CN108546756A - A kind of single nucleotide polymorphism gene tester - Google Patents
A kind of single nucleotide polymorphism gene tester Download PDFInfo
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- CN108546756A CN108546756A CN201810469171.7A CN201810469171A CN108546756A CN 108546756 A CN108546756 A CN 108546756A CN 201810469171 A CN201810469171 A CN 201810469171A CN 108546756 A CN108546756 A CN 108546756A
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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
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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
Abstract
The invention discloses a kind of single nucleotide polymorphism gene testers,Including present Research,Research significance,Analyzing detecting method and cholesterol ester transfer protein (CETP) gene analysis test method of SNP,The present Research is compared with a limited number of protein sequencings and DNA sequence analysis method,The basic skills of snp analysis is up to as many as tens kinds,Snp analysis is used for medical diagnosis on disease,Can gene diagnosis be accurately fast increased to single base level,At present,Conventional detection SNP technologies have been widely used for pharmacogenetics research and promote the universal field of genetic diseases diagnosis,Miscellaneous snp analysis method,The research for reflecting SNP is concerned in biology and medical domain,However any type method can not possibly independently meet requirement of the genome times afterwards comprehensively to snp analysis.
Description
Technical field
The present invention relates to mononucleotide technical field of gene detection, specifically a kind of single nucleotide polymorphism genetic test side
Method.
Background technology
The completion of human genome sequencing plan, indicates that the mankind have walked people's genome times afterwards comprehensively, the epoch are with genome
The research of middle individual inheritance difference is attached most importance to, and individual inheritance difference most common form is single nucleotide polymorphism (SNP), exactly
The variation of these SNP causes the mankind in stature, the bodily form, the colour of skin, the neurological susceptibility to disease, disease phenotype and reacts drug therapy
The difference more or less etc. all various aspects can be also preced with to analyze with the mankind by cholesterol detection transesterify albumen (CETP) gene
The main function of contact between worry, CETP genes is transfer neutral lipid such as cholesteryl ester (CE) and the glycerine between lipoprotein
Three esters (TG), that is, be catalyzed CE from HDL2 (form of the bulky grain of HDL-C) to very low density lipoprotein (VLDL), LDLs and in
Between density lipoprotein (IDL) transhipment, while being also catalyzed the transhipments of TG round about with equimolecular ratio, CETP is known
The uniquely albumen with this function, in numerous gene mutations for leading to human diseases, cause of disease hypotype and drug-tolerant gene mutation,
Single base mutation accounts for sizable ratio, and the exploration of detection method is always the important topic in gene diagnosis research, especially
Detection to known mutations will carry out one of the important means of basic research and clinical gene diagnosis.
Invention content
The purpose of the present invention is to provide a kind of single nucleotide polymorphism gene testers, to solve above-mentioned background technology
The problem of middle proposition.
To achieve the above object, the present invention provides the following technical solutions：
A kind of single nucleotide polymorphism gene tester, including the concept of SNP, present Research, research significance and SNP
Analyzing detecting method, the present Research are SNP points compared with a limited number of protein sequencings and DNA sequence analysis method
Up to as many as tens kinds, the principle of main snp analysis method is the basic skills of analysis:(1) base variation is utilized to generate newly
Or eliminate existing endonuclease recognized site；(2) shadow of the base complement to primer extension reaction caused by utilizing base variation
It rings；(3) utilize nucleotide variation to nucleic acid；Research significance is in all polygenic variations, and SNP is research gene genetic and change different time
A kind of efficient quantization mark, about 3,000,000,000 bases in human genome, SNP site exist with million order of magnitude,
Either using SNP as the physical token of quick scanning full-length genome, or to full-length genome related or drug metabolism to disease
Relevant SNP carries out selective scanning, be required to make the SNP site of detection further from million grades of quantity drop to it is tens thousand of or
It is thousands of, therefore, disease associated analysis is carried out using SNP, only by carrying out SNP linkage disequilibriums point to multigenic disease
Analysis, could reinforce the specific aim of disease prevention and treatment, cholesterol ester transfer protein (CETP) gene analysis test be by
There is specific polymorphism in gene, cause restriction enzyme digestion sites to change, disappear or generate new site, if using certain
Digestion with restriction enzyme genomic DNA then generates the DNA fragmentation with normal gene group different length, detects this DNA after digestion
The size and number of segment judges whether there is specific polymorphism.
As a further solution of the present invention：The snp analysis method depends on being to look for unknown SNP, or analysis is
The SNP known, the analysis to unknown SNP are completed today mainly by sequencing, to known SNP, need according to be to single site or
To multiple Locus Analysis in Shoots and experiment purpose difference, and different analysis methods is selected, it is main to the analysis method of a large amount of SNP sites
There are 3 kinds, i.e. oligonucleotides snp analysis chip, single residue extends SNP chip and porous plate technology, such as using the in due course of 6 orifice plates
The single base polymorphisms analysis method of PCR (PCR), method workable for the snp analysis to single-point is relatively
More, most of bases and clinical research for the purpose of single-point or SNP site few in number, selection is not required to the side of fluorescence detection
Method can generally meet the requirement of molecular pharmacology and gene diagnosis.
As further scheme of the invention：The snp analysis method restrictive internally-cut enzyme segment length polymorphism
It analyzes (RFLP), this side, which dispels, is only applicable to the SNP that analysis is located at restriction enzyme digestion sites, and step is：What need to be analyzed
The upstream and downstream of single base polymorphisms designs 2 primers, is used in combination this to amplify associated nucleic acid segment to primer, then to the nucleic acid fragment
With digestion with restriction enzyme, whether analysis allele contains restriction enzyme site, to obtain the product of different length, finally leads to
It crosses gel electrophoresis to detach enzymolysis product, from nucleic acid fragment length shown in gel electrophoresis it can be learnt that whether the segment is disappeared
Change, if PCR product is digested or is not digested completely, the respectively homozygote of different genotype；If only portion of product is disappeared
Change, then its genotype be heterozygote, using when RFIP it may be noted that design of primers according to required enlarged fragment length or digestion with
Length afterwards determines that the gel of rather high concentration is suitable for the shorter segment of analysis length, and relatively low intensity of gel is then advantageous
In the long segment of analysis length；Gel strength is different length by designing enzymolysis product generally between 1% to 2%
DNA fragmentation, a reaction can analyze several different SNP polymorphisms simultaneously.
Compared with prior art, the beneficial effects of the invention are as follows：Mentality of designing of the present invention is novel, passes through design of primers foundation
The length of required enlarged fragment digests later length decision, and the gel of rather high concentration is suitable for the shorter piece of analysis length
Section, relatively low intensity of gel are then conducive to the long segment of analysis length；Gel strength leads to generally between 1% to 2%
The DNA fragmentation that design enzymolysis product is different length is crossed, a reaction can analyze several different SNP polymorphisms simultaneously, can
Accurately analysis detects single nucleotide polymorphism gene, convenient for causing the mankind in stature, the bodily form, skin the variation of SNP
Color, the treatment to the neurological susceptibility, disease phenotype of disease, while the monoclonal antibody for studying anti-CETP can block the transhipment of neutral lipid, subtract
The composition for having lacked CE and TG in LDL keeps LDL anti-oxidant, and macrophage also reduces the intake of the LDL (ox-LDL) of oxidation, from
And reduce the danger that atherosclerosis occurs, producing corresponding drug is treated, and the health of people is protected.
Specific implementation mode
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
In the embodiment of the present invention, a kind of single nucleotide polymorphism gene tester, including the concept of SNP, present Research,
The analyzing detecting method of research significance and SNP, the concept of the SNP refer to being made due to conversion, transversion, slotting people, missing etc.
Different Individual is chromosome there are the polymorphism of variety classes base, i.e. SNP on genomic DNA specific nucleotide position
Single base on some site of DNA sequence dna, can express out the individual difference to vary with each individual, if in large number of crowd
Middle occurrence frequency is more than 1%, you can the idiovariation event for being considered more ancient SNP, rather than occurring recently；Present Research
Be compared with a limited number of protein sequencings and DNA sequence analysis method, the basic skills of snp analysis up to tens kinds it
More, the principle of main snp analysis method is:(1) base variation is utilized to generate new or eliminate existing restriction endonuclease and identify position
Point；(2) influence of the base complement to primer extension reaction caused by utilizing base variation；(3) utilize nucleotide variation to nucleic acid；
Research significance is in all polygenic variations, and SNP is that research gene genetic indicates with a kind of different time efficient quantization is become, in the mankind
About 3,000,000,000 bases in genome, SNP site exist with million order of magnitude, either complete using SNP as quick scanning
The physical token of genome, or to full-length genome, the relevant SNP of related or drug metabolism carries out selective scanning to disease,
It needs the SNP site of detection is made further to drop to from million grades of quantity tens thousand of or thousands of, therefore, disease is carried out using SNP
Sick correlation analysis could reinforce disease prevention and treatment only by carrying out SNP linkage disequilibrium values to multigenic disease
Specific aim, snp analysis is used for medical diagnosis on disease, can be quick and gene diagnosis is accurately increased to single base level, currently,
Conventional detection SNP technologies have been widely used for pharmacogenetics research and promote the universal field of genetic diseases diagnosis, type various
Snp analysis method, the research for reflecting SNP is concerned in biology and medical domain, however any type method is not
Requirement of the genome times afterwards comprehensively to snp analysis, cholesterol ester transfer protein (CETP) genetic analysis may independently be met
Detection is to cause restriction enzyme digestion sites to change, disappear or generate new site since gene has specific polymorphism,
If generating the DNA fragmentation with normal gene group different length with certain digestion with restriction enzyme genomic DNA, after digestion,
The size and number for detecting this DNA fragmentation judges whether there is specific polymorphism, and preferably this method is combined with round pcr, is being set
When counting PCR amplification experiment, primer is located at the both sides at gene pleiomorphism position, first expands target gene, and making it easier to detection is
Since specific polymorphism causes existing restriction endonuclease sites to change, then can first be expanded with corresponding digestion with restriction enzyme
Increase production object, then observed into row agarose gel electrophoresis, is judged with normal control according to product clip size or quantity.
Snp analysis method restrictive internally-cut enzyme segment length polymorphism analysis (RFLP) is in the single base that need to be analyzed
The upstream and downstream of polymorphism designs 2 primers, this is used in combination to amplify associated nucleic acid segment to primer, then limits the nucleic acid fragment
Property endonuclease digestion, analysis allele whether contain restriction enzyme site, to obtain the product of different length, finally by gel
Electrophoresis detaches enzymolysis product, from nucleic acid fragment length shown in gel electrophoresis it can be learnt that whether the segment is digested, if
PCR product is digested or is not digested completely, then the respectively homozygote of different genotype；If only portion of product is digested,
Its genotype be heterozygote, using when RFIP it may be noted that design of primers according to required enlarged fragment length or digestion it is later
Length determines, the gel of rather high concentration is suitable for the shorter segment of analysis length, and relatively low intensity of gel is then conducive to point
Analyse the long segment of length；Gel strength is generally between 1% to 2%, by designing the DNA that enzymolysis product is different length
Segment, a reaction can analyze several different SNP polymorphisms simultaneously, and the method analyzed the PCR product is polymerization
Enzyme chain reaction is combined with restriction fragment length polymorphism analysis, specific by being used comprising the PCR product of purpose SNP site
Restriction enzyme makees digestion, then include the SNP site its individual PCR product will produce be cut into two or three segments or
Motionless three kinds of situations are cut, in the present invention for the sites -971G/A, after PCR amplification, obtain the PCR of 309 bases length
Product selects AvaI restriction enzymes (Shanghai biotechnology Engineering Co., Ltd) to carry out the PCR product of 309 base length
Digestion, when producing the segment of 210 and 99bp, two kinds of length after cutting, the allele in the sites explanation -971G/A is G, when
When the PCR product of only 309 bases length, the allele in the sites explanation -971G/A is A, when existing simultaneously 309,210 and 99bp
When the segment of three kinds of length, the genotype in the sites explanation -971G/A is G/A heterozygotes.
The present invention operation principle be：2 primers are designed in the upstream and downstream for the single base polymorphisms that need to be analyzed, are used in combination this right
Primer amplifies associated nucleic acid segment, and then to the nucleic acid fragment digestion with restriction enzyme, whether analysis allele contains
Restriction enzyme site detaches enzymolysis product finally by gel electrophoresis, with obtaining the product of different length from gel electrophoresis
Shown nucleic acid fragment length if PCR product is digested or is not digested completely, is distinguished it can be learnt that whether the segment is digested
For the homozygote of different genotype；If only portion of product is digested, genotype is heterozygote, using when RFIP it may be noted that
The design of primers length later according to the length of required enlarged fragment or digestion determines that the gel of rather high concentration is suitable for analyzing
The shorter segment of length, relatively low intensity of gel are then conducive to the long segment of analysis length；Gel strength is generally 1%
To between 2%, by designing the DNA fragmentation that enzymolysis product is different length, a reaction can analyze several different SNP simultaneously
Polymorphism, cholesterol ester transfer protein (CETP) gene analysis test are caused restricted since gene has specific polymorphism
Endonuclease digestion site changes, disappears or generates new site, if with certain digestion with restriction enzyme genomic DNA, enzyme
The DNA fragmentation with normal gene group different length is generated after cutting, and is detected the size and number of this DNA fragmentation, is judged whether have
Specific polymorphism.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Profit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent requirements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiment being appreciated that.
Claims (3)
1. a kind of single nucleotide polymorphism gene tester, includes the analyzing detecting method of present Research, research significance, SNP
With cholesterol ester transfer protein (CETP) gene analysis test method, which is characterized in that the present Research be with for number it is limited
Protein sequencing compared with DNA sequence analysis method, the basic skills of snp analysis is up to as many as tens kinds, main SNP points
The principle of analysis method is:(1) base variation is utilized to generate new or eliminate existing endonuclease recognized site；(2) base is utilized
Influence of the base complement caused by variation to primer extension reaction；(3) utilize nucleotide variation to nucleic acid；Research significance is all
In polygenic variation, SNP is that research gene genetic indicates with a kind of different time efficient quantization is become, in human genome about
3000000000 bases, SNP site exist with million order of magnitude, either using SNP as the physics mark of quick scanning full-length genome
Will, or to full-length genome, the relevant SNP of related or drug metabolism carries out selective scanning to disease, is required to make detection
SNP site further drops to from million grades of quantity tens thousand of or thousands of, therefore, disease associated analysis is carried out using SNP,
It, will only by the way that multigenic disease progress SNP linkage disequilibrium values, the specific aim of disease prevention and treatment could be reinforced
Snp analysis is used for medical diagnosis on disease, can gene diagnosis fast and be accurately increased to single base level, currently, conventional detection SNP
Technology has been widely used for pharmacogenetics research and promotes the universal field of genetic diseases diagnosis, miscellaneous snp analysis side
Method, the research for reflecting SNP are concerned in biology and medical domain, however any type method can not possibly be independently full
Therefore requirement of the sufficient genome times afterwards comprehensively to snp analysis is either looked for new, or confirm known SNP site, traditional
For DNA sequencing method still in the status for being difficult to substitute, cholesterol ester transfer protein (CETP) gene analysis test is due to base
Because having specific polymorphism, restriction enzyme digestion sites are caused to change, disappear or generate new site, if being limited with certain
Property endonuclease digestion genomic DNA, then generated after digestion with the DNA fragmentation of normal gene group different length, detect this DNA fragmentation
Size and number, judge whether that there is specific polymorphism.
2. a kind of single nucleotide polymorphism gene tester according to claim 1, which is characterized in that the SNP points
Analysis method depends on being to look for unknown SNP, or analyze known SNP, the analysis to unknown SNP, today mainly by sequencing
It completes, to known SNP, needs according to being different to single site or to multiple Locus Analysis in Shoots and experiment purpose, and select different
Analysis method mainly have 3 kinds to the analysis method of a large amount of SNP sites, i.e., oligonucleotides snp analysis chip, single residue extend
SNP chip and porous plate technology.
3. a kind of a kind of preparation method of single nucleotide polymorphism gene tester as described in claim 1-2 is any,
It is characterized in that, the snp analysis method restrictive internally-cut enzyme segment length polymorphism analysis (RFLP), this side, which dispels, to be only applicable to
Analysis is located at the SNP of restriction enzyme digestion sites, and step is：2 are designed in the upstream and downstream for the single base polymorphisms that need to be analyzed
A primer is used in combination this to amplify associated nucleic acid segment to primer, then to the nucleic acid fragment digestion with restriction enzyme, analysis etc.
Whether position gene contains restriction enzyme site, to obtain the product of different length, is carried out to enzymolysis product finally by gel electrophoresis
Separation, from nucleic acid fragment length shown in gel electrophoresis it can be learnt that whether the segment is digested, if PCR product digested completely or
It is not digested, then the respectively homozygote of different genotype；If only portion of product is digested, genotype is heterozygote.
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| CN201810469171.7A CN108546756A (en) | 2018-05-16 | 2018-05-16 | A kind of single nucleotide polymorphism gene tester |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020034752A1 (en) * | 2000-05-11 | 2002-03-21 | Ordovas Jose M. | CETP TaqIB polymorphism as risk factor for development of coronary heart disease |
| CN1891823A (en) * | 2005-07-05 | 2007-01-10 | 北京诺赛基因组研究中心有限公司 | CETP gene with specific mononucleotide pleiomorphism, and its detecting method and use |
-
2018
- 2018-05-16 CN CN201810469171.7A patent/CN108546756A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020034752A1 (en) * | 2000-05-11 | 2002-03-21 | Ordovas Jose M. | CETP TaqIB polymorphism as risk factor for development of coronary heart disease |
| CN1891823A (en) * | 2005-07-05 | 2007-01-10 | 北京诺赛基因组研究中心有限公司 | CETP gene with specific mononucleotide pleiomorphism, and its detecting method and use |
Non-Patent Citations (1)
| Title |
|---|
| 彭翠英等: "单核苷酸多态性及其检测方法", 《中华检验医学杂志》 * |
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