CN108060214A - A kind of combination primer of mankind's gall stone associated gene mutation examination and its application - Google Patents
A kind of combination primer of mankind's gall stone associated gene mutation examination and its application Download PDFInfo
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- CN108060214A CN108060214A CN201810122702.5A CN201810122702A CN108060214A CN 108060214 A CN108060214 A CN 108060214A CN 201810122702 A CN201810122702 A CN 201810122702A CN 108060214 A CN108060214 A CN 108060214A
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Abstract
Combination primer and its application the invention discloses a kind of mankind's gall stone associated gene mutation examination, are related to technical field of gene detection, and the combination primer of mankind's gall stone associated gene mutation examination is divided into combination forward primer and combination reverse primer；Mankind's gall stone associated gene mutation kit for screening, including combination primer and LightScanner Master Mix.The present invention proposes a kind of method of mankind's gall stone associated gene mutation examination, it is based on high-resolution melting curve method, can simultaneously the common gall stone related gene of examination 3 mutational sites, include rs1260326, rs1256049, rs11887534 common mutations site of related gene on chr2, chr14 chromosome, have the characteristics that flux is high, at low cost, detection time is short.
Description
Technical field：
The present invention relates to technical field of gene detection, and in particular to a kind of group of mankind's gall stone associated gene mutation examination
Close primer and its application.
Background technology：
Gall stone is a kind of ancient and common benign disease, and become international important public health problem.West
The epidemiology survey of square developed country shows that incidence is 10%-15%, and the average attack rate of China at this stage is 8%,
There is the morbidity that investigation shows 25% gall stone to be influenced by inherent cause.
Through Primary Study mankind gall stone may with chr2, the rs1260326 of related gene on chr14 chromosomes,
The polymorphism in rs1256049, rs11887534 site has certain association.And currently for the research of these gene loci polymorphisms
Mainly Sanger PCR sequencing PCRs, a certain section of region that this method can only be directed to some sample are detected, and detection efficiency is low, into
This height.
Based on this, combination primer and its application the present invention provides a kind of mankind's gall stone associated gene mutation examination.
The content of the invention：
In view of the deficienciess of the prior art, there is provided a kind of mankind's gall stone dependency basis for first purpose of the present invention
Because of the combination primer of Mutation Screening, second purpose there is provided a kind of mankind's gall stone associated gene mutation kit for screening,
There is provided a kind of mankind's gall stone related gene fluorescent quantitative PCR detection method, the 4th purposes to be to provide for 3rd purpose
The application of said combination primer and kit.
The technical problems to be solved by the invention are realized using following technical solution：
A kind of combination primer of mankind's gall stone associated gene mutation examination, the combination primer are divided into combination forward primer
With combination reverse primer；
The combination forward primer includes the forward primer sequence SEQ ID in rs1260326 sites on detection chr2 chromosomes
NO:1st, the forward primer sequence SEQ ID NO in rs11887534 sites:5 and detection chr14 chromosomes on rs1256049 sites
Forward primer sequence SEQ ID NO:3；
The combination reverse primer includes the reverse primer sequences SEQ ID in rs1260326 sites on detection chr2 chromosomes
NO:2nd, the reverse primer sequences SEQ ID NO in rs11887534 sites:6 and detection chr14 chromosomes on rs1256049 sites
Reverse primer sequences SEQ ID NO:4；
SEQ ID NO:1F:TGGCACACAGCACCGTGGGTCAGACCTTGC
SEQ ID NO:2R:GGTGAGAGTCCAGCCGTGACAAAGGGACCC
SEQ ID NO:3F:ACTTGGTCGAACAGGCTGAGCTCCACAAAG
SEQ ID NO:4R:GCTTTCAAAAGCAGAAGTAGGAGGCTGAGA
SEQ ID NO:5F:AGAGGGCTGCCGAAAGGGGCCACTCCCCAG
SEQ ID NO:6R:ATACCTCGGTGAGTGAGCAATGGGAAGTCG。
A kind of mankind's gall stone associated gene mutation kit for screening, including combination primer and LightScanner
Master Mix。
A kind of detection method of mankind's gall stone associated gene mutation kit for screening, comprises the following steps：
1) sample collection, DNA extractions；
2) combination primer according to equal proportion is mixed, as a primer pond after mixing, and configures reaction system, utilization is glimmering
Fluorescent Quantitative PCR technology Amplification primer, amplification condition are：95 DEG C of pre-degeneration 10min；Then by 95 DEG C of denaturation 10s, 65 DEG C are moved back
The program of fiery 10s, 72 DEG C of extension 30s carry out 20 Xun Huans, and often completing one cycle annealing temperature reduces by 0.5 DEG C, until 55 DEG C,
Stop cooling, carry out 25 Xun Huans again afterwards；The DNA fragmentation amplified is to expand library；
3) DNA fragmentation amplified carries out high-resolution melting curve process according to high-resolution melting curve condition, melts
Solution step carries out immediately after PCR cycle, and program is：95 DEG C of 1min are warming up to, 40 DEG C of 1min is then cooled to, then heats up
To 65 DEG C of 1s, from 65 DEG C of continuous warmings to phosphor collection is carried out in a manner of 25 times/DEG C during 95 DEG C, 40 are finally cooled to
℃。
The reaction system is as follows：
It is described combination primer for 3 pairs combination primers, upstream combination primer combine primer with downstream between annealing temperature 3
Within DEG C, and the △ G absolute values of the primer dimer formed between primer pair and primer pair are less than 5.
The combination primer, the kit are in terms of preparing for mankind's gall stone associated gene mutation screening agent
Using.
For a better understanding of the present invention, the definition and explanation of relational language is provided below.
" high-resolution melting curve method " as used herein, the term is primarily referred to as based on nucleic acid molecules physical property
Difference.The segment length of different nucleic acid molecules, G/C content, GC distributions etc. are different, therefore any double chain DNA molecule is adding
Can all there are shape and the position of oneself melting curve during thermal denaturation.The basic principle of high-resolution melting curve technology is exactly basis
The difference of melting curve distinguishes sample.
Design mainly follows：It is compared with software, ensures that the annealing temperature between primer pair is not much different, preferably 3
Within DEG C.The absolute value of the △ G of the primer dimer formed between primer pair and primer pair is less than 5.The length of combination primer exists
20-40bp so in synthetic primer, will be selected compared with High Purity standard.
The beneficial effects of the invention are as follows：
(1) present invention proposes a kind of method of mankind's gall stone associated gene mutation examination, is melted based on high-resolution
Solution curve method (fluorescent quantitative PCR technique), can simultaneously the common gall stone related gene of examination 3 mutational sites, including
Rs1260326, rs1256049, rs11887534 common mutations site of related gene on chr2, chr14 chromosome have logical
The characteristics of amount is high, at low cost, detection time is short.
(2) it is high-throughput.The result obtained by generation sequencing is single base as a result, of the invention to carry out once
Method can at least detect 30 parts of samples, so that it can be widely used in the census operations of gall stone related gene.
(3) it is inexpensive.Using the sequenator of independent research and the reagent of independent research, price will be well below import
Instrument and reagent so that sequencing cost substantially reduces, so as to which its testing cost is greatly reduced.
Specific embodiment：
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Specific embodiment is closed, the present invention is further explained.
Design of primers is as follows：
In order to ensure that sample is not polluted by food or beverage, it please don't feed and drink water in 30min before sampling.
1) genomic DNA is extracted from Patients with Cholelithiasis buccal swab sample.
2) different combination primers according to equal proportion is mixed, a primer pond is used as after mixing.Using 96 orifice plates or
384 orifice plates configure reaction system, seal plate film, and of short duration centrifugation makes the solution on tube wall be collected into tube bottom；(pay attention to：During pad pasting,
Using Roche LightCycler 480 II specially with scraper plate, film is made to be adjacent to PCR plate.)
Reaction system is as follows：
480 II real-time fluorescence quantitative PCR systems of Roche Light Cycler, start；
It is expanded with this primer according to the method for quantitative fluorescent PCR, amplification condition is as follows：95 DEG C of pre-degeneration 10min；So
Afterwards by 95 DEG C of denaturation 10s, 65-55 DEG C of annealing (each cycles and declines 0.5 DEG C) 10s, and the program of 72 DEG C of extension 30s carries out 45 and follows
Ring；The DNA fragmentation amplified is to expand library,；
3) DNA fragmentation in step 2) carries out high-resolution solubility curve process according to high-resolution melting curve condition,
The melting step of amplified production carries out immediately after PCR cycle, and program is：95 DEG C of 1min are warming up to, are then cooled to 40
DEG C 1min, then it is warming up to 65 DEG C of 1s.From 65 DEG C of continuous warmings to carrying out phosphor collection (25 time/DEG C) during 95 DEG C.Finally,
It is cooled to 40 DEG C.
The basic principles, main features and the advantages of the invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (6)
1. a kind of combination primer of mankind's gall stone associated gene mutation examination, it is characterised in that：The combination primer is divided into group
Close forward primer and combination reverse primer；
The combination forward primer includes the forward primer sequence SEQ ID NO in rs1260326 sites on detection chr2 chromosomes:
1st, the forward primer sequence SEQ ID NO in rs11887534 sites:5 and detection chr14 chromosomes on rs1256049 sites just
To primer sequence SEQ ID NO:3；
The combination reverse primer includes the reverse primer sequences SEQ ID NO in rs1260326 sites on detection chr2 chromosomes:
2nd, the reverse primer sequences SEQ ID NO in rs11887534 sites:6 and detection chr14 chromosomes on rs1256049 sites it is anti-
To primer sequence SEQ ID NO:4；
SEQ ID NO:1F:TGGCACACAGCACCGTGGGTCAGACCTTGC
SEQ ID NO:2R:GGTGAGAGTCCAGCCGTGACAAAGGGACCC
SEQ ID NO:3F:ACTTGGTCGAACAGGCTGAGCTCCACAAAG
SEQ ID NO:4R:GCTTTCAAAAGCAGAAGTAGGAGGCTGAGA
SEQ ID NO:5F:AGAGGGCTGCCGAAAGGGGCCACTCCCCAG
SEQ ID NO:6R:ATACCTCGGTGAGTGAGCAATGGGAAGTCG。
2. a kind of mankind's gall stone associated gene mutation kit for screening, it is characterised in that：Including combination described in claim 1
Primer and LightScanner Master Mix.
3. the detection method of mankind's gall stone associated gene mutation kit for screening as claimed in claim 2, which is characterized in that
Comprise the following steps：
1) sample collection, DNA extractions；
2) combination primer according to equal proportion is mixed, as a primer pond after mixing, and configures reaction system, determined using fluorescence
Round pcr Amplification primer is measured, amplification condition is：95 DEG C of pre-degeneration 10min；Then by 95 DEG C of denaturation 10s, 65 DEG C of annealing
The program of 10s, 72 DEG C of extension 30s carry out 20 Xun Huans, and often completing one cycle annealing temperature reduces by 0.5 DEG C, until 55 DEG C, stop
Only cool down, carry out 25 Xun Huans again afterwards；The DNA fragmentation amplified is to expand library；
3) DNA fragmentation amplified carries out high-resolution melting curve process according to high-resolution melting curve condition, melts step
Suddenly carried out immediately after PCR cycle, program is：95 DEG C of 1min are warming up to, 40 DEG C of 1min is then cooled to, then is warming up to 65
DEG C 1s from 65 DEG C of continuous warmings to phosphor collection is carried out in a manner of 25 times/DEG C during 95 DEG C, is finally cooled to 40 DEG C.
4. the detection method of mankind's gall stone associated gene mutation kit for screening according to claim 3, feature exist
In the reaction system is as follows：
5. the kit described in combination primer according to claim 1, claim 2, the kit described in claim 3
Detection method, it is characterised in that：The combination primer is 3 pairs of combination primers, between upstream combination primer combines primer with downstream
Annealing temperature within 3 DEG C, and the △ G absolute values of the primer dimer formed between primer pair and primer pair be less than 5.
6. the kit described in combination primer described in claim 1, claim 2 is being prepared for mankind's gall stone dependency basis
Because of the application in terms of Mutation Screening reagent.
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| CN201810122702.5A CN108060214A (en) | 2018-02-07 | 2018-02-07 | A kind of combination primer of mankind's gall stone associated gene mutation examination and its application |
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| CN201810122702.5A CN108060214A (en) | 2018-02-07 | 2018-02-07 | A kind of combination primer of mankind's gall stone associated gene mutation examination and its application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234406A (en) * | 2018-10-30 | 2019-01-18 | 深圳市万众基因转化医学研究院 | A kind of quick detection combination primer of mankind's skin quality and its 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 |
| CN109295211A (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 alopecia areata associated gene mutation screening and its application |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921830A (en) * | 2010-03-29 | 2010-12-22 | 苏州工业园区为真生物医药科技有限公司 | Rapid detection of EGFR (Epidermal Growth Factor Receptor) gene mutation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234406A (en) * | 2018-10-30 | 2019-01-18 | 深圳市万众基因转化医学研究院 | A kind of quick detection combination primer of mankind's skin quality and its 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 |
| CN109295211A (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 alopecia areata associated gene mutation screening and its application |
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