CN108866168A - A kind of primer and probe for people's mthfr gene SNP detection - Google Patents
A kind of primer and probe for people's mthfr gene SNP detection Download PDFInfo
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Abstract
The present invention provides a kind of primer and probe for people's mthfr gene SNP detection, including：The probe that upstream primer, downstream primer, the probe of FAM modification of genotype T, the VIC of genotype C are modified, the upstream primer sequence:5'-TGACCTGAAGCACTTGAAGGA-3'；Downstream primer sequence：5'-AAGAAAAGCTGCGTGATGATGA-3'；The probe sequence of the FAM modification of genotype T：5'-FAM-TCTGCGGGAGTCG-MGB-3'；The probe sequence of the VIC modification of genotype C：5'-VIC-CTGCGGGAGCCGA-MGB-3'.The present invention high-throughput can detect its SNP site, as a result be easy interpretation.It can detecte 96 or 384 samples every time, and the subsequent analysis for not needing to carry out PCR product substantially reduces detection cycle while saving testing cost, improves detection efficiency.
Description
Technical field
The present invention relates to field of biotechnology, specially a kind of primer and probe for people's mthfr gene SNP detection.
Background technique
Folic acid is particularly important methyl donor in vivo, participates in DNA synthesis and repairs, maintain intracellular normal methyl groupization and
Genome stability.The enzyme for participating in folic acid metabolism mainly has 5,10- methylenetetrahydrofolate reductase (5,10-
Methylenetetrahydrofolate reductase, MTHFR), methionine synthetase reductase (5-
Methyltetrahydrofolate-homocysteine methyltransferase reductase, MTRR) and first sulphur ammonia
Acid enzyme (5-methyltetrahydrofolate-homocysteine methyltransferase, MTR), etc..Folic acid
Certain enzyme gene Mutations may will affect folate level in circulation in metabolic pathway.Folic acid metabolism enzyme related genes variants draw
The corresponding enzymatic activity reduction risen can suppress homocysteine to be converted into methionine, lead to low folic acid mass formed by blood stasis and high homotype half
Cystine mass formed by blood stasis.On the one hand, MTHFR is catalyzed the methyl group for the 5-methyltetrahydrofolate that 5,10-CH2-THFA generates
Methionine is generated, then methionine is transformed into S-adenosylmethionine under the action of adenylase, and S- adenosine first
Methyllanthionine is methyl donor general in vivo.On the other hand, MTHFR catalysis reaction substrate 5,10-CH2-THFA with
Multiple metabolic pathways such as thymidine and purine synthesis are related, have extremely important effect for DNA synthesis.
The mutation of folic acid metabolism enzyme can directly result in DNA synthesis and repair generate obstacle, intracellular aberrant methylation and
Genome it is unstable, become a major reason of tumour occurrence and development and the weight of related neoplasms medication (such as 5-FU)
It will foundation.Meanwhile the polymorphism of folic acid metabolism enzyme also influences absorption of the pregnant woman to folic acid, the detection of related gene can be effectively reduced
Newborn Birth-defects risk reduces parent and suffers from pregnant woman's macrocytic anemia, cardiovascular and cerebrovascular disease, malignant tumour, gestation height
The risk of the Pregnancy Complications such as blood pressure, pre-eclampsia, and prevent fetus and generate embryo's neural tube malformation, harelip, congenital
The diseases such as heart malformations.
Research confirms that being metabolized closely related gene with methyl biostearin (folic acid and vitamin B12) mainly has
MTHFR, MTRR and MTR etc., wherein MTHFR plays more crucial effect during folic acid metabolism.Mthfr gene is located at people
On class No.1 chromosome 1p36.3, full-length genome 15.8kb, including 11 exons and 10 intrones, entirely encode the head of district
1980bp.MTHFR is the key enzyme during folic acid metabolism, 5,10-MTHF can be transformed into 5-MTHF, to participate in methionine
The methylation of metabolic cycles and DNA.
Main SNP site is C677T (rs1801133) on mthfr gene, and the site mthfr gene C677T is the 677th
Position nucleotide cytosine (C) is replaced by thymidine (T), and the 222nd amino acids of enzyme is caused to become valine by alanine,
To influence MTHFR activity, causes enzymatic activity and thermal stability to decline, change simultaneously the metabolism of cysteine.If being taken with individual
Its MTHFR activity is 100% when band rs1801133C/C genotype, and the activity for carrying T/C genotype is then the 71% of C/C, base
Because type is only the 34% of T/T type.Thus, there is very big difference in the demand of the folic acid in pregnancy period in Different Individual, if gene
The individual of type position T/T type then needs to supplement more one times of folic acid compared with normal individual.In addition, the site mthfr gene C677T is polymorphic
The neurological susceptibilities of Several Kinds of Malignancy such as property and lung cancer, gastric cancer, colorectal cancer, liver cancer, nasopharyngeal carcinoma are related.Clinically, MTHFR base
Because the polymorphic detection in the site C677T has been widely accepted.Studies have shown that the polymorphism of mthfr gene C677T will affect folic acid
Concentration and its intracellular distribution simultaneously influence the growth of tumour cell and the sensibility to chemotherapy.In addition, MTHFR base
Generation phase because of C677T also with the diseases such as cerebral apoplexy, congenital heart disease, hyperlipemia, neural tube defect, habitual abortion
It closes.
Summary of the invention
Technical problem solved by the invention is to provide a kind of primer and probe for people's mthfr gene SNP detection,
To solve the problems in above-mentioned background technique.
Technical problem solved by the invention is realized using following technical scheme：One kind is examined for people's mthfr gene SNP
The primer and probe of survey, including：The VIC modification of upstream primer, downstream primer, the probe of the FAM modification of genotype T, genotype C
Probe, the upstream primer sequence: 5'- TGACCTGAAGCACTTGAAGGA-3'；Downstream primer sequence：5'-
AAGAAAAGCTGCGTGATGATGA-3'；The probe sequence of the FAM modification of genotype T：5'-FAM- TCTGCGGGAGTCG-
MGB-3'；The probe sequence of the VIC modification of genotype C：5'-VIC- CTGCGGGAGCCGA-MGB-3', proportion are：Upstream
The probe of the VIC modification of the probe 0.25ul, genotype C of the FAM modification of primer 0.25ul, downstream primer 0.25ul, genotype T
0.25ul, sterile water：3ul.
The primer and probe each component concentration are upstream primer 5umol, the FAM of downstream primer 5umol, genotype T are repaired
The probe 5umol of the VIC modification of the probe 5umol, genotype C of decorations.
A kind of progress mthfr gene SNP detection method, includes the following steps：
（1）Determine MTHFR primer and probe；MTHFR primer and probe are:Upstream primer sequence: 5'-
TGACCTGAAGCACTTGAAGGA-3'；Downstream primer sequence：5'- AAGAAAAGCTGCGTGATGATGA-3'；Genotype T's
The probe sequence of FAM modification：5'-FAM- TCTGCGGGAGTCG-MGB-3'；The probe sequence of the VIC modification of genotype C：5'-
VIC- CTGCGGGAGCCGA-MGB-3'；
(2) PCR reaction is carried out to genomic DNA using the primer and probe of MTHFR；
(3) PCR carries out fluorescence reading after reaction, and carries out Genotyping.
The PCR reaction condition is：15min at 95 DEG C, 95 DEG C of 20sec, 1min at 60 DEG C, 40 circulations, at 72 DEG C
5min。
It is compared to open technology, there are following advantages by the present invention：It is provided by the invention to be examined using Taqman MGB probe
The method for surveying people's mthfr gene polymorphism high-throughput can detect its SNP site, as a result be easy interpretation.Every time can detecte 96 or
384 samples, and the subsequent analysis for not needing to carry out PCR product substantially reduces detection while saving testing cost
Period improves detection efficiency.The primer and probe high specificity that the present invention uses not will receive the interference of other genes, drop
Low PCR product pollution causes the risk of false positive, and result interpretation is easier, and can be realized to people's mthfr gene SNP site
High throughput detection is carrying out having wide application value in mthfr gene detection to people.
Specific embodiment
In order to make, technological means of the invention, creation characteristic, workflow, application method reach purpose and effect is easy to bright
White understanding, below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " installation ", " company ", " connection "
It shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or be integrally connected to be mechanical connection,
It is also possible to be electrically connected;It can be directly connected, can also indirectly connected through an intermediary, it can company inside two elements
It is logical, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.
Embodiment 1
A kind of progress mthfr gene SNP detection method, includes the following steps：
(1) in view of the error of pipettor and suction nozzle, general each 384 orifice plate presses 400 Response calculation systems, prepares mixing
Liquid.
(2) a blank control NTC is arranged in each 384 orifice plate, and the known TT type mthfr gene group of setting two compares, and one
A known CC type mthfr gene group control, a known TC type mthfr gene group control.
(3) using the volley of rifle fire in every 1 μ L DNA sample of Kong Zhongjia.
(4) it is sealed after the completion of with sealed membrane, plate centrifuge centrifugation.
(5) ABI Q7 real-time fluorescence quantitative PCR system is opened, parameter is set, reaction condition is as follows：
15min at 95 DEG C, 20sec at 95 DEG C, 1min at 60 DEG C, 40 circulations, 5min at 72 DEG C, 60 DEG C of whens, read fluorescence.
Data analysis is carried out using 7500 real time fluorescent quantitative software of ABI.According to control amplification, parting generates three
Kind genotype：
The first genotype：TT, with TT type to impinging upon on an axis；
Second of genotype：CC, with CC type to impinging upon on an axis；
The third genotype：TC, with TC type to impinging upon on an axis.
Probe and one couple of PCR primers containing a pair of of double labelling in Taqman probe PCR reaction system, are contaminated with two kinds of fluorescence
Expect that Fam, Hex (Vic) mark both probes respectively, there is a fluorescence radiation group (reporter dye in TaqMan probe
Abbreviation R group) an and fluorescent quenching group (quencher abbreviation Q group), what the two groups on complete probe leaned on
Close, the fluorescence that R group issues, then can be by Q group absorptions within the scope of the absorbing wavelength of Q group, therefore will not generate fluorescence,
When probe is in conjunction with template, and upstream primer (5 ' ends) extends to the place of probe combination, polymerase is utilized
(Taqpolymerase) 5 prime excision enzyme activity of 5 ' -3 ' degrades probe, while R group is dissociated to get off from probe, makes it
Group separates with quenching, at this moment will generate fluorescence, detects signal finally by terminal read plate to determine whether there is SNP
Point.
The above shows and describes the basic principle, main features and advantages of the invention.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 improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.Claimed range of the invention by appended claims and
Its equivalent thereof.
Claims (4)
1. a kind of primer and probe for people's mthfr gene SNP detection, it is characterised in that：Including：Upstream primer, downstream are drawn
The probe of the VIC modification of object, the probe of the FAM modification of genotype T, genotype C, the upstream primer sequence: 5'-
TGACCTGAAGCACTTGAAGGA-3'；Downstream primer sequence：5'- AAGAAAAGCTGCGTGATGATGA-3'；Genotype T's
The probe sequence of FAM modification：5'-FAM- TCTGCGGGAGTCG-MGB-3'；The probe sequence of the VIC modification of genotype C：5'-
VIC- CTGCGGGAGCCGA-MGB-3', proportion are：The FAM of upstream primer 0.25ul, downstream primer 0.25ul, genotype T
The probe 0.25ul of the VIC modification of the probe 0.25ul, genotype C of modification, sterile water：3ul.
2. a kind of primer and probe for people's mthfr gene SNP detection according to claim 1, it is characterised in that：Institute
State the probe of the FAM modification of primer and probe each component concentration for upstream primer 5umol, downstream primer 5umol, genotype T
The probe 5umol of the VIC modification of 5umol, genotype C.
3. a kind of progress mthfr gene SNP detection method, it is characterised in that：Include the following steps：
（1）Determine MTHFR primer and probe；MTHFR primer and probe are:Upstream primer sequence: 5'-
TGACCTGAAGCACTTGAAGGA-3'；Downstream primer sequence：5'- AAGAAAAGCTGCGTGATGATGA-3'；Genotype T's
The probe sequence of FAM modification：5'-FAM- TCTGCGGGAGTCG-MGB-3'；The probe sequence of the VIC modification of genotype C：5'-
VIC- CTGCGGGAGCCGA-MGB-3'；
(2) PCR reaction is carried out to genomic DNA using the primer and probe of MTHFR；
(3) PCR carries out fluorescence reading after reaction, and carries out Genotyping.
4. a kind of progress mthfr gene SNP detection method according to claim 3, it is characterised in that：The PCR reaction
Condition is：15min at 95 DEG C, 95 DEG C of 20sec, 1min at 60 DEG C, 40 circulations, 5min at 72 DEG C.
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Cited By (1)
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| CN112980950A (en) * | 2021-01-06 | 2021-06-18 | 广州瑞熹生物科技有限公司 | Kit for detecting 15 gene mutation sites related to sensitivity of radiotherapy and chemotherapy of rectal cancer and application thereof |
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