CN110042158A - The detection method in ferro element absorption associated gene mutation site - Google Patents
The detection method in ferro element absorption associated gene mutation site Download PDFInfo
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- CN110042158A CN110042158A CN201910334950.0A CN201910334950A CN110042158A CN 110042158 A CN110042158 A CN 110042158A CN 201910334950 A CN201910334950 A CN 201910334950A CN 110042158 A CN110042158 A CN 110042158A
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- single base
- base extension
- primer
- ferro element
- detection method
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
Abstract
The invention belongs to fields of biomedicine, it solves unidirectional Single base extension to be difficult to accurately determine variant sites, disclose a kind of detection method in ferro element absorption associated gene mutation site, target fragment is obtained by the Single-tube multiplex-PCR amplified reaction of the target site specific primer of design, then Single base extension technology is recycled to carry out two-way extension to the target fragment, it the gene loci to be detected such as obtains, accurately measures target dna sequence finally by the gene loci to be detected such as nucleic acid mass spectral analysis；Wherein, the Single base extension technology is to carry out two-way extension using target fragment described in Single base extension primer pair；It is intended to the technology by two-way Single base extension, related gene is absorbed to ferro element and carries out variation inspection, to provide safely instruction for individual clinical application.
Description
Technical field
The present invention relates to field of biomedicine, in particular to a kind of ferro element absorbs the detection side in associated gene mutation site
Method.
Background technique
Genomics is studies have shown that the assimilation effect of ferro element in vivo and the phenotype of TMPRSS6 gene are closely related.
The amplified fragments of TMPRSS6 gene are TMPRSS6-1；
Therefore, the variation situation of related gene is detected, people's reasonable diet can be effectively helped, guarantees nutrient balance.
Associated nucleic acid mass spectrum detection currently on the market is carried out using unidirectional Single base extension technology.And
Unidirectional Single base extension is difficult to accurately determine variant sites.
The present invention is directed to the technologies by two-way Single base extension, absorb related gene to ferro element and carry out variation inspection,
To provide safely instruction for individual clinical application.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, the technical problem to be solved by the present invention is that providing a kind of ferro element suction
Receive the detection method and detection kit in associated gene mutation site.
The technical solution adopted by the present invention is that: a kind of ferro element absorbs the detection method in associated gene mutation site, passes through
The Single-tube multiplex-PCR amplified reaction of the target site specific primer of design obtains target fragment, then single base is recycled to prolong
It stretches technology and two-way extension is carried out to the target fragment, the gene loci to be detected such as obtain, finally by nucleic acid mass spectral analysis etc.
Gene loci to be detected accurately measures target dna sequence；
Wherein, the Single base extension technology is to carry out two-way prolong using target fragment described in Single base extension primer pair
It stretches.
Preferably, the target site specific primer includes:
The specific primer of related gene TMPRSS6-1 segment is absorbed for expanding ferro element, the specific primer is just
To primer nucleotide sequence as shown in SEQ ID No.1, the nucleotide sequence of the reverse primer of the specific primer such as SEQ
Shown in ID No.2.
Preferably, the Single base extension primer includes:
The Single base extension primer of related gene TMPRSS6-1 segment, the Single base extension are absorbed for expanding ferro element
The nucleotide sequence of primer is as shown in SEQ ID No.3.
Preferably, the response procedures of Single-tube multiplex-PCR amplification are as follows: 95 DEG C 2 minutes, 95 DEG C 30 seconds, 56 DEG C 20 seconds, 72
DEG C 60 seconds, 45 circulations, 72 DEG C 5 minutes, 4 DEG C of preservations.
The beneficial effects of the present invention are:
Target fragment is obtained by the Single-tube multiplex-PCR amplified reaction of the target site specific primer of design, then again
Using Single base extension technology, by two-way extension, the gene loci to be detected such as obtain, it is accurate finally by nucleic acid mass spectral analysis
Measure target dna sequence；The present invention is directed to the technologies by two-way Single base extension, absorb related gene to ferro element and become
Different inspection, to provide safely instruction for individual clinical application.
Specific embodiment
The present invention will be further described in detail below with reference to the embodiments, to enable those skilled in the art referring to specification
Text can be implemented accordingly.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein are not precluded one or more
The presence or addition of a other elements or combinations thereof.
A kind of ferro element of the invention absorbs the detection method in associated gene mutation site, comprising the following steps:
1) sample DNA is extracted,
1, the sample 10000rpm in centrifuge tube is centrifuged 2min, removes supernatant, stays precipitating spare.
2,350 μ l Buffer MCL (MCL buffer) and 20 μ lProteinase K (eggs are added in Xiang Shangshu centrifuge tube
White enzyme K), concussion mix, 65 DEG C of water-bath 20min, or mix.
3,350 μ l Buffer MA (MA buffer) and 25 μ l SanMag are added in the backward centrifuge tube of water-bath completion
Beads (adsorptivity magnetic bead), vibrates or is mixed by inversion, be stored at room temperature 3min, or mix.
4, centrifuge tube is placed in 30s on magnetic frame, after being drawn to tube wall completely to SanMag Beads, inhales and abandon supernatant, from
Centrifuge tube is taken out on magnetic frame.
5,700 μ l70% ethyl alcohol are added into centrifuge tube, suction is played or put vibration and mix, centrifuge tube is placed on magnetic frame
30s inhales and abandons supernatant, centrifuge tube is taken out from magnetic frame.
6, it is primary to repeat step 5, room temperature is uncapped dry 10min no liquid residual in managing.
7,50 μ l TE Buffer (TE buffer) (pH8.0) are added into centrifuge tube, or mix.
8, it takes out centrifuge tube and is placed in 30s on magnetic frame, it is careful to inhale after being drawn on tube wall completely to SanMag Beads
Take supernatant to new centrifuge tube, i.e. acquisition genomic DNA.
Be related to main agents consumptive material: (all reagents used above are all inside this kit to DNA extraction agent box
) (Order NO.B518766), pipettor (step 2,3,4,5,6,7,8), pipette tips (step 2,3,4,5,6,7,8), small-sized
Centrifuge (step 1), magnetic frame (step 4,5,8), vortex oscillator (step 2,3,5,6,7).
2) specific primer is designed,
The gene sequence information is inquired and downloaded on the website NCBI, and the upstream and downstream in site to be measured chooses the special of 20nt
Property sequence is as amplimer.Design principle is: the DNA fragmentation length 100bp-300bp comprising site to be measured avoids sending out
Clamping structure and repetitive sequence.After design is completed, one section of fixed sequence program ACGTTGGATG is added at the end primer 5'.
3) Single base extension primer is designed,
The design of Single base extension primer selects base position upstream to be measured under also in designing in the gene order
The base being close to is swum, and according to Mass Spectrometer Method range (- 9000 Dalkon Shield of 4000 Dalkon Shield), design primer length, design principle
First base of primer amplification is base to be measured, and avoids hairpin structure and repetitive sequence.
4) nucleic acid mass spectral analysis,
1,1 μM of (each primer) PCR primer mixture is prepared, the inside includes each SNP site in multiple reaction
Forward (forward direction) and reverse (reversed) primer.
2, PCR is prepared with 2ml, pipe and mixes liquid, DNA sample and control are not added in wherein.
3,2ul DNA sample is added, vortex oscillator mixes, and sticks sealed membrane.
4,96 orifice plates are put into PCR instrument and carry out following thermal cycle: 95 DEG C 2 minutes, 95 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 60
Second, 45 circulation, 72 DEG C 5 minutes, 4 DEG C heat preservation.
5, the extension primer in each site is diluted to 100Um, calculates the ratio in system, Um is that part per million is dense
Degree prepares iPLEX extension primer and mixes liquid.
6, iPLEX is prepared in 1.5mL pipe extends mixed liquid.
7, each hole is added 2 μ l iPLEX and extends mixed liquid and mixed.
8, plate is sealed with film, be vortexed concussion and centrifugation (4000rpm5 seconds).
9,96 orifice plates are put into PCR instrument and carry out following thermal cycle:
10,41ul water is added in each hole for having sample of sample plane to be then centrifuged for.
11,96 orifice plates are put into mass spectrograph, open point sample program, carries out automatic printing operation.
12, flight mass spectrum software is opened, parameter is set, is detected.
13, it after detection is introduced, is automatically generated in software as a result, exporting.
Be related to main agents consumptive material: the centrifuges (step 3,8,10) of 96 hole PCR plates, vortex oscillator (step 3,8),
PCR instrument (thermo) (step 4,9), MassARRAY mass spectrograph (agena) (step 11,12,13), PCR kit (Agena
Bioscience article No.: 11327) (step 2), Single base extension kit (Agena Bioscience article No.: 10165) (step
It is rapid 6), pipettor (steps 1 and 2,3,5,6,7,10), pipette tips (steps 1 and 2,3,5,6,7,10), 96 orifice plates (step 2,3,4,7,
8,9,10,11), sealed membrane (step 3,8).
Table 1 is the sequence table of specific primer；Table 2 is Single base extension primer sequence table；
1. specific primer sequence table of table
2. Single base extension primer sequence table of table
| Amplified fragments | Primer | Sequence number | 5'-3' |
| TMPRSS6-1 | TMPRSS6-1-E | SEQ ID No.3 | CCAAAGGACCTGTGCAGCGAGG |
Sequence table
<110>Hangzhou Yun Ding gene biological Science and Technology Ltd.
<120>ferro element absorbs the detection method and detection kit in associated gene mutation site
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>forward primer of homo sapiens (Homo sapiens) TMPRSS6-1
<400> 1
acgttggatg atgtgcagtt gatcccacag 30
<210> 2
<211> 30
<212> DNA
<213>reverse primer of homo sapiens (Homo sapiens) TMPRSS6-1
<400> 2
acgttggatg atccttcttg cccttgcggt 30
<210> 3
<211> 22
<212> DNA
<213>the Single base extension primer of homo sapiens (Homo sapiens) TMPRSS6-1
<400> 3
ccaaaggacc tgtgcagcga gg 22
Claims (4)
1. the detection method that a kind of ferro element absorbs associated gene mutation site, it is characterised in that: pass through the target site of design
The Single-tube multiplex-PCR amplified reaction of specific primer obtains target fragment, then recycles Single base extension technology to the mesh
Standard film section carries out two-way extension, the gene loci to be detected such as obtains, finally by the gene loci to be detected such as nucleic acid mass spectral analysis
Accurate measurement target dna sequence；
Wherein, the Single base extension technology is to carry out two-way extension using target fragment described in Single base extension primer pair.
2. the detection method that ferro element according to claim 1 absorbs associated gene mutation site, which is characterized in that described
Target site specific primer includes:
The specific primer of related gene TMPRSS6-1 segment is absorbed for expanding ferro element, the forward direction of the specific primer is drawn
The nucleotide sequence of object is as shown in SEQ ID No.1, the nucleotide sequence of the reverse primer of the specific primer such as SEQ ID
Shown in No.2.
3. the detection method in Human Iron element absorption associated gene mutation site according to claim 1, which is characterized in that
The Single base extension primer includes:
The Single base extension primer of related gene TMPRSS6-1 segment, the Single base extension primer are absorbed for expanding ferro element
Nucleotide sequence as shown in SEQ ID No.3.
4. the detection method that ferro element according to claim 1 absorbs associated gene mutation site, which is characterized in that described
Single-tube multiplex-PCR amplification response procedures are as follows: 95 DEG C 2 minutes, 95 DEG C 30 seconds, 56 DEG C 20 seconds, 72 DEG C 60 seconds, 45 circulation, 72 DEG C 5
Minute, 4 DEG C of preservations.
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| CN201910334950.0A CN110042158A (en) | 2019-04-24 | 2019-04-24 | The detection method in ferro element absorption associated gene mutation site |
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Citations (5)
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|---|---|---|---|---|
| CN107227376A (en) * | 2017-08-08 | 2017-10-03 | 杭州祥音生物医药科技有限公司 | For Primer composition, kit and the methods and applications of the susceptible mutation of gene for detecting influence absorption of trace elements ability |
| CN107760780A (en) * | 2017-12-05 | 2018-03-06 | 天津脉络医学检验有限公司 | A kind of amplimer and application for detecting Iron in Children and absorbing gene pleiomorphism |
| CN108977499A (en) * | 2018-06-26 | 2018-12-11 | 苏州道尔盾基因科技有限公司 | A kind of detection method and kit of mankind's alcohol metabolism ability gene mutation site |
| CN108977515A (en) * | 2018-06-26 | 2018-12-11 | 苏州道尔盾基因科技有限公司 | A kind of methods of genotyping and its application based on SNP site nucleic acid Mass Spectrometer Method |
| CN108977518A (en) * | 2018-07-24 | 2018-12-11 | 为康（苏州）基因科技有限公司 | The detection method and detection kit in a kind of folic acid metabolism associated gene mutation site |
-
2019
- 2019-04-24 CN CN201910334950.0A patent/CN110042158A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107227376A (en) * | 2017-08-08 | 2017-10-03 | 杭州祥音生物医药科技有限公司 | For Primer composition, kit and the methods and applications of the susceptible mutation of gene for detecting influence absorption of trace elements ability |
| CN107760780A (en) * | 2017-12-05 | 2018-03-06 | 天津脉络医学检验有限公司 | A kind of amplimer and application for detecting Iron in Children and absorbing gene pleiomorphism |
| CN108977499A (en) * | 2018-06-26 | 2018-12-11 | 苏州道尔盾基因科技有限公司 | A kind of detection method and kit of mankind's alcohol metabolism ability gene mutation site |
| CN108977515A (en) * | 2018-06-26 | 2018-12-11 | 苏州道尔盾基因科技有限公司 | A kind of methods of genotyping and its application based on SNP site nucleic acid Mass Spectrometer Method |
| CN108977518A (en) * | 2018-07-24 | 2018-12-11 | 为康（苏州）基因科技有限公司 | The detection method and detection kit in a kind of folic acid metabolism associated gene mutation site |
Non-Patent Citations (2)
| Title |
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| STEFANIA BERTONCINI ET AL.: ""A Novel SNaPshot Assay to Detect Genetic Mutations Related to Iron Metabolism"", 《GENETIC TESTING AND MOLECULAR BIOMARKERS》 * |
| 翟静等: "《生物化学》", 31 August 2014, 江苏凤凰科学技术出版社 * |
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