CN103103259A - Method, kit and primers for determining whether two predetermined loci of nucleic acid sample mutate or not - Google Patents
Method, kit and primers for determining whether two predetermined loci of nucleic acid sample mutate or not Download PDFInfo
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- CN103103259A CN103103259A CN2012105924168A CN201210592416A CN103103259A CN 103103259 A CN103103259 A CN 103103259A CN 2012105924168 A CN2012105924168 A CN 2012105924168A CN 201210592416 A CN201210592416 A CN 201210592416A CN 103103259 A CN103103259 A CN 103103259A
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- 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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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a method, a kit and primers for determining whether two predetermined loci of a nucleic acid sample mutate or not. The method comprises the following steps of: amplifying a nucleic acid sample by carrying out PCR (polymerase chain reaction) by using a first primer and a second primer, and adding a nucleic acid probe into a system for amplifying by carrying out PCR, wherein the nucleic acid probe corresponds to a nucleic acid sequence, and the nucleic acid sequence corresponds to at least one part of the two predetermined loci; and detecting the light emission of a reaction system to determine whether the predetermined loci of the nucleic acid sample mutate or not. The proximal 3' end of the first primer comprises a base group which corresponds to one of the mutated predetermined loci, and the proximal 3' end of the second primer comprises a base group which corresponds to the other mutated predetermined loci; the nucleic acid probe comprises a light-emitting group and an adjusting group, wherein the light-emitting group is formed on the 5' end of the nucleic acid probe, and the adjusting group is formed on the 3' end of the nucleic acid probe and is capable for adjusting the light emitting by the light-emitting group. The method can be used for effectively determining whether the two predetermined loci of the nucleic acid sample mutate or not.
Description
Technical field
The present invention relates to biomedical sector.Particularly, the present invention relates to method, test kit and the primer whether two predetermined sites of definite kernel acid sample have known mutations.
Background technology
ApoE is a kind of lipophorin, its genetic polymorphism mainly is controlled by two SNP site rs429358 and rs7412, these two SNP sites have determined three kinds of allelotypes of ApoE by different nucleic acid combinations: ε 2(rs429358 is T, rs7412 is T), ε 3(rs429358 is T, rs7412 is C), ε 4(rs429358 is C, rs7412 is C), this three types allelotrope three kinds of ApoE protein subunits of encoding respectively, 112 and 158 that are its protein sequence of ApoE2(are halfcystine), 112 of its protein sequence of ApoE3(is halfcystine, 158 is arginine), 112 and 158 of its protein sequence of ApoE4(are arginine).Therefore, the ApoE gene has ε 2/ ε 2, ε 3/ ε 3 and ε 4/ ε 4 three kinds of isozygoty subtype and ε 2/ ε 3, ε 2/ ε 4 and 4 three kinds of heterozygosis subtypes of ε 3/ ε.At present the method for ApoE gene type mainly contains: traditional DNA sequencing method, alleles-specific oligonucleotide probe hybrid method, polymerase chain reaction-Single strand conformation polymorphism, polymerase chain reaction-Restriction fragment length polymorphism method, multiplex amplification be obstructed sudden change system round pcr method, high resolving power solubility curve method and Two Colour Fluorescence molecular probe method etc.
Yet whether two predetermined sites of definite kernel acid sample have the method for known mutations at present, still remain to be improved.
Summary of the invention
The present invention is intended to solve at least one of technical problem that exists in prior art.For this reason, the present invention proposes the method whether effective two predetermined sites of definite kernel acid sample have known mutations.
Whether two predetermined sites that in one aspect of the invention, the present invention proposes a kind of definite kernel acid sample have the method for known mutations.according to embodiments of the invention, the method comprises: utilize the first primer and the described sample of nucleic acid of the second primer pair to carry out pcr amplification, and add nucleic acid probe in described pcr amplification system, the nucleotide sequence of at least a portion between corresponding two predetermined sites of described nucleic acid probe, and the detection reaction system is luminous, in order to determine whether the predetermined site of described sample of nucleic acid exists known mutations, wherein, nearly 3 ' end of described the first primer comprises the base corresponding with one of described two predetermined sites known mutations, nearly 3 ' end of described the second primer comprises and described two bases that another known mutations of predetermined site is corresponding, one of described the first primer and second primer are as forward primer, another of described the first primer and described the second primer is as reverse primer, described nucleic acid probe has: luminophore, described luminophore is formed at 5 ' end of described nucleic acid probe, regulate group, described adjusting group is formed at 3 ' end of described nucleic acid probe, and described adjusting group can be regulated the luminous of described luminophore.
Method according to the embodiment of the present invention, the fluorescence that can produce by detecting the pcr amplification system, there is corresponding known mutations simultaneously in two predetermined sites of definite sample of nucleic acid that detects effectively, and further can effectively determine the allelic gene typing of ApoE, have very important significance for prevention, early diagnosis and the disease prognosis with ApoE genotypic correlation disease such as senile dementia and cardiovascular and cerebrovascular.
According to some embodiments of the present invention, the method whether two predetermined sites of above-mentioned definite kernel acid sample have known mutations can also have following additional technical feature:
According to one embodiment of present invention, described sample of nucleic acid derives from tissue or cell, recombinant nucleic acid or genetically modified organism histocyte, preferably adopts mankind's complete genome DNA.According to embodiments of the invention, can effectively determine mutation type to the sample of nucleic acid in above-mentioned source, thereby can effectively analyze the individuality that sample of nucleic acid is provided.
According to one embodiment of present invention, described predetermined site is SNP rs429358 and rs7412.Thus, utilize according to the embodiment of the present invention, effectively whether have known mutations at two predetermined site SNP rs429358 and rs7412 in the definite kernel acid sample, thereby can effectively determine the ApoE allelic gene typing.
According to one embodiment of present invention, nearly 3 ' end of described the first primer comprises the base corresponding with the known mutations of rs429358, and nearly 3 ' end of described the second primer comprises the base corresponding with the known mutations of rs7412.Thus, utilization detects sample of nucleic acid according to the method whether two predetermined sites of definite sample of nucleic acid of the embodiment of the present invention have known mutations, primer can be with template according to the basepairing rule selective binding when PCR reacts, thereby can amplify specifically the not isoallele of ApoE, and further can effectively determine the allelic type of ApoE.
According to one embodiment of present invention, 3 ' end base of described the first primer is the base corresponding with the known mutations of rs429358, and 3 ' end base of described the second primer is the base corresponding with the known mutations of rs7412.According to one embodiment of present invention, described the first primer is as forward primer, and described the second primer is as reverse primer.According to one embodiment of present invention, described the first primer be selected from following one of at least: 5 '-CGGACATGGAGGACGTGT-3 ', 5 '-GCGGACATGGAGGACGTGT-3 ', 5 '-CGCGGACATGGAGGACGTGT-3 ', 5 '-CGGACATGGAGGACGTGC-3 ', 5 '-GCGGACATGGAGGACGTGC-3 ', 5 '-CGCGGACATGGAGGACGTGC-3 '.Described the second primer be selected from following one of at least: 5 '-TGGTACACTGCCAGGCA-3 ', 5 '-CTGGTACACTGCCAGGCA-3 ', 5 '-CCTGGTACACTGCCAGGCA-3 ', 5 '-TGGTACACTGCCAGGCG-3 ', 5 '-CTGGTACACTGCCAGGCG-3 ', 5 '-CCTGGTACACTGCCAGGCG-3 '.Thus, utilization detects sample of nucleic acid according to the method whether two predetermined sites of definite sample of nucleic acid of the embodiment of the present invention have known mutations, in the PCR reaction process, primer can be with template according to the basepairing rule selective binding, thereby can amplify specifically the not isoallele of ApoE, and further can effectively determine the allelic type of ApoE.
According to one embodiment of present invention, the length of described nucleic acid probe is 20 Nucleotide.According to one embodiment of present invention, the sequence of described nucleic acid probe is 5 '-CAGCTCCTCGGTGCTCTGGC-3 '.According to one embodiment of present invention, the luminophore of described nucleic acid probe is fluorophor.According to one embodiment of present invention, described fluorophor is FAM.According to one embodiment of present invention, described adjusting group is quenching group.According to one embodiment of present invention, described quenching group is BHQ1.Thus, utilization detects sample of nucleic acid according to the method whether two predetermined sites of definite sample of nucleic acid of the embodiment of the present invention have known mutations, have or not fluorescent signal by detecting in amplification system corresponding to each genotype, can judge the ApoE gene that whether contains this hypotype in testing sample.
Aspect another, the present invention proposes a kind of two test kits whether predetermined site has known mutations of definite kernel acid sample of the present invention.According to embodiments of the invention, whether two predetermined sites that described test kit can be applied to foregoing definite kernel acid sample have the method for known mutations.Thus, utilization detects sample of nucleic acid according to the test kit whether two predetermined sites of definite sample of nucleic acid of the embodiment of the present invention have known mutations, whether two of the definite kernel acid sample predetermined sites have known mutations effectively, and further can effectively determine the allelic gene typing of ApoE, have very important significance for prevention and the early diagnosis of senile dementia and cardiovascular and cerebrovascular diseases.In addition, the described feature and advantage of the method whether front has known mutations for two predetermined sites of definite kernel acid sample, also applicable this test kit natch, repeat no more.
Of the present invention aspect another, the present invention proposes the PCR primer whether two predetermined sites of one group of definite kernel acid sample have known mutations.According to embodiments of the invention, described PCR primer can be applied to method and the test kit whether two predetermined sites of foregoing definite kernel acid sample have known mutations.Thus, in the pcr amplification process, primer can be with template according to the basepairing rule selective binding, and pcr amplification can effectively increase according to pre-determined direction, and can amplify specifically not isoallele.In addition, whether the front has the feature and advantage of the description of the method for known mutations and test kit the primer for two predetermined sites of definite kernel acid sample, and also applicable this PCR primer natch, repeat no more.
Two of the definite kernel acid sample predetermined sites method of whether having known mutations can realize that following advantages is one of at least according to an embodiment of the invention:
1, whether two of the definite kernel acid sample predetermined sites have the method for known mutations according to an embodiment of the invention, and the method is easy and simple to handle, only needs two steps of pcr amplification and fluoroscopic examination;
2, whether two of the definite kernel acid sample predetermined sites have the method for known mutations according to an embodiment of the invention, and the method easily realizes automated operation, can greatly reduce the personal errors in process hand-manipulated, makes the result that obtains more accurate;
3, whether two of the definite kernel acid sample predetermined sites have the method for known mutations according to an embodiment of the invention, and the method saves time fast, completes the time of whole detection less than 1.5 hours, consuming timely are less than existing method;
4, whether two of the definite kernel acid sample predetermined sites have the method for known mutations according to an embodiment of the invention, and the cost of the method is lower, only needs a fluorescent probe and conventional PCR system to get final product;
5, whether two of the definite kernel acid sample predetermined sites have the method for known mutations according to an embodiment of the invention, the detected result of the method is directly perceived, can qualitatively judge whether have the corresponding ApoE allelotype of this amplification system primer pair in sample to be tested according to having or not of fluorescent signal, need not loaded down with trivial details analytic process.
Additional aspect of the present invention and advantage part in the following description provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 is that the different primers of two predetermined sites of definite kernel acid sample according to an embodiment of the invention method of whether having known mutations is to the different allele specific amplification principle schematic of ApoE, in the figure, 1 expression contains the ApoE gene DNA fragment schematic diagram of SNP rs42358 and rs7412;
Fig. 2 is the ApoE gene PCR amplification system schematic diagram of two predetermined sites of definite kernel acid sample according to an embodiment of the invention method of whether having known mutations, in the figure, 1 expression ApoE gene DNA template antisense strand, 2 expression forward amplimers, 3 expression couplings have the DNA molecular probe of fluorophor (F) and quenching group (Q), 4 expression ApoE gene DNA template positive-sense strands, the 5 reverse amplimers of expression, 6 expression archaeal dna polymerases;
Fig. 3 is the fluorescent probe principle of work schematic diagram whether two predetermined sites of definite kernel acid sample according to an embodiment of the invention have the method for known mutations, in the figure, 1 expression ApoE gene DNA template antisense strand, 2 expression forward amplimers, the DNA molecular probe that 3 expression fluorophors (F) break away from, 4 expression ApoE gene DNA template positive-sense strands, the 5 reverse amplimers of expression, 6 expression archaeal dna polymerases;
Fig. 4 is that the example of two predetermined sites of definite kernel acid sample according to an embodiment of the invention method of whether having known mutations detects the fluorescent signal value of reading figure as a result;
Fig. 5 is the column type analysis chart that the example of two predetermined sites of definite kernel acid sample according to an embodiment of the invention method of whether having known mutations detects the fluorescent signal value of reading.
Embodiment
The below describes embodiments of the invention in detail, and the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or the element with identical or similar functions from start to finish.Be exemplary below by the embodiment that is described with reference to the drawings, only be used for explaining the present invention, and can not be interpreted as limitation of the present invention.
In description of the invention, it will be appreciated that, term " thickness ", " on ", orientation or the position relationship of the indication such as D score be based on orientation shown in the drawings or position relationship, only the present invention for convenience of description and simplified characterization, rather than indicate or hint that the device of indication or element must have specific orientation, construct and operation with specific orientation, therefore can not be interpreted as limitation of the present invention.
Whether two predetermined sites that in one aspect of the invention, the present invention proposes a kind of definite kernel acid sample have the method for known mutations.According to embodiments of the invention, the method whether two predetermined sites of definite kernel acid sample have known mutations can comprise the following steps:
S100: utilize the first primer and the second primer pair sample of nucleic acid to carry out pcr amplification, and add nucleic acid probe in the pcr amplification system
In this step, PCR primer of nucleotide sequence design for one of two predetermined sites of sample of nucleic acid: the first primer, PCR primer of nucleotide sequence design for two another predetermined sites of predetermined site of sample of nucleic acid: the second primer, and add nucleic acid probe, then sample of nucleic acid is carried out pcr amplification.Thus, obtain the pcr amplification sample, in order to further it is carried out fluoroscopic examination.
the contriver finds, when pcr amplification, if nearly 3 ' end of the first primer comprises the base corresponding to known mutations of one of two predetermined sites with sample of nucleic acid, nearly 3 ' end of the second primer comprises the base corresponding with the known mutations of another predetermined site of sample of nucleic acid, and first one of primer and second primer as forward primer, another of the first primer and described the second primer is as reverse primer, primer can be with template according to the basepairing rule selective binding when pcr amplification, pcr amplification can effectively increase according to pre-determined direction, and can amplify specifically not isoallele.
According to embodiments of the invention, 3 ' end base of the first primer is the base corresponding with the known mutations of rs429358, and 3 ' end base of the second primer is the base corresponding with the known mutations of rs7412.According to embodiments of the invention, the first primer is as forward primer, and the second primer is as reverse primer.According to embodiments of the invention, the first primer be selected from following one of at least: 5 '-CGGACATGGAGGACGTGT-3 ', 5 '-GCGGACATGGAGGACGTGT-3 ', 5 '-CGCGGACATGGAGGACGTGT-3 ', 5 '-CGGACATGGAGGACGTGC-3 ', 5 '-GCGGACATGGAGGACGTGC-3 ', 5 '-CGCGGACATGGAGGACGTGC-3 '.Described the second primer be selected from following one of at least: 5 '-TGGTACACTGCCAGGCA-3 ', 5 '-CTGGTACACTGCCAGGCA-3 ', 5 '-CCTGGTACACTGCCAGGCA-3 ', 5 '-TGGTACACTGCCAGGCG-3 ', 5 '-CTGGTACACTGCCAGGCG-3 ', 5 '-CCTGGTACACTGCCAGGCG-3 '.Thus, during pcr amplification, primer can be with template according to the basepairing rule selective binding, and pcr amplification can effectively increase according to predetermined 5 ' to 3 ' direction, and can amplify specifically not isoallele.
According to a particular embodiment of the invention, the nucleotide sequence that is T or C for SNP rs429358 designs respectively a PCR forward primer (hereinafter referred to as p112T or p112C), and the nucleotide sequence that is T or C for SNP rs7412 designs respectively a PCR reverse primer (hereinafter referred to as p158T or p158C).Difference genotypic according to ApoE can be learnt, nucleotide sequence between ApoE different genotype SNP rs429358 and rs7412 can be by following primer pair specific amplified in PCR reaction: primer p112T and p158T can specific amplified ε 2 type genes, primer p112T and the p158C ε 3 type genes that can increase specifically, and primer p112C and the p158C ε 4 type genes that can increase specifically.Thus, PCR can amplify not isoallele specifically, thereby whether two of the definite kernel acid sample predetermined sites have known mutations effectively, further the allelic type of ApoE in the definite kernel acid sample effectively.
In this step, has specific nucleic acid fluorescent probe for one section of the design of the nucleotide sequence between two predetermined sites of sample of nucleic acid, nucleotide sequence corresponding at least a portion between corresponding two predetermined sites of nucleic acid probe.
The contriver finds, if nucleic acid probe has: luminophore, luminophore is formed at 5 ' end of described nucleic acid probe, regulate group, regulate the 3 ' end that group is formed at described nucleic acid probe, and described adjusting group can be regulated the luminous of described luminophore, when containing the corresponding nucleotide sequence of nucleic acid probe by pcr amplification, probe can specific combination to amplification template, this moment, the 5 prime excision enzyme activity of nucleic acid polymerase can downcut probe 5 ' end group group, luminophore is separated with the adjusting group, thereby send detectable signal.
According to a particular embodiment of the invention, the luminophore of nucleic acid probe is fluorophor, and preferred fluorophor is FAM.According to embodiments of the invention, the adjusting group of nucleic acid probe is fluorophor, and preferably regulating group is BHQ1.According to embodiments of the invention, the length of nucleic acid probe is not particularly limited, and for example, the length of nucleic acid probe can be 20 Nucleotide.According to a particular embodiment of the invention, has specific nucleic acid fluorescent probe for one section of the design of the nucleotide sequence between SNP rs429358 and rs7412.According to one embodiment of present invention, the sequence of nucleic acid probe is 5 ' FAM-CAGCTCCTCGGTGCTCTGGC-BHQ13 '.Thus, when detecting the ApoE genotype of sample of nucleic acid, only need to add the nucleic acid fluorescent probe of design as stated above in the pcr amplification system, utilize each self-corresponding different primers of E2/E3/E4 genotype pcr amplification DNA profiling to be measured respectively.After PCR reaction finishes, detect in amplification system corresponding to each genotype and have or not fluorescent signal, can judge the ApoE gene that whether contains this hypotype in the determined nucleic acid sample.
According to embodiments of the invention, the type of sample of nucleic acid and source also are not particularly limited, and for example, can be at least a portion of human gene group DNA, can derive from anyone body tissue or cell, recombinant nucleic acid or genetically modified organism histocyte.Thus, utilization is according to the method for the embodiment of the present invention, whether two of the definite kernel acid sample predetermined sites have known mutations effectively, further can effectively determine the allelic gene typing of ApoE, have very important significance for prevention and the early diagnosis of senile dementia and cardiovascular and cerebrovascular diseases.
The implication of the term that uses in the present invention " sample of nucleic acid " refers at least a portion any human gene group DNA of relating to, and the sample of nucleic acid of detection can derive from anyone body tissue or cell, recombinant nucleic acid or genetically modified organism histocyte.In the present invention, unless stated otherwise, " first with, second " is only the present invention for convenience of description, can not be interpreted as limitation of the present invention.In the present invention, unless stated otherwise, " near 3' end " refers to, apart from the base of 3' end be pre-determined range with interior base, for example distance is 10,9,8,7,6,5,4,3,2,1 or 0 (being the 3' end).
S200: detection reaction system luminous, so that whether the predetermined site of definite kernel acid sample exists known mutations
In this step, resulting pcr amplification sample in step S100 is carried out fluoroscopic examination, when fluoroscopic examination, whether has the corresponding ApoE allelic gene typing of this pcr amplification system primer pair in the determined nucleic acid sample according to having or not fluorescent signal to qualitatively judge in the pcr amplification system.Thus, whether predetermined site that can the definite kernel acid sample exists known mutations, and further can effectively determine the allelic type of ApoE.
According to a particular embodiment of the invention, the pcr amplification sample of human genome DNA is carried out fluoroscopic examination, if only fluorescent signal detected in ε 2 amplification systems, the people ApoE genotype to be measured of this sample source is ε 2/ ε 2; If in like manner only at ε 3(or ε 4) fluorescent signal detected in amplification system, the people ApoE genotype to be measured of this sample source is ε 3/ ε 3(or ε 4/ ε 4); If fluorescent signal detected simultaneously in ε 2 and ε 3 amplification systems, the people ApoE genotype to be measured of this sample source is ε 2/ ε 3; If in like manner simultaneously fluorescent signal detected in ε 2 and ε 4 amplification systems, the people ApoE genotype to be measured of this sample source is ε 2/ ε 4; If in like manner simultaneously at ε 2 and ε 4(or ε 3 and ε 4) fluorescent signal detected in amplification system, the people ApoE genotype to be measured in this sample source is ε 2/ ε 4(or ε 3/ ε 4).Thus, whether two of the definite kernel acid sample predetermined sites have known mutations effectively, further can effectively determine the allelic gene typing of ApoE, have very important significance for prevention, early diagnosis and the disease prognosis of senile dementia and cardiovascular and cerebrovascular diseases.
According to embodiments of the invention, the means of pcr amplification and fluoroscopic examination also are not particularly limited, and can carry out pcr amplification and fluoroscopic examination by any known method and apparatus.for example according to a particular embodiment of the invention, utilize the 7500Fast Real-Time PCR system of U.S. Applied Biosystems company, can be with the PCR thermal cycling, fluoroscopic examination and various analysis software combine, can dynamically observe the situation that in each each reaction tubes that circulates of PCR, pcr amplification product increases gradually, after finishing, the PCR experiment can obtain result at once, need not gel electrophoresis analysis, need not purified pcr product, has time province, highly sensitive, the advantages such as accuracy is good, again for example, person skilled can be according to the difference of concrete actual needs, primer and PCR temperature etc. is adjusted accordingly, do not repeat them here, these are all within the scope of the present invention.
Aspect another, the present invention proposes a kind of two test kits whether predetermined site has known mutations of definite kernel acid sample of the present invention.According to a particular embodiment of the invention, whether this test kit two predetermined sites that can be applied to foregoing definite kernel acid sample have the method for known mutations.Thus, utilization detects sample of nucleic acid according to the test kit whether two predetermined sites of definite sample of nucleic acid of the embodiment of the present invention have known mutations, whether two of the definite kernel acid sample predetermined sites have known mutations effectively, and further can effectively determine the allelic gene typing of ApoE.In addition, the described feature and advantage of the method whether front has known mutations for two predetermined sites of definite kernel acid sample, also applicable this test kit natch, repeat no more
Of the present invention aspect another, the present invention proposes the PCR primer whether two predetermined sites of one group of definite kernel acid sample have known mutations.According to a particular embodiment of the invention, this primer can be applied to method and the test kit whether two predetermined sites of foregoing definite kernel acid sample have known mutations.Thus, in the pcr amplification process, primer can be with template according to the basepairing rule selective binding, and pcr amplification can effectively increase according to pre-determined direction, and can amplify specifically not isoallele.In addition, whether the front has the feature and advantage of the description of the method for known mutations and test kit the primer for two predetermined sites of definite kernel acid sample, and also applicable this PCR primer natch, repeat no more.
Method, test kit and the primer whether two predetermined sites of definite sample of nucleic acid that this law is bright have a known mutations can be applied in prevention and the early diagnosis aspect of senile dementia and cardio-cerebrovascular diseases; person skilled can certainly be extended to it other its Application Areas; do not repeat them here, these are all within the scope of the present invention.
Below by specific embodiment, the present invention will be described, need to prove that these embodiment are only for illustration purpose, and can not be construed to by any way limitation of the present invention.In addition, if not otherwise specified, the equipment that adopts and material are commercially available in the following example.
General method:
1 material and reagent:
1) DNA primer p112T:5 '-GCGGACATGGAGGACGTGT-3 ', Tm=59.5 ℃, GC content=63.2%, the 5.6nM/OD/ pipe adds aseptic deionized water 55.6 μ l and is mixed with 100 μ M reaction solutions, in 4 ℃ of storages; 5 '-CGGACATGGAGGACGTGT-3 ', Tm=57.2 ℃, GC content=61.1%, the 5.9nM/OD/ pipe adds aseptic deionized water 58.9 μ l and is mixed with 100 μ M reaction solutions, in 4 ℃ of storages.Primer all synthesizes in Shanghai Bo Shang Bioisystech Co., Ltd, and the purifying mode is: the PAGE purifying.
2) DNA primer p112C:5 '-CGGACATGGAGGACGTGC-3 ', Tm=59.5 ℃, GC content=66.7%, the 5.9nM/OD/ pipe adds aseptic deionized water 59 μ l and is mixed with 100 μ M reaction solutions, in 4 ℃ of storages.Primer all synthesizes in Shanghai Bo Shang Bioisystech Co., Ltd, and the purifying mode is: the PAGE purifying.
3) DNA primer p158T:5 '-CCTGGTACACTGCCAGGCA-3 ', Tm=59.5 ℃, GC content=63.2%, the 5.7nM/OD/ pipe adds aseptic deionized water 57.2 μ l and is mixed with 100 μ M reaction solutions, in 4 ℃ of storages.Primer all synthesizes in Shanghai Bo Shang Bioisystech Co., Ltd, and the purifying mode is: the PAGE purifying.
4) DNA primer p158C:5 '-CTGGTACACTGCCAGGCG-3 ', Tm=59.5 ℃, GC content=66.7%, the 6nM/OD/ pipe adds aseptic deionized water 60 μ l and is mixed with 100 μ M reaction solutions, in 4 ℃ of storages.Primer all synthesizes in Shanghai Bo Shang Bioisystech Co., Ltd, and the purifying mode is: the PAGE purifying.
5) fluorescent DNA molecular probe: 5 ' FAM-CAGCTCCTCGGTGCTCTGGC-BHQ13 ', Tm=65 ℃, GC content=70%, the 5.1nM/OD/ pipe adds aseptic deionized water 51 μ l and is mixed with 100 μ M reaction solutions, in 4 ℃ of lucifuge storages.The fluorescent DNA molecular probe synthesizes in the English Weihe River prompt base (Shanghai) trade Co., Ltd, and the purifying mode is: the HPLC purifying.
6) PCR premixed liquid: the TaqMan Genotyping Master Mix(that is purchased from U.S. Applied Biosystems company comprises archaeal dna polymerase, dNTPs, ROX reference fluorescence), ROX reference fluorescence can not add amplification in PCR reaction, it can be corrected in the PCR reaction as the fluorescence internal reference fluorescent signal that causes due to concentration and stereomutation and change.
7) aseptic deionized water: deionized water is taken from Britain ELGA pure water instrument (model purelab3000), and after high pressure steam sterilization gained.
8) 1.5ml centrifuge tube: be purchased from U.S. Axygen company.
9) PCR plate:
Optical96-Well Reaction Plate is purchased from U.S. Applied Biosystems company.
10) PCR shrouding film:
96-Well Optical Adhesive Film is purchased from U.S. Applied Biosystems company.
11) Dispoable medical asepsis injector: Dispoable medical asepsis injector (5ml specification) is purchased from Xinxiang City, the Henan space peace medical material company limited of defending.
12) human blood extracting genome DNA related reagent:
A, erythrocyte cracked liquid: take 3.735g ammonium chloride, Tutofusin tris (Tris) 1.3g is dissolved in water and be diluted to 500ml, 0.22m filter membrane (being purchased from U.S. Millipore company) filtration sterilization is in 4 ℃ of preservations;
B, Nuclei Lysis Solution(are purchased from U.S. Promega company);
C, Precipitation Solution(are purchased from U.S. Promega company).
13) ε 2/ ε 2DNA known sample: the DNA sample of pcr amplification and sequence verification from ε 2/ ε 2 genomic dnas.
14) ε 3/ ε 3DNA known sample: the DNA sample of pcr amplification and sequence verification from ε 3/ ε 3 genomic dnas.
15) ε 4/ ε 4DNA known sample: the DNA sample of pcr amplification and sequence verification from ε 4/ ε 4 genomic dnas.
16) ε 2/ ε 3DNA known sample: the DNA sample of pcr amplification and sequence verification from ε 2/ ε 3 genomic dnas.
17) ε 2/ ε 4DNA known sample: the DNA sample of pcr amplification and sequence verification from ε 2/ ε 4 genomic dnas.
16) ε 3/ ε 4DNA known sample: the DNA sample of pcr amplification and sequence verification from ε 3/ ε 4 genomic dnas.
2 instrument and equipments:
1) pipettor: micropipet (10 μ L, 20 μ L, 100 μ L, 1000 μ L) is purchased from German Eppendorf company.
2) whizzer: small-sized high speed centrifugal machine (model: 5424), be purchased from German Eppendorf company.
3) whizzer: Universal32R type temperature control supercentrifuge is purchased from German Hettich zentrifugen company.
4) turbine mixer: be purchased from Haimen, Jiangsu its woods Bel instrument manufacturing company limited.
5) the multi-functional readout instrument of full wavelength scanner formula: the multi-functional readout instrument of Thermo Scientific Varioskan Flash full wavelength scanner formula is purchased from Thermo Scientific company.
6) Real-Time PCR instrument: 7500Fast Real-Time PCR system is purchased from U.S. Applied Biosystems company.
7) analysis software: 7500software V2.0.6 is purchased from U.S. Applied Biosystems company.
(see in detail the following examples 1-3) in the following example, the step of method whether two predetermined sites of definite kernel acid sample have known mutations is as follows:
1 design of primers:
The DNA sequence dna of ApoE gene is provided by American National biotechnology information center (NCBI) website, according to the DNA sequence dna information that comprises ApoE gene polymorphism sites SNP rs429358 and SNP rs7412, primer is carried out following design:
1) be the forward pcr amplification primer (p112T) of T for SNP rs429358 site nucleic acid form, its DNA sequence dna is: 5 '-GCGGACATGGAGGACGTGT-3 ' and 5 '-CGGACATGGAGGACGTGT-3 ';
2) be the forward pcr amplification primer (p112C) of C for SNP rs429358 site nucleic acid form, its DNA sequence dna is: 5 '-CGGACATGGAGGACGTGC-3 ';
3) be the inverse PCR amplimer (p158T) of T for SNP rs7412 site nucleic acid form, its DNA sequence dna is: 5 '-CCTGGTACACTGCCAGGCA-3 ';
4) be the inverse PCR amplimer (p158C) of C for SNP rs7412 site nucleic acid form, its DNA sequence dna is: 5 '-CTGGTACACTGCCAGGCG-3 '.
2 fluorescent DNA molecular probe designs:
The ApoE gene DNA sequence information that provides according to American National biotechnology information center (NCBI) website, DNA sequence dna between this gene SNP rs429358 site and SNP rs7412 site is: tgcggccgcctggtgcagtaccgcggcgaggtgcaggccatgctcggccagagcac cgaggagctgcgggtgcgcctcgcctcccacctgcgcaagctgcgtaagcggctcc tccgcgatgccgatgacctgcagaagcgc, choose one section complementary nucleic acid Sequence as probe in this sequence, this sequence is 5 '-CAGCTCCTCGGTGCTCTGGC-3 '.Select fluorescent probe, choose FAM fluorophor and BHQ1 quenching group, the FAM fluorophor is coupled at 5 ' end of complementary nucleic acid sequence, and the BHQ1 quenching group is coupled at 3 ' end of complementary nucleic acid sequence, and fluorescent probe is: 5 ' FAM-CAGCTCCTCGGTGCTCTGGC-BHQ13 '.
Following part is set forth take human blood genomic dna sample as test sample:
3 operating processes
1) sample collection:
Use Dispoable medical asepsis injector (5ml) to extract people's venous blood 1ml in the pipe that contains 1.5mg EDETATE SODIUM anti-coagulant, place 4 ℃ of refrigerator storages stand-by.
2) human blood extracting genome DNA:
A, take out the human blood that gathers and mixing gently from 4 ℃ of refrigerators, then get the 300ul blood transfer in the 1.5ml centrifuge tube that contains the 900ul erythrocyte cracked liquid with pipettor, turn upside down 5-6 time.
B, room temperature place 10 minutes (during put upside down 2-3 time), and under room temperature condition, 13000-16000g is centrifugal 20 seconds.
C, suck supernatant, remaining approximately 10-20ul is in the pipe end.
D, the resuspended white corpuscle of vortex vibration add Nuclei Lysis Solution, pressure-vaccum 5-6 time, and optional step adds the RNA enzyme mixing of 1.5ul, places 15 minutes under 37 ℃ of conditions.
E, add 100ul Precipitation Solution, vortex vibration 10-20 second.
Under f, room temperature condition, 13000-16000g is centrifugal 10 minutes.
G, transfer supernatant add the 300ul Virahol to new 1.5ml centrifuge tube, put upside down gently up and down until the cotton-shaped DNA of adularescent occurs.
Under h, room temperature condition, 13000-16000g is centrifugal 1 minute.
I, suck supernatant, add 70% ethanol, DNA is washed in turning upside down gently several times, and under room temperature condition, 13000-16000g is centrifugal 1 minute.
J, suck ethanol gently, the 1.5ml centrifuge tube is upside down on clean thieving paper 10-15 minute.
K, add 100ul aseptic deionized water dissolving DNA, 65 ℃ of water-baths 1 hour also can 4 ℃ be spent the night.
L, utilize DNA measurement of concetration program in the multi-functional readout instrument of full wavelength scanner formula to measure the concentration of the DNA that puies forward, get a part of DNA solution and be diluted to 2ng/ml with aseptic deionized water, detect template as follow-up PCR, surplus DNA solution is deposited in 2～8 ℃ or-20 ℃.
3) PCR reaction system preparation:
According to three polymorphisms of ApoE gene, with three kinds of detection reagent (ε 2 reagent, ε 3 reagent and ε 4 reagent), be respectively used to exist in the crowd 6 kinds of different ApoE genotype ε 2/ ε 2, ε 3/ ε 3, ε 4/ ε 4, ε 2/ ε 3, the detection of ε 2/ ε 4 and ε 3/ ε 4.6 kinds of known ApoE genotype DNA known sample (ε 2/ ε 2DNA known sample, ε 3/ ε 3DNA known sample, ε 4/ ε 4DNA known sample, ε 2/ ε 3DNA known sample, ε 2/ ε 4DNA known sample and ε 3/ ε 4DNA known sample) in contrast
Wherein ε 2 reagent comprise:
The forward primer p112T (this example amplifying nucleic acid sequence is 5 '-GCGGACATGGAGGACGTGT-3 ') of a, corresponding A poE ε 2 allelotype SNP site rs429358SNP nucleic acid forms.
This example amplifying nucleic acid sequence of reverse primer p158T(of b, corresponding A poE ε 2 allelotype SNP site rs7412 reverse primer nucleic acid forms is 5 '-CCTGGTACACTGCCAGGCA-3 ').
C, fluorescent probe (this example middle probe form is 5 ' FAM-CAGCTCCTCGGTGCTCTGGC-BHQ13 ').
D, PCR premixed liquid TaqMan Genotyping Master Mix.
The concrete dosage of its each composition is:
TaqMan Genotyping Master Mix 4.925μl
Fluorescent probe (100 μ M) 0.05 μ l
Wherein ε 3 reagent comprise:
The forward primer p112T (this example amplifying nucleic acid sequence is 5 '-CGGACATGGAGGACGTGT-3 ') of a, corresponding A poE ε 3 allelotype SNP site rs429358SNP nucleic acid forms.
This example amplifying nucleic acid sequence of reverse primer p158C(of b, corresponding A poE ε 3 allelotype SNP site rs7412 reverse primer nucleic acid forms is 5 '-CTGGTACACTGCCAGGCG-3 ').
C, fluorescent probe (this example middle probe form is 5 ' FAM-CAGCTCCTCGGTGCTCTGGC-BHQ13 ').
D, PCR premixed liquid TaqMan Genotyping Master Mix.
The concrete dosage of its each composition is:
TaqMan Genotyping Master Mix 4.925μl
Fluorescent probe (100 μ M) 0.05 μ l
Wherein ε 4 reagent comprise:
The forward primer p112C (this example amplifying nucleic acid sequence is 5 '-CGGACATGGAGGACGTGC-3 ') of a, corresponding A poE ε 4 allelotype SNP site rs429358SNP nucleic acid forms.
This example amplifying nucleic acid sequence of reverse primer p158C(of b, corresponding A poE ε 4 allelotype SNP site rs7412 reverse primer nucleic acid forms is 5 '-CTGGTACACTGCCAGGCG-3 ').
C, fluorescent probe (this example middle probe form is 5 ' FAM-CAGCTCCTCGGTGCTCTGGC-BHQ13 ').
D, PCR premixed liquid TaqMan Genotyping Master Mix.
The concrete dosage of its each composition is:
TaqMan Genotyping Master Mix 4.925μl
Fluorescent probe (100 μ M) 0.05 μ l
Use ApoE genotype tests reagent to need 10 μ l reaction systems just signal can fully be detected.
The PCR reaction system of ε 2 gene tests is:
DNA sample to be measured (2ng/ μ l): 5 μ l
The PCR reaction system of ε 3 gene tests is:
DNA sample to be measured (2ng/ μ l): 5 μ l
The PCR reaction system of ε 4 gene tests is:
DNA sample to be measured (2ng/ μ l): 5 μ l
4) pcr amplification reaction:
Be that the genomic dna (2ng/ μ l) that human blood extracts adds respectively ε 2 with the genotypic DNA known sample of known ApoE with testing sample, in 10 μ l PCR reaction systems of ε 3 and ε 4 gene tests.With the negative control of the deionized water of sterilizing as whole PCR system.Prepare all PCR reaction systems in the PCR plate after, with PCR shrouding membrane closure good PCR plate, remain on PCR plate hole wall liquid with Universal32R type temperature control supercentrifuge centrifugal to the pipe end bubble removing side by side.Then, this PCR plate is put into 7500Fast Real-Time PCR system instrument, open 7500software V2.0.6 software, select the Genotyping module in software, the Detection of content that correspondence is put into every hole on the PCR plate is set in Plate setup.Then PCR response procedures and reaction system are set in Run method, these projects after setting completed, just but working procedure, first read in advance the background fluorescence intensity level before this program acquiescence PCR reaction and carry out pcr amplification to eliminate the background fluorescence value again under 60 ℃ of conditions.After treating that the PCR reaction is completed, instrument can be collected fluorescence data automatically under 60 ℃ of conditions.Table 1 is the standard thermal cycling program of this example PCR reaction process.
Table 1PCR response procedures
| Process | Temperature (℃) | Time | Circulation |
| Reading of data | 60 | 1min | |
| Activate the DNA synthetic enzyme | 95 | 10min | |
| Sex change | 95 | 15s | 40 |
| Annealing/extension | 60 | 1min | |
| Reading of data | 60 | 1min |
5) data analysis and conclusion:
Derive fluorescence numerical value after Real-Time PCR instrument runs through fluorescent value, result as shown in Figure 4.Be in the DNA known sample of ε 2/ ε 2 in the ApoE genotype, the ε 2 positive results of gene test system, its fluorescent value is 2.23623729.ε 3, the ε 4 negative results of gene test system, and its fluorescent value is respectively 0.218611 and 0.51656318; Be in the DNA known sample of ε 3/ ε 3 in the ApoE genotype, the ε 3 positive results of gene test system, its fluorescent value is 2.50921822.ε 2, the ε 4 negative results of gene test system, and its fluorescent value is respectively 0.1933465 and 0.40723515; Be in the DNA known sample of ε 4/ ε 4 in the ApoE genotype, the ε 4 positive results of gene test system, its fluorescent value is 2.59521461.ε 2, the ε 3 negative results of gene test system, and its fluorescent value is respectively 0.17685843 and 0.22782183.As the aseptic deionized water sample of negative control, at ε 2, in ε 3 and ε 4 gene test systems, its fluorescent value is respectively 0.17888165,0.22742152 and-0.0582402.Like this, negative findings fluorescent value maximum in this example is 0.51656318, and the fluorescent value of all positive findingses is all much larger than the negative findings fluorescent value of maximum.In this cut off value that defines positive as fluorescent signal with 2 multiple value of maximum negative findings fluorescent value in negative findings, in this example, this cut off value is 1.03312636.
Can make column diagram according to the fluorescent value in Fig. 4, column diagram at positive and the standardized straight line in negative findings cut off value place, can find out intuitively all positive findingses all above straight line, and all negative findingses is all below straight line as shown in Figure 5.In this example, DNA sample to be measured obtains result in ε 2 and ε 3 gene test systems and is judged as positive findings according to above-mentioned analytical procedure, is judged as negative findings and obtain result in ε 4 gene test systems according to above-mentioned analytical procedure.Learn thus, in this example, the ApoE genotype of DNA sample to be measured is ε 2/ ε 3 types.
In an embodiment, as shown in Figure 1, primer p112T is only the nucleotide sequence coupling of T with SNP rs429358, primer p112C is only the nucleotide sequence coupling of C with SNP rs429358, primer p158T is only the nucleotide sequence coupling of T with SNP rs7412, and primer p158C is only the nucleotide sequence coupling of C with SNP rs7412.Thus, the PCR system that contains primer pair p112T/p158T only can increase SNP rs429358 be T simultaneously SNP rs7412 be the ApoE allelotrope (being ε 2) of T; The PCR system that contains primer pair p112T/p158C only can increase SNP rs429358 be T simultaneously SNP rs7412 be the ApoE allelotrope (being ε 3) of C; The PCR system that contains primer pair p112C/p158C only can increase SNP rs429358 be C simultaneously SNPrs7412 be the ApoE allelotrope (being ε 4) of C.So, utilize above-mentioned three kinds of primer pairs, can amplify specifically the not isoallele of ApoE, and the allelic type of people ApoE in definite DNA sample.
In an embodiment, as shown in Figure 2, two sections of the DNA molecular probe 3 that fluorophor (F) breaks away from respectively coupling fluorophor and quenching group are arranged, can absorb the excitation energy of fluorophor due to quenching group this moment, therefore, the background fluorescence signal can only be detected in system.The DNA molecular probe 3 that fluorophor (F) breaks away from can mate with the special complementation of ApoE gene DNA template antisense strand 1, and matching sequence is between the DNA sequence dna of forward amplimer 2 and reverse amplimer 5 correspondences, after PCR expansion reaction beginning, the DNA molecular probe 3 that forward amplimer 2 and fluorophor (F) break away from will with 1 combination of ApoE gene DNA template antisense strand, oppositely amplimer 5 will with 4 combinations of ApoE gene DNA template positive-sense strand, beginning amplification template DNA under the effect of archaeal dna polymerase 6.
in an embodiment, as shown in Figure 3, archaeal dna polymerase 6 in the PCR reaction process from forward amplimer 2 or oppositely amplimer 5, take ApoE gene DNA template antisense strand 1 or ApoE gene DNA template positive-sense strand 4 as template, the extension of initiate dna, run into the DNA molecular probe 3 of fluorophor (F) disengaging that is combined on ApoE gene DNA template antisense strand 1 in the process that mediated dna extends when archaeal dna polymerase 6, archaeal dna polymerase 6 will utilize 5 ' end of the DNA molecular probe 3 that its 5 prime excision enzyme activity breaks away from from fluorophor (F) to cut away one by one Nucleotide on probe, cause fluorophor break away from and separate with quenching group from probe, can detect the fluorescent signal that fluorophor inspires this moment, and determine that thus test sample contains the corresponding ApoE allelic gene type of this primer pair.
Although the above has illustrated and has described embodiments of the invention; be understandable that; above-described embodiment is exemplary; can not be interpreted as limitation of the present invention; those of ordinary skill in the art is not in the situation that break away from principle of the present invention and aim can change above-described embodiment within the scope of the invention, modification, replacement and modification, within these all drop on the scope of the present invention.
Claims (7)
1. whether two of a definite kernel acid sample predetermined sites have the method for known mutations, it is characterized in that, comprising:
Utilize the first primer and the described sample of nucleic acid of the second primer pair to carry out pcr amplification, and add nucleic acid probe in described pcr amplification system, the nucleotide sequence of at least a portion between corresponding two predetermined sites of described nucleic acid probe; And
Detection reaction system luminous, in order to determine whether the predetermined site of described sample of nucleic acid exists known mutations,
Wherein,
Nearly 3 ' end of described the first primer comprises the base corresponding with one of described two predetermined sites known mutations,
Nearly 3 ' end of described the second primer comprises and described two bases that another known mutations of predetermined site is corresponding,
One of described the first primer and second primer be as forward primer, and another of described the first primer and described the second primer be as reverse primer,
Described nucleic acid probe has:
Luminophore, described luminophore are formed at 5 ' end of described nucleic acid probe,
Regulate group, described adjusting group is formed at 3 ' end of described nucleic acid probe, and described adjusting group can regulate the luminous of described luminophore,
Randomly, described sample of nucleic acid derives from tissue or cell, recombinant nucleic acid or genetically modified organism histocyte,
Randomly, described predetermined site is SNP rs429358 and rs7412,
Randomly, described nearly 3 ' end of stating the first primer comprises the base corresponding with the known mutations of rs429358, and nearly 3 ' end of described the second primer comprises the base corresponding with the known mutations of rs7412,
Randomly, 3 ' end base of described the first primer is the base corresponding with the known mutations of rs429358, and 3 ' end base of described the second primer is the base corresponding with the known mutations of rs7412,
Randomly, described the first primer is as forward primer, and described the second primer is as reverse primer,
Randomly, described the first primer be selected from following one of at least:
5’-CGGACATGGAGGACGTGT-3’
5’-GCGGACATGGAGGACGTGT-3’
5’-CGCGGACATGGAGGACGTGT-3’
5’-CGGACATGGAGGACGTGC-3’
5’-GCGGACATGGAGGACGTGC-3’
5’-CGCGGACATGGAGGACGTGC-3’，
Described the second primer be selected from following one of at least:
5’-TGGTACACTGCCAGGCA-3’
5’-CTGGTACACTGCCAGGCA-3’
5’-CCTGGTACACTGCCAGGCA-3’
5’-TGGTACACTGCCAGGCG-3’
5’-CTGGTACACTGCCAGGCG-3’
5’-CCTGGTACACTGCCAGGCG-3’，
Randomly, the length of described nucleic acid probe is 20 Nucleotide.
2. method according to claim 1, is characterized in that, the sequence of described nucleic acid probe is:
5’-CAGCTCCTCGGTGCTCTGGC-3’。
3. method according to claim 1, is characterized in that, the luminophore of described nucleic acid probe is fluorophor,
Randomly, described fluorophor is FAM,
Randomly, described adjusting group is quenching group,
Randomly, described quenching group is BHQ1.
4. two of a definite kernel acid sample test kits whether predetermined site has known mutations, is characterized in that, comprising:
The first primer, nearly 3 ' end of described the first primer comprises the base corresponding with one of described two predetermined sites known mutations,
The second primer, nearly 3 ' end of described the second primer comprises and described two bases that another known mutations of predetermined site is corresponding,
Nucleotide sequence corresponding at least a portion between nucleic acid probe, described nucleic acid probe and two predetermined sites, and described nucleic acid probe has:
Luminophore, described luminophore are formed at 5 ' end of described nucleic acid probe,
Regulate group, described adjusting group is formed at 3 ' end of described nucleic acid probe, and described adjusting group can regulate the luminous of described luminophore,
Randomly, described predetermined site is SNP rs429358 and rs7412,
Randomly, described nearly 3 ' end of stating the first primer comprises the base corresponding with the known mutations of rs429358, and nearly 3 ' end of described the second primer comprises the base corresponding with the known mutations of rs7412,
Randomly, 3 ' end base of described the first primer is the base corresponding with the known mutations of rs429358, and 3 ' end base of described the second primer is the base corresponding with the known mutations of rs7412,
Randomly, described the first primer is as forward primer, and described the second primer is as reverse primer,
Randomly, described the first primer be selected from following one of at least:
5’-CGGACATGGAGGACGTGT-3’
5’-GCGGACATGGAGGACGTGT-3’
5’-CGCGGACATGGAGGACGTGT-3’
5’-CGGACATGGAGGACGTGC-3’
5’-GCGGACATGGAGGACGTGC-3’
5’-CGCGGACATGGAGGACGTGC-3’，
Described the second primer be selected from following one of at least:
5’-TGGTACACTGCCAGGCA-3’
5’-CTGGTACACTGCCAGGCA-3’
5’-CCTGGTACACTGCCAGGCA-3’
5’-TGGTACACTGCCAGGCG-3’
5’-CTGGTACACTGCCAGGCG-3’
5’-CCTGGTACACTGCCAGGCG-3’，
Randomly, the length of described nucleic acid probe is 20 Nucleotide.
5. test kit according to claim 4, is characterized in that, the sequence of described nucleic acid probe is:
5’-CAGCTCCTCGGTGCTCTGGC-3’。
6. test kit according to claim 4, is characterized in that, the luminophore of described nucleic acid probe is fluorophor,
Randomly, described fluorophor is FAM,
Randomly, described adjusting group is quenching group,
Randomly, described quenching group is BHQ1.
7. two of one group of definite kernel acid sample PCR primers whether predetermined site has known mutations, is characterized in that, comprising:
The first primer, nearly 3 ' end of described the first primer comprises the base corresponding with one of described two predetermined sites known mutations,
The second primer, nearly 3 ' end of described the second primer comprises and described two bases that another known mutations of predetermined site is corresponding,
Nucleotide sequence corresponding at least a portion between nucleic acid probe, described nucleic acid probe and two predetermined sites, and described nucleic acid probe has:
Luminophore, described luminophore are formed at 5 ' end of described nucleic acid probe,
Regulate group, described adjusting group is formed at 3 ' end of described nucleic acid probe, and described adjusting group can regulate the luminous of described luminophore,
Randomly, described predetermined site is SNP rs429358 and rs7412,
Randomly, described nearly 3 ' end of stating the first primer comprises the base corresponding with the known mutations of rs429358, and nearly 3 ' end of described the second primer comprises the base corresponding with the known mutations of rs7412,
Randomly, 3 ' end base of described the first primer is the base corresponding with the known mutations of rs429358, and 3 ' end base of described the second primer is the base corresponding with the known mutations of rs7412,
Randomly, described the first primer is as forward primer, and described the second primer is as reverse primer,
Randomly, described the first primer be selected from following one of at least:
5’-CGGACATGGAGGACGTGT-3’
5’-GCGGACATGGAGGACGTGT-3’
5’-CGCGGACATGGAGGACGTGT-3’
5’-CGGACATGGAGGACGTGC-3’
5’-GCGGACATGGAGGACGTGC-3’
5’-CGCGGACATGGAGGACGTGC-3’，
Described the second primer be selected from following one of at least:
5’-TGGTACACTGCCAGGCA-3’
5’-CTGGTACACTGCCAGGCA-3’
5’-CCTGGTACACTGCCAGGCA-3’
5’-TGGTACACTGCCAGGCG-3’
5’-CTGGTACACTGCCAGGCG-3’
5’-CCTGGTACACTGCCAGGCG-3’。
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| WO2014101276A2 (en) * | 2012-12-31 | 2014-07-03 | 厦门人瑞生物医药科技有限公司 | Method, kit and primers for determining whether two predetermined sites in nucleic acid sample have known mutations |
| CN106011298A (en) * | 2015-11-12 | 2016-10-12 | 厦门人瑞生物医药科技有限公司 | ApoE kit, primers and use thereof |
| CN109097457A (en) * | 2017-06-20 | 2018-12-28 | 深圳华大智造科技有限公司 | The method for determining predetermined site mutation type in sample of nucleic acid |
| CN110029159A (en) * | 2019-02-26 | 2019-07-19 | 成都华青精准医疗科技有限公司 | A kind of nucleotide sequence group and its application for the detection of ApoE Genotyping |
| CN110114458A (en) * | 2016-09-22 | 2019-08-09 | 豪夫迈·罗氏有限公司 | POL6 polymerase mutants |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014101276A2 (en) * | 2012-12-31 | 2014-07-03 | 厦门人瑞生物医药科技有限公司 | Method, kit and primers for determining whether two predetermined sites in nucleic acid sample have known mutations |
| WO2014101276A3 (en) * | 2012-12-31 | 2014-08-28 | 厦门人瑞生物医药科技有限公司 | Method, kit and primers for determining whether two predetermined sites in nucleic acid sample have known mutations |
| CN106011298A (en) * | 2015-11-12 | 2016-10-12 | 厦门人瑞生物医药科技有限公司 | ApoE kit, primers and use thereof |
| CN110114458A (en) * | 2016-09-22 | 2019-08-09 | 豪夫迈·罗氏有限公司 | POL6 polymerase mutants |
| CN109097457A (en) * | 2017-06-20 | 2018-12-28 | 深圳华大智造科技有限公司 | The method for determining predetermined site mutation type in sample of nucleic acid |
| CN110029159A (en) * | 2019-02-26 | 2019-07-19 | 成都华青精准医疗科技有限公司 | A kind of nucleotide sequence group and its application for the detection of ApoE Genotyping |
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