CN103571932A - Detection method for single nucleotide polymorphism based on heat-resistant nuclease HII - Google Patents
Detection method for single nucleotide polymorphism based on heat-resistant nuclease HII Download PDFInfo
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- CN103571932A CN103571932A CN201210260940.5A CN201210260940A CN103571932A CN 103571932 A CN103571932 A CN 103571932A CN 201210260940 A CN201210260940 A CN 201210260940A CN 103571932 A CN103571932 A CN 103571932A
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- molecular beacon
- hii
- single nucleotide
- nucleotide polymorphism
- nuclease
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q2521/00—Reaction characterised by the enzymatic activity
- C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
- C12Q2521/327—RNAse, e.g. RNAseH
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- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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- C12Q2525/121—Modifications characterised by incorporating both deoxyribonucleotides and ribonucleotides
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- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/30—Oligonucleotides characterised by their secondary structure
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- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
- C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
Abstract
The invention brings forward a detection method for single nucleotide polymorphism based on heat-resistant nuclease HII. According to the method, the heat-resistant nuclease HII is used to cut off a molecular beacon with ribonucleotide, and fluorescence signals are accumulated for detection of double-stranded DNA, wherein the molecular beacon with ribonucleotide has one ribonucleic acid at its central part and pairing nucleotides at its two sides, the two sides are respectively labeled by a fluorescence emission group and a fluorescence quenching group, and the whole molecular beacon forms a stem-loop structure and does not release fluorescence. The detection method provided by the invention can detect double-stranded nucleic acids and amplify detection signals and has high sensitivity.
Description
Technical field
The present invention relates to technical field of biological, refer to especially a kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII.
Background technology
Detection of nucleic acids and snp analysis have been very large industries.These technology not only obtain application widely in scientific research, also more and more obvious in the importance in the fields such as clinical diagnosis, food inspection, forensic identification.Detection of nucleic acids now and snp analysis technology kind are various, and its taproot is nucleic acid hybridization detection technique, nucleic acid sequence analysis technology and polymerase chain reaction (PCR).Detect flux and highly sensitive, and simple to operate, detection time is short is the developing direction of gene and SNP detection.
The Constant Temperature Detection that molecular beacon (molecular beacon) is DNA provides possibility, and success is for detection of multiple pathogenic microorganism.Molecular beacon is the loop-stem structure that a kind of short oligonucleotide strand forms.Oligonucleotide chain one end is fluorophor mark, and the other end is quenching group mark.Because the nucleotide sequence at two ends is complementary, mutually near there is energy resonance in its fluorophor and quenching group, and the photon that after being excited, fluorophor produces is quenched group cancellation.If detected nucleic acid molecule can with the single-stranded loop area hybridization of molecular beacon, the stem structure of molecular beacon is destroyed, increased the space length between fluorophor and quenching group, can not produce fluorescent energy resonance transfer, just can detect the fluorescent signal that after being excited, fluorophor produces.But in whole testing process, the power of fluorescent signal depends on the content of detected DNA molecular, do not exist signal to amplify, therefore, directly by the detection technique of molecular beacon hybridization target nucleic acid, its sensitivity is undesirable.
RNase HII is a kind of endoribonuclease, and it can be hydrolyzed the RNA phosphodiester bond hybridizing on DNA chain specifically, can decompose the RNA chain in RNA/DNA hybridization system.This enzyme can not digest strand or double-stranded DNA.The people such as J.Rizzo in 2002, use first molecular beacon method to detect Yeast Nucleic Acid enzyme kinetics and enzyme is cut mechanism [Molecular and Cellular Probes, 2002,16:277 – 283].But have no so far the method that detects nucleic acid molecule about ribonuclease H II binding molecule beacon.
Summary of the invention
The present invention is directed to the defect of prior art, propose a kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII, this detection method can detect double-strandednucleic acid amplification detection signal, and sensitivity is higher.
Technical scheme of the present invention is achieved in that
A method for detecting single nucleotide polymorphism based on heat stable nuclease HII, is used heat stable nuclease HII() cut off the molecular beacon with ribonucleotide, fluorescent signal is accumulated, detect double-stranded DNA; Wherein, the described molecular beacon with ribonucleotide is middle with a Yeast Nucleic Acid, and the Nucleotide of both sides is paired with each other, and fluorescent emission group and fluorescent quenching group mark are used respectively in both sides, and whole molecular beacon forms loop-stem structure, without fluorescence, discharges.In the middle of molecular beacon, with ribonucleotide, heat stable nuclease HII can cut off molecular beacon, amplifies fluorescent signal.Nuclease HII derives from heat resistant microbe, so detection reaction can at high temperature carry out with the form of thermal cycling, can effectively detect double-stranded DNA sample.
As preferred technical scheme, described detection method also comprises and pcr amplification coupling, and detection reaction and nucleic acid amplification reaction are coupled, and have improved detection sensitivity.
As preferred technical scheme, described 33 Nucleotide of molecular beacon length, middle with a ribonucleotide, 21 middle Nucleotide match with detecting sample DNA, and 6 Nucleotide of both sides are paired with each other.
As preferred technical scheme, described fluorescent emission group can be used FAM, TET etc.; Described fluorescent quenching group can be with DACBYL etc.
As preferred technical scheme, described molecular beacon is
Amp-T：
5’FAM-CGCGCCAGTAAGGGAAruAGGAAGTACTGGCGCG-DABCYL、
Amp-A：
5’FAM-CGCGCCAGTAAGGGAAraAGGAAGTACTGGCGCG-DABCYL、
Amp-C：
5 ' FAM-CGCGCCAGTAAGGGAArcAGGAAGTACTGGCGCG-DABCYL or
Amp-G：
5’FAM-CGCGCCAGTAAGGGAArgAGGAAGTACTGGCGCG-DABCYL。
As preferred technical scheme, described detection method specifically comprises:
Step I, prepares the molecular beacon of a ribonucleotide of Intermediate Gray;
Step II, the foundation of detection reaction system;
Primer sequence, dNTP, Taq archaeal dna polymerase, Tth nuclease HII, molecular beacon and the detection sample DNA of amplification target dna are added in the enzymatic reaction system of Taq to preparation reaction mixture;
Step III, fluorescent signal iodine;
By the reaction mixture in step II, put into quantitative real time PCR Instrument, carry out pcr amplification and genotype tests;
Step IV, determines gene pleiomorphism;
According to the difference of fluorescent signal between different samples, determine the single nucleotide polymorphism that detects sample site to be measured.
Detection method of the present invention can detect double-strandednucleic acid amplification detection signal, and mainly by heat stable nuclease HII, by centre, the molecular beacon with a ribonucleotide cuts off in centre for it, and fluorescent signal is accumulated, and reaches the effect that signal amplifies.The method can be measured for detection of nucleic acids and gene type.Combine with quantitative real time PCR Instrument, can realize online high throughput testing.The method can be carried out the Molecular Detection of food contamination microorganism, environmental microorganism, pathogenic micro-organism etc., the Molecular Detection diagnosis of important inherited disease, and HLA gene type etc.
The present invention has compared significant progress with the detection of nucleic acids of existing gene molecule beacon with genotyping technique.Major advantage is as follows:
(1) molecular beacon of the present invention is middle can cut off molecular beacon with a ribonucleotide, amplifies fluorescent signal;
(2) detection reaction of the present invention and nucleic acid amplification reaction are coupled, and have improved detection sensitivity;
(3) nuclease HII derives from heat resistant microbe, so detection reaction can at high temperature carry out with the form of thermal cycling, can effectively detect double-stranded DNA sample.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, to the accompanying drawing of required use in embodiment or description of the Prior Art be briefly described below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skills, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is a kind of method for detecting single nucleotide polymorphism schematic diagram based on heat stable nuclease HII of the present invention;
1, the first fluorescent signal amplifies loop, and the 2, second fluorescent signal amplifies loop.
Fig. 2 is the detection signal of four kinds of molecular beacons of the present invention.
Embodiment
Below the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Embodiment based in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
The inventive method as shown in Figure 1, first designs synthetic mesophase with the molecular beacon of a ribonucleotide, utilizes heat-resisting nuclease HII to cut off the molecular beacon with ribonucleotide, and fluorescent signal is accumulated, and reaches the effect that signal amplifies.The foundation of detection reaction system: add, measure sample DNA, PCR component, molecular beacon and nuclease HII in reaction system.Fluorescent signal iodine: detect in quantitative real time PCR Instrument, determine the single nucleotide polymorphism of sample.
The first step, pcr amplification target nucleic acid to be checked district; Second step, molecular beacon and target nucleic acid hybridization pairing form double-stranded collochore, and thermostability ribonuclease H II cuts off molecular beacon at ribonucleotide place, and molecular beacon discharges fluorescent signal; The 3rd step, the molecular beacon 3 ' of fracture holds half molecule to disintegrate down from single stranded DNA template, and free single stranded DNA template can instruct the molecular beacon endonuclease reaction of next round (fluorescent signal amplifies loop 1); Simultaneously, it is free hydroxyl that the molecular beacon 5 ' of fracture is held 3 ' end of half molecule, half molecule of being combined with single stranded DNA template can extend into the double-stranded DNA with 3 ' long end, after next round PCR, generate short flat terminal double link DNA, these two double-stranded DNAs all can instruct the molecular beacon endonuclease reaction (fluorescent signal amplifies loop 2) of next round.If the ribonucleotide of molecular beacon forms base mismatch pair with the corresponding base of target nucleic acid, the ability of thermostability ribonuclease H II cut-out molecular beacon lowers greatly, net result is to only have the molecular beacon of minute quantity to be cut off, compare with the molecular beacon of correct pairing the fluorescent signal that now discharges extremely a little less than, therefore can determine SNP kind by the fluorescence signal intensity difference discharging.
Embodiment 1
For ammonia benzyl resistant gene, utilize the ribonucleotide molecular beacon of design and the nuclease HII of Tth, to carry out ammonia benzyl resistant gene polymorphism somatotype and detect, concrete implementation step is as follows:
The first step, prepares the molecular beacon of a ribonucleotide of Intermediate Gray.Long 33 Nucleotide of molecular beacon, middle with a ribonucleotide, the 300-321 position oligonucleotide ligand pair of middle 21 Nucleotide and ammonia benzyl resistance gene DNA, 6 Nucleotide of both sides are paired with each other, fluorescent emission group and fluorescent quenching group mark are used respectively in both sides, whole molecular beacon forms loop-stem structure, without fluorescence, discharges.Article 4, molecular beacon is as follows:
Amp-T：
5’FAM-CGCGCCAGTAAGGGAAruAGGGAAGTACTGGCGCG-DABCYL
Amp-A：
5’FAM-CGCGCCAGTAAGGGAAraAGGAAGTACTGGCGCG-DABCYL
Amp-C：
5’FAM-CGCGCCAGTAAGGGAArcAGGAAGTACTGGCGCG-DABCYL
Amp-G：
5’FAM-CGCGCCAGTAAGGGAArgAGGAAGTACTGGCGCG-DABCYL
Second step, the foundation of detection reaction system.The primer sequence that relates to synthetic amplification ammonia benzyl resistant gene, and by dNTP, Taq archaeal dna polymerase, Tth nuclease HII, a kind of molecular beacon, DNA sample add in the enzymatic reaction system of Taq, preparation reaction mixture.Article 4, molecular beacon adds respectively and contains the reaction system that detects sample DNA, corresponding 4 reactions of sample.Primer sequence is as follows:
Amp-F:5’atgagtattc?aacatttccg?t
Amp-R:5’cgtcaatacg?ggataatacc
The 3rd step, fluorescent signal iodine.By 4 reaction mixtures for detection sample in second step, put into quantitative real time PCR Instrument, carry out pcr amplification and genotype tests.
The 4th step, determines gene pleiomorphism.The fluorescent signal of quantitative 4 detection reaction, according to the sequence-specific of 4 reaction systems and molecular beacon, determines the single nucleotide polymorphism (as shown in Figure 2) that detects sample site to be measured.
The fluorescent emission group of above-mentioned molecular beacon can also be used TET, and this is to well known to a person skilled in the art alternative, at this, does not repeat.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (6)
1. the method for detecting single nucleotide polymorphism based on heat stable nuclease HII, is characterized in that, uses heat stable nuclease HII to cut off the molecular beacon with ribonucleotide, and fluorescent signal is accumulated, and detects double-stranded DNA; Wherein, the described molecular beacon with ribonucleotide is middle with a Yeast Nucleic Acid, and the Nucleotide of both sides is paired with each other, and fluorescent emission group and fluorescent quenching group mark are used respectively in both sides, and whole molecular beacon forms loop-stem structure, without fluorescence, discharges.
2. a kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII according to claim 1, is characterized in that, described detection method also comprises and pcr amplification coupling.
3. a kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII according to claim 1 and 2, is characterized in that, described fluorescent emission group is FAM or TET; Described fluorescent quenching group is DACBYL.
4. a kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII according to claim 3, it is characterized in that, long 33 Nucleotide of described molecular beacon, middle with a ribonucleotide, 21 middle Nucleotide match with detecting sample DNA, and 6 Nucleotide of both sides are paired with each other.
5. a kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII according to claim 4, is characterized in that, described molecular beacon is
Amp-T：
5’FAM-CGCGCCAGTAAGGGAAruAGGAAGTACTGGCGCG-DABCYL、
Amp-A：
5’FAM-CGCGCCAGTAAGGGAAraAGGAAGTACTGGCGCG-DABCYL、
Amp-C：
5 ' FAM-CGCGCCAGTAAGGGAArcAGGAAGTACTGGCGCG-DABCYL or
Amp-G：
5’FAM-CGCGCCAGTAAGGGAArgAGGAAGTACTGGCGCG-DABCYL。
6. a kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII according to claim 1, is characterized in that, described detection method specifically comprises:
Step I, prepares the molecular beacon of a ribonucleotide of Intermediate Gray;
Step II, the foundation of detection reaction system;
Primer sequence, dNTP, Taq archaeal dna polymerase, Tth nuclease HII, molecular beacon and the detection sample DNA of amplification target dna are added in the enzymatic reaction system of Taq to preparation reaction mixture;
Step III, fluorescent signal iodine;
By the reaction mixture in step II, put into quantitative real time PCR Instrument, carry out pcr amplification and genotype tests;
Step IV, determines gene pleiomorphism;
According to the difference of fluorescent signal between different samples, determine the single nucleotide polymorphism that detects sample site to be measured.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755402A (en) * | 2016-12-22 | 2017-05-31 | 远见生物科技（上海）有限公司 | A kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII |
| CN107385017A (en) * | 2017-06-14 | 2017-11-24 | 苏州承美生物科技有限公司 | A kind of molecular beacon probe and kit for detecting circulating tumor cell |
| CN107937486A (en) * | 2017-12-25 | 2018-04-20 | 苏州旷世骏弛生物科技有限公司 | A kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII |
| CN113201593A (en) * | 2021-05-25 | 2021-08-03 | 广州普世君安生物科技有限公司 | Primer probe combination product for detecting ACTN3 genotype |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181519A (en) * | 2011-02-16 | 2011-09-14 | 上海交通大学 | Method for detecting gene polymorphism SNP (single nucleotide polymorphism) site by utilizing cyclization of RNase H (ribonuclease H) |
| CN102212615A (en) * | 2011-04-01 | 2011-10-12 | 上海交通大学 | Method for detecting single-nucleotide polymorphism based on high-temperature resistant RNase HII |
-
2012
- 2012-07-25 CN CN201210260940.5A patent/CN103571932A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181519A (en) * | 2011-02-16 | 2011-09-14 | 上海交通大学 | Method for detecting gene polymorphism SNP (single nucleotide polymorphism) site by utilizing cyclization of RNase H (ribonuclease H) |
| CN102212615A (en) * | 2011-04-01 | 2011-10-12 | 上海交通大学 | Method for detecting single-nucleotide polymorphism based on high-temperature resistant RNase HII |
Non-Patent Citations (2)
| Title |
|---|
| JINGLI HOU等: "A Method for HLA Genotyping Using the Specific Cleavage of DNA-rN1-DNA/DNA with RNase HII from Chlamydia pneumoniae", 《OLIGONUCLEOTIDES》 * |
| XI-PENG LIU等: "A novel single nucleotide polymorphism detection of a double-stranded DNA target by a ribonucleotide-carrying molecular beacon and thermostable RNase HII", 《ANALYTICAL BIOCHEMISTRY》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755402A (en) * | 2016-12-22 | 2017-05-31 | 远见生物科技（上海）有限公司 | A kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII |
| CN107385017A (en) * | 2017-06-14 | 2017-11-24 | 苏州承美生物科技有限公司 | A kind of molecular beacon probe and kit for detecting circulating tumor cell |
| CN107937486A (en) * | 2017-12-25 | 2018-04-20 | 苏州旷世骏弛生物科技有限公司 | A kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII |
| CN113201593A (en) * | 2021-05-25 | 2021-08-03 | 广州普世君安生物科技有限公司 | Primer probe combination product for detecting ACTN3 genotype |
| CN113201593B (en) * | 2021-05-25 | 2024-01-23 | 广州盛世中豪运动科技有限公司 | Primer probe combination product for detecting ACTN3 genotype |
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Application publication date: 20140212 |