matter of restructuring
The melting curve of grain is compared, and the mutation type in sample is determined according to this, and be directed to every kind of genotype according to HRM analysis results
Select 1 sample at random carries out Sanger sequence verifications in raw work biology (Shanghai).
, referring to table 1 and Fig. 2, JAG2 gene SNP sites rs741859 is wild in 30 samples of data display for HRM analysis results
Type has 17, G>A heterozygous mutants have 8, G>A homozygous mutants 5.
Sanger sequencing results are referring to Fig. 3：Wherein a is the sequencing result of sample number 7, and b is the sequencing of sample number 14
As a result, c is the sequencing result of sample number 28；It is completely the same with the result that the inventive method is detected.
Analysis result of the application method of the present invention of table 1 to sample
| Sample number | Genotype | Sample number | Genotype |
| 1 | Homozygous mutant | 16 | Wild type |
| 2 | Wild type | 17 | Heterozygous mutant |
| 3 | Wild type | 18 | Wild type |
| 4 | Wild type | 19 | Homozygous mutant |
| 5 | Heterozygous mutant | 20 | Wild type |
| 6 | Heterozygous mutant | 21 | Homozygous mutant |
| 7 | Wild type | 22 | Wild type |
| 8 | Wild type | 23 | Heterozygous mutant |
| 9 | Heterozygous mutant | 24 | Wild type |
| 10 | Homozygous mutant | 25 | Heterozygous mutant |
| 11 | Wild type | 26 | Wild type |
| 12 | Wild type | 27 | Wild type |
| 13 | Wild type | 28 | Homozygous mutant |
| 14 | Heterozygous mutant | 29 | Heterozygous mutant |
| 15 | Wild type | 30 | Wild type |
For a person skilled in the art, technical scheme that can be as described above and design, make other each
Plant corresponding change and deform, and all these changes and deforms the protection model that should all belong to the claims in the present invention
Within enclosing.
Claims (10)
1. a kind of kit of detection JAG2 gene SNP site rs741859 genotype：It is characterized in that including：Standard items, draw
Thing, the qPCR amplifing reagents containing fluorescent dye；The standard items are comprising JAG2 rs741859 (c*1053G>A) site
Wild-type DNA-sequence and comprising JAG2 rs741859 (c*1053G>A) the homozygous mutant DNA sequence dna in site；The primer is used
In amplification standard items and detected sample.
2. the kit of detection JAG2 gene SNP site rs741859 genotype according to claim 1, its feature exists
In：The wild-type DNA-sequence is sequence W；The homozygous mutant DNA sequence dna is sequence M.
3. the kit of detection JAG2 gene SNP site rs741859 genotype according to claim 2, its feature exists
In：The standard items are the plasmid for being connected with sequence W and the plasmid for being connected with sequence M.
4. the kit of detection JAG2 gene SNP site rs741859 genotype according to claim 2, its feature exists
In：The primer is JAG2-F and JAG2-R.
5. the kit of detection JAG2 gene SNP site rs741859 genotype according to claim 1, its feature exists
In：What the qPCR amplifing reagents containing fluorescent dye made a living that work bioengineering (Shanghai) limited company produced contains
The Green-2-Go qPCR Mastermix kits of EvaGreen.
6. a kind of preparation side of the kit of detection JAG2 gene SNP site rs741859 genotype as claimed in claim 1
Method, it is characterised in that comprise the following steps：
1) design of primers and synthesis
According to JAG2 rs741859 gene orders, using the softwares of Primer express 3, the softwares of Primer Premier 5 with
And the softwares of Oligo 7, design one group of primer；
2) standard items are prepared
A, synthesis contain JAG2 rs741859 (c*1053G>A) wild-type DNA-sequence in site and homozygous mutant DNA sequence dna；
B, wild-type DNA-sequence and homozygous mutant DNA sequence dna are connected with plasmid respectively；Plasmid after connection is imported into bacterium
In；Cultivate bacterium and obtain positive colony；
The positive colony that C, Amplification Culture step B are obtained, extracts plasmid and obtains standard items；
3) choose or prepare the qPCR amplifing reagents containing fluorescent dye.
7. the preparation method of the kit of detection JAG2 gene SNP site rs741859 genotype according to claim 6,
It is characterized in that the plasmid described in step B is pMD18-T plasmids.
8. the preparation method of the kit of detection JAG2 gene SNP site rs741859 genotype according to claim 7,
It is characterized in that：The wild-type DNA-sequence is sequence W；The homozygous mutant DNA sequence dna is sequence M.
9. the preparation method of the kit of detection JAG2 gene SNP site rs741859 genotype according to claim 8,
It is characterized in that：Sequence W or sequence M are connected with pMD18-T plasmids respectively in step B, the linked system for being used for：
The reaction time of connection is 10-16 hours, and reaction temperature is 16 DEG C.
10. the preparation side of the kit of detection JAG2 gene SNP site rs741859 genotype according to claim 6
Method, it is characterised in that：The concrete operations of step C are that positive colony is inoculated in the LB fluid nutrient mediums containing ammonia benzyl resistance,
Temperature is that 37 DEG C of mixing speeds are that 220rpm cultivates 14-16h；Plasmid is extracted from thalline again and obtains standard items.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589785A (en) * | 2013-09-05 | 2014-02-19 | 谢小冬 | Application of NOTCH3 and JAG2 gene SNP ((Single Nucleotide Polymorphism)) loci |
| US20160032389A1 (en) * | 2008-11-03 | 2016-02-04 | Life Technologies Corporation | Method, Kits, and Reaction Mixtures For High Resolution Melt Genotyping |
| CN105441545A (en) * | 2015-12-17 | 2016-03-30 | 苏州百源基因技术有限公司 | Method for detecting FOXH1 gene SNP locus rs750472 genotype |
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2017
- 2017-02-28 CN CN201710113052.3A patent/CN106868136A/en active Pending
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|---|---|---|---|---|
| US20160032389A1 (en) * | 2008-11-03 | 2016-02-04 | Life Technologies Corporation | Method, Kits, and Reaction Mixtures For High Resolution Melt Genotyping |
| CN103589785A (en) * | 2013-09-05 | 2014-02-19 | 谢小冬 | Application of NOTCH3 and JAG2 gene SNP ((Single Nucleotide Polymorphism)) loci |
| CN105441545A (en) * | 2015-12-17 | 2016-03-30 | 苏州百源基因技术有限公司 | Method for detecting FOXH1 gene SNP locus rs750472 genotype |
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| CARILLO S等: "Nested high-resolution melting curve analysis a highly sensitive, reliable, and simple method for detection of JAK2 exon 12 mutations--clinical relevance in the monitoring of polycythemia", 《J MOL DIAGN》 * |
| 张婷婷: "中国西北人群NOTCH3，JAG2基因多态性与非综合征性唇腭裂的关联研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
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Application publication date: 20170620 |