CN109022554A - A kind of method of one kettle way rolling circle amplification detection single nucleotide polymorphism - Google Patents
A kind of method of one kettle way rolling circle amplification detection single nucleotide polymorphism Download PDFInfo
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Abstract
The invention discloses a kind of methods of one kettle way rolling circle amplification detection single nucleotide polymorphism.It is one that the reaction of conventional rolling circle amplification is closed three by one kettle way rolling circle amplification, the raw materials such as padlock probe, the dNTPs that amplification is needed constitute solution A, required buffer, ligase, polymerase and target sequence mixing constitute B solution, solution A and B solution are mixed, the time required for being expanded at a certain temperature with reaction under certain buffer system.The present invention establishes fast and convenient one kettle way rolling circle amplification new method, provides a kind of method of simple and quick, high sensitivity single nucleotide polymorphism, has the characteristics that easy to operate, analysis time is short, specific height, is more suitable in situ detection.
Description
Technical field
The present invention relates to a kind of methods of one kettle way rolling circle amplification detection single nucleotide polymorphism, belong to molecular biosciences
And field of nucleic acid chemistry.
Background technique
Rolling circle amplification (RCA) is a kind of constant temperature nucleic acid amplification method set up for 1998, and nature is simulated in its proposition
The rolling-circle replication process of boundary's microorganism cyclic DNA, and researcher has found that this DNA replication dna mode is present in many viruses
In.The appearance of the technique of gene detection and padlock-probe partial dna detection technique that are mediated with ligase, rolling circle amplification
Development speed improves rapidly.RCA technology is at a constant temperature using single-stranded cyclic DNA as the linear single stranded amplification of template progress, in generation
DNA made of thousand cyclic DNA copy linkings is single-stranded, does not need specific thermal cycler, and have high sensitivity, specificity
Good, feature easy to operate.However, there is also some shortcomings for RCA technology: 1. reaction step is more, takes a long time.2. selecting
Lower, the poor compatibility of property.3. being difficult to realize in situ detection.Therefore, RCA method that is simple, quick, being suitble in situ detection is established
With highly important theory significance and practical value.
One kettle way (one-pot) reaction is prepared in nanoparticle, and organic synthesis field has been widely used, in biochemical analysis
Also applied etc. many fields it is more and more, for example, the selective colorimetric multicore glycosides based on Au nanoparticle probes such as Storhoff
Acid detection realizes the one kettle way detection of target in the presence of four single base defects, but one kettle way is anti-applied to RCA amplification
Answer that there is not been reported.
For the deficiency of conventional RCA technology and the concept of one kettle way, one kettle way rolling circle amplification detection monokaryon glycosides is established in proposition
The method of sour polymorphism, conventional RCA need the operation of three steps, cumbersome time-consuming；It is one that one kettle way RCA, which closes three, simple and convenient.This side
The foundation of method helps to improve miRNA theory and detection technique, expands biomedical and clinical detection, and grind in biology and medicine
Study carefully field to have great importance.
Summary of the invention
The object of the present invention is to provide a kind of methods of one kettle way rolling circle amplification detection single nucleotide polymorphism.
Specific steps are as follows:
(1) one kettle way rolling circle amplification: by 1 μ L concentration be the padlock probe of 1 μm of ol/L, 1 μ L concentration is the four of 10 mmol/L
The distilled water (DEPC water) of the pyrocarbonic acid diethyl ester processing of the mixture (dNTPs) and 8 μ L of kind deoxyribonucleoside triphosphate is mixed
It is combined into 10 μ L A premixed liquids, then by 2 μ L 10 × Taq DNA ligase Buffer, 2 μ L10 × Bst DNA polymerases
Buffer, 1 μ L concentration are the target sequence to be measured of 10 nmol/L, the Taq DNA ligase of 0.5 μ L 40U/ μ L, 0.5 μ L 8U/ μ L
Bst archaeal dna polymerase and 4 μ L DEPC water mixing 10 μ L B premixed liquids of composition, then A premixed liquid and B premixed liquid are mixed
Uniformly, with liquid-transfering gun blow and beat uniformly, in mixed 20 μ L solution contain 40 mmol/L Tris-HCl (pH7.6), 25
Mmol/L KAc, 10 mmol/L Mg (Ac)2, 10 mmol/L dithiothreitol (DTT)s (DTT), 1 mmol/L nicotinamide adenine two
Nucleotide (NAD), 10 mmol/L KCl, 10 mmol/L (NH4)2SO4, 2 mmol/L MgSO4, mass percent concentration is
0.2% Triton X-100,20U Taq DNA ligase, the Bst archaeal dna polymerase 100nmol/L padlock probe of 4U and 500 μ
The dNTPs of mol/L, 0.5 nmol/L target nucleic acid;The above operation carries out in ice face, then small as reaction 6 at 55 DEG C
When, inactivate enzyme to terminate amplified reaction within 20 minutes at 80 DEG C.
(2) SNP calls measure: taking 5 μ L of amplified production, the 16 × Loading of μ L in step (1)
I working solution (100 ×) of Buffer and 0.6 μ L Syber Green, is placed at room temperature for 5 minutes, and point sample is to mass percent after encapsulating
Concentration be 1% Ago-Gel in, run glue when with Tris boric acid (TBE) buffer (0.04 mol/L Tris- boric acid, 0.001
Mol/L EDTA, pH=8.0), it runs glue 40 minutes under 100V voltage, is imaged under gel imager, save picture.
(3) 2 μ L of amplified production, the 2 μ L Syber Green I in step (1) single nucleotide polymorphism fluorescence detection: are taken
(20 ×) solution is diluted to 100 μ L with 10 mmol/L phosphate buffer solution (pH7.4) solution, keeps 10-15 points at room temperature
Clock is measured with the microcolorimetric ware of 350 μ L, and selective exitation wavelength is 495 nm when fluorescence measurement, and spectrum recording interval is from 505
Nm to 700 nm measures its fluorescence signal intensity at 534 nm.
The advantages of the method for the present invention, is as follows:
It is one that one kettle way RCA, which closes three, simple and convenient.Will amplification need target sequence, padlock probe, ligase, polymerase and
The mixing of the raw materials such as dNTP realizes step amplification with reaction under buffer system at a certain temperature.Pass through test various combination enzyme
Activity, the selective difference of more different enzymes establish fast and convenient one kettle way RCA new method；It is examined in conjunction with sensitive SG fluorescence
The modes such as survey, strand replacement reaction further increase the amplification efficiency of one kettle way RCA.This method has the characteristics of easy operation, contracting
Short analysis time improves the selectivity of padlock probe, and is suitable for augmentation detection in situ.
Detailed description of the invention
Fig. 1 is the schematic diagram of one kettle way Rolling Circle Amplification methods of the present invention.
Fig. 2 is DNA mismatch site of the present invention figure.
Fig. 3 is Taq of embodiment of the present invention DNA ligase and Bst DNA polymerase method of fractional steps electrophoretogram.
Fig. 4 is the fluorescent value figure of Taq of embodiment of the present invention DNA ligase and Bst DNA polymerase method of fractional steps product.
Fig. 5 is the electrophoretogram of SNP calls detection in the embodiment of the present invention one.
Fig. 6 is the fluorescent value figure of product in the embodiment of the present invention one.
Specific embodiment
It elaborates below with reference to Figure of description and specific embodiment to the present invention.
Embodiment:
(1) be by 1 μ L concentration the padlock probe of 1 μm of ol/L, the dNTP that 1 μ L concentration is 10 mmol/L and 8 μ L coke acid diethyl
Ester processing distilled water (DEPC water) mixing composition 10 μ L A premixed liquids, then by 2 μ L 10 × Taq DNA ligase Buffer,
2 μ L10 × Bst DNA polymerase Buffer, the Taq that 1 μ L concentration is the target sequence to be measured of 10 nmol/L, 0.5 μ L 40U/ μ L
DNA ligase, the Bst archaeal dna polymerase of 0.5 μ L 8U/ μ L and 4 μ L DEPC water mixing 10 μ L B premixed liquids of composition, then will
A premixed liquid and B premixed liquid are uniformly mixed, and are blown and beaten uniformly with liquid-transfering gun, 40 mmol/L are contained in mixed 20 μ L solution
Tris-HCl (pH7.6), 25 mmol/L KAc, 10 mmol/L Mg (Ac)2, 10 mmol/L dithiothreitol (DTT)s (DTT), 1
Mmol/L nicotinamide adenine dinucleotide (NAD), 10 mmol/L KCl, 10 mmol/L (NH4)2SO4, 2 mmol/L
MgSO4, mass percent concentration is 0.2% Triton X-100,20U Taq DNA ligase, the Bst archaeal dna polymerase of 4U
The dNTP of 100 nmol/L padlock probes and 500 μm of ol/L, 0.5 nmol/L target nucleic acid, the above operation are enterprising in ice face
Row, then as reacting at 55 DEG C 6 hours, inactivates enzyme to terminate amplified reaction in 20 minutes at 80 DEG C.
(2) detection of single nucleotide polymorphism fluorescence:
2 μ L of amplified production, 2 μ L Syber Green, I (20 ×) solution in step (1) are taken, with 10 mmol/L phosphate-buffereds
Solution (pH7.4) solution is diluted to 100 μ L, is kept for 10-15 minutes at room temperature, is measured with the microcolorimetric ware of 350 μ L, fluorescence is surveyed
Selective exitation wavelength is 495 nm when amount, and spectrum recording interval measures its fluorescence letter from 505 nm to 700 nm at 534 nm
Number intensity.
(3) SNP calls measure:
Take 5 μ L of amplified production, 1 μ 6 × Loading of the L Buffer and 0.6 μ L Syber Green, I working solution in step (1)
(100 ×) are placed at room temperature for 5 minutes, in the Ago-Gel that point sample is 1% to mass concentration after encapsulating, run slow with 1 × TBE when glue
Fliud flushing (0.04 mol/L Tris- boric acid, 0.001 mol/L EDTA, pH=8.0), runs glue 40 minutes under 100V voltage,
It is imaged under gel imager, saves picture.
As shown in Figure 1A, conventional RCA reaction is generally divided into three steps: target sequence and padlock probe hybridization, ligase connect
It connects padlock probe cyclization and polymerase is expanded.Specific steps are as follows: first by target sequence and padlock probe thermal denaturation, then
5 ' ends of shown probe and 3 ' terminal sequences hybridize with the both ends of target sequence respectively, form one between 5 ' ends and 3 ' ends at this time and lack
Mouthful, the DNA ligase of addition, which repairs this notch and forms phosphodiester bond, makes padlock-probe connection cyclization, only in target sequence and
It can just work in the case where padlock-probe complete complementary；Then, target sequence is as primer, ring-shaped probe as template,
Start RCA reaction under the action of polymerase with high strand-displacement activity, forming one has thousands of a repetitive sequences
Single stranded DNA.
One kettle way RCA principle as shown in Figure 1B is similar with routine RCA principle, and difference is that one kettle way reacts conventional RCA
Closing three is one, and connection reaction and amplified reaction are simultaneously or timesharing carries out, the raw materials structure such as padlock probe, dNTPs that amplification is needed
At solution A, required buffer, ligase, polymerase and target sequence mixing constitute B solution, and solution A and B solution are mixed,
Time required for being expanded at a certain temperature with reaction under certain buffer system.
Fig. 2 is DNA mismatch site figure, and other than the number of base complete complementary of DNA-7a and padlock probe, mispairing is all
It cannot complete complementary therewith.
Fig. 3 is Taq DNA ligase and Bst DNA polymerase method of fractional steps electrophoretogram, as seen from Figure 3, method of fractional steps 7a
Amplified production is shown with all mispairing (including 7g).
As seen from Figure 4, method of fractional steps mispairing and complete complementary fluorescence intensity it is very close, illustration method selectivity
It is very poor.
As seen from Figure 5, one kettle way 7a massive amplification, only 7b, 7d are expanded on a small quantity.
As shown in fig. 6, the fluorescence intensity of 7a is 4.2 times of 7b, and 10.9 times of 7c, 11.8 times of 7d, the choosing of illustration method
Selecting property is fine.
The above is only the preferred embodiments of the invention, protection scope of the present invention is not limited merely to above-described embodiment,
It is within the scope of the invention with present inventive concept without the various process programs of substantial differences.
Claims (2)
1. a kind of method of one kettle way rolling circle amplification detection single nucleotide polymorphism, it is characterised in that specific steps are as follows:
(1) one kettle way rolling circle amplification: by 1 μ L concentration is the padlock probe of 1 μm of ol/L, 1 μ L concentration is 10 mmol/L four kinds it is de-
Distilled water, that is, DEPC water mixing group of the pyrocarbonic acid diethyl ester of mixture, that is, dNTPs of oxygen ribonucleotide triphosphate and 8 μ L processing
At 10 μ L A premixed liquids, then by 2 μ L 10 × Taq DNA ligase Buffer, 2 μ L10 × Bst DNA polymerases
Buffer, 1 μ L concentration are the target sequence to be measured of 10 nmol/L, the Taq DNA ligase of 0.5 μ L 40U/ μ L, 0.5 μ L 8U/ μ L
Bst archaeal dna polymerase and 4 μ L DEPC water mixing 10 μ L B premixed liquids of composition, then A premixed liquid and B premixed liquid are mixed
Uniformly, it is blown and beaten uniformly with liquid-transfering gun, the above operation carries out in ice face, then reacts 6 hours as at 55 DEG C, at 80 DEG C
Inactivate enzyme within 20 minutes to terminate amplified reaction；
(2) SNP calls measure: taking 5 μ L of amplified production, 1 μ 6 × Loading of the L Buffer in step (1)
With 0.6 μ 100 × Syber of L Green, I working solution, 5 minutes are placed at room temperature for, the agar that point sample is 1% to mass concentration after encapsulating
In sugared gel, with 1 × tbe buffer liquid, i.e. 0.04 mol/L Tris- boric acid when running glue, 0.001 mol/L EDTA, pH=8.0,
It runs glue 40 minutes under 100V voltage, is imaged under gel imager, save picture；
(3) 2 μ L of amplified production, 2 μ 20 × Syber of the L Green I in step (1) single nucleotide polymorphism fluorescence detection: are taken
Solution is diluted to 100 μ L with 10 mmol/L phosphate buffer solution pH7.4 solution, is kept for 10-15 minutes at room temperature, with 350
The microcolorimetric ware of μ L measures, and selective exitation wavelength is 495 nm when fluorescence measurement, and spectrum recording interval is from 505 nm to 700
Nm measures its fluorescence signal intensity at 534 nm.
2. the method for one kettle way rolling circle amplification detection single nucleotide polymorphism as described in claim 1, it is characterised in that
Containing 40 mmol/L Tris-HCl of pH7.6 in the mixed 20 μ L solution of step (1), 25 mmol/L KAc, 10
mmol/L Mg(Ac)2, 10 mmol/L dithiothreitol (DTT)s, that is, DTT, 1 mmol/L nicotinamide adenine dinucleotide, that is, NAD, 10
Mmol/L KCl, 10 mmol/L (NH4)2SO4, 2 mmol/L MgSO4, mass percent concentration is 0.2% Triton X-
100,20U Taq DNA ligases, the dNTP of Bst archaeal dna polymerase 100 the nmol/L padlock probe and 500 μm of ol/L of 4U,
0.5 nmol/L target nucleic acid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112110968A (en) * | 2020-03-19 | 2020-12-22 | 北京师范大学 | Aggregation-induced fluorescence molecule modified nucleotide and application thereof in DNA sequencing and SNPs detection |
Citations (2)
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| CN102827929A (en) * | 2012-08-01 | 2012-12-19 | 浙江大学 | Method for detecting nucleic acid |
| CN106399542A (en) * | 2016-10-26 | 2017-02-15 | 沈阳优宁生物科技有限公司 | Rolling circle constant-temperature amplification quick detection primer and kit for mycoplasma pneumoniae |
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- 2018-07-18 CN CN201810793176.5A patent/CN109022554A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827929A (en) * | 2012-08-01 | 2012-12-19 | 浙江大学 | Method for detecting nucleic acid |
| CN106399542A (en) * | 2016-10-26 | 2017-02-15 | 沈阳优宁生物科技有限公司 | Rolling circle constant-temperature amplification quick detection primer and kit for mycoplasma pneumoniae |
Non-Patent Citations (2)
| Title |
|---|
| HEIKO KUHN ET AL.: "Rolling-circle amplification under topological constraints", 《NUCLEIC ACIDS RESEARCH》 * |
| 周国华: "《SNP检测技术与个体化药物治疗》", 28 February 2015, 苏州大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112110968A (en) * | 2020-03-19 | 2020-12-22 | 北京师范大学 | Aggregation-induced fluorescence molecule modified nucleotide and application thereof in DNA sequencing and SNPs detection |
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Application publication date: 20181218 |