CN108707649A - A kind of method of hyperbranched one kettle way rolling circle amplification detection single nucleotide polymorphism - Google Patents
A kind of method of hyperbranched one kettle way rolling circle amplification detection single nucleotide polymorphism Download PDFInfo
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- CN108707649A CN108707649A CN201810792602.3A CN201810792602A CN108707649A CN 108707649 A CN108707649 A CN 108707649A CN 201810792602 A CN201810792602 A CN 201810792602A CN 108707649 A CN108707649 A CN 108707649A
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- rolling circle
- circle amplification
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- single nucleotide
- nucleotide polymorphism
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The invention discloses a kind of methods that hyperbranched one kettle way rolling circle amplification detects single nucleotide polymorphism.In conjunction with the reaction of hyperbranched one kettle way rolling circle amplification and unmarked fluoroscopic examination, a kind of high sensitivity and specific good method for detecting single nucleotide polymorphism are established.The branched rolling circle amplification reaction of connection reaction and the guiding of strong target dna of specificity is utilized in method, the second primer is introduced on the basis of one kettle way, it can effectively be expanded and be produced the DNA product of different length by the hyperbranched rolling circle amplification reaction of one kettle way, highly sensitive SYBR Green I dyestuffs are then added and carry out fluorescence measurement, the sensitivity of one kettle way rolling circle amplification is improved, method can be used for the Quantitative detection of single nucleotide polymorphism.
Description
Technical field
The present invention relates to a kind of methods of hyperbranched one kettle way rolling circle amplification single nucleotide polymorphism, belong to molecular biology
And field of nucleic acid chemistry.
Background technology
Single nucleotide polymorphism (SNPs) is most common variation type in human genome, they are various medical genetics
It learns research and provides strong tool.Single nucleotide polymorphism has following features：（1）It is large number of, it is widely distributed to account for about
The 1 ‰ of human genome nucleotide, sum reach 3,000,000 it is even more, it is widely more than first two genetic marker.
（2）Highly stable, the SNPs for being especially in code area is highly stable.（3）It is general suitable for fast and automatically changing analysis
SNPs is the label of two condition, need not analyze the length of segment, this is conducive to the automated analysis of SNPs and detection.（4）It is suitable
In Genotyping, the frequency of its allele is can be evaluated whether in anyone group.Therefore, develop high selectivity, sensitivity is good
The method of detection SNPs has great significance to the gene diagnosis of genetic disease and study of pathogenesis.
Hyperbranched rolling circle amplification is that introducing Article 2 is identical with padlock probe sequence anti-in linear rolling circle amplification system
It is complementary with rolling circle amplification product and carry out DNA chain that is oppositely extending and replacing downstream generation to primer i.e. the second primer, to
The double-stranded DNA and single stranded DNA of different length are formd, this cycle carries out successively, the speed ratio of hyperbranched rolling circle amplification reaction
Linear amplification is many soon, may be implemented 109Exponential amplification again, can further increase the sensitivity of detection.
The method that one kettle way rolling circle amplification combination fluorescence electrophoresis detects SNPs, although realizing the highly selective of detection SNPs
It measures, but sensitivity need to be improved.For the low problem of one kettle way detection sensitivity, establishes hyperbranched one kettle way rolling ring and expand
Increasing technology.The second primer is added on the basis of one kettle way rolling circle amplification（2nd primer）, the amplification of rolling circle amplification in one pot
Product is that template is reversely expanded, and highly sensitive SYBR Green I dyestuffs are added and carry out fluorescence measurement, establish hyperbranched
One kettle way rolling circle amplification.Hyperbranched amplification technique improves the sensitivity of one kettle way rolling circle amplification, and this method is not only simple
Fast, and high sensitivity, detection limit is low, successfully detects 1% frequency of mutation.
Invention content
The object of the present invention is to provide a kind of methods that hyperbranched one kettle way rolling circle amplification detects single nucleotide polymorphism.
The specific steps are：
（1）By the padlock probe of a concentration of 1 μm of ol/L of 1 μ L, the dNTPs of 1 a concentration of 10mmol/L of μ L, a concentration of 4 μ of 1 μ L
The A premixs of the 10 μ L of distilled water (DEPC water) mixing composition of the pyrocarbonic acid diethyl ester of the 2nd primer of mol/L and 7 μ L processing
Liquid, then 2 μ L 10 × Taq DNA ligase buffer solutions, 2 μ L 10 × phi29 DNA polymerase buffers, 1 μ L is dense
Degree is the phi29 of the target sequence to be measured of 10 nmol/L, the Taq DNA ligases of 0.5 μ L 40U/ μ L, 0.5 μ L 10U/ μ L
The B premixed liquids of 10 μ L of archaeal dna polymerase and 4 μ L DEPC water mixing composition, then mix A premixed liquids and B premixed liquids equal
It is even, 70 mmol/L Tris-HCl (pH 7.6), 25 mmol/L KAc, 10 mmol/L are contained in mixed 20 μ L solution
Mg(Ac)2, 1 mmol/L nicotinamide adenine dinucleotide (NAD), 10 mmol/L MgCl2, 10 mmol/L (NH4)2SO4, 14 mmol/L dithiothreitol (DTT)s (DTT), the Triton X-100,500 μm of ol/L that mass percent concentration is 0.2%
DNTPs, 0.2 μm of ol/L2nd primer, 20 U Taq DNA ligases, 5 U phi29 archaeal dna polymerases and 0.5
Nmol/L target dnas, the above operation are completed on ice, are subsequently placed at 30 DEG C and are reacted 6 hours, make within 10 minutes at 65 DEG C
Enzyme is inactivated to terminate amplified reaction.
（2）SNP calls measure：Take step（1）In 5 μ L of amplified production, 16 × Loading of μ L
I working solution of Buffer and 0.6 μ L Syber Green（100×）, 5 minutes are placed at room temperature for, point sample is to mass percent after encapsulating
In the Ago-Gel of concentration 1%, with 1 × tbe buffer liquid when running glue（0.04 mol/L Tris- boric acid, 0.001 mol/L
EDTA, pH=8.0）, glue is run under 100V voltages 40 minutes, be imaged under gel imager, preserve picture.
（3）Single nucleotide polymorphism fluoroscopic examination：Take step（1）In 2 μ L of amplified production, be added 2 μ L Syber
Green I (20 ×) solution, with 10 mmol/L phosphate buffer solutions（pH7.4）Solution is diluted to 100 μ L, keeps at room temperature
10 minutes, measured with the microcolorimetric ware of 350 μ L, selective exitation wavelength is 495 nm when fluorescence measurement, spectrum recording interval from
505 nm to 700 nm measure 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 rolling circle amplification, which closes three, simple and convenient.By the target sequence of amplification needs, padlock probe, ligase, polymerization
The raw materials such as enzyme and dNTPs mix, and realize step amplification with reaction under buffer system at a certain temperature.Hyperbranched amplification technique carries
The high sensitivity of one kettle way rolling circle amplification, this method is not only simple fast, and high sensitivity, and detection limit is low.
Description of the drawings
Fig. 1 is the schematic diagram that the hyperbranched one kettle way rolling circle amplification of the present invention detects single nucleotide polymorphism.
Fig. 2 is the sequence chart in DNA mismatch site and the second primer.
Fig. 3 is the agarose gel electrophoresis figure in the embodiment of the present invention.
Fig. 4 is that one kettle way and hyperbranched one kettle way rolling circle amplification detect the glimmering of single nucleotide polymorphism in the embodiment of the present invention
Light spectrum.
Fig. 5 is single nucleotide polymorphism frequency of mutation detection figure in the embodiment of the present invention.
The fluorescence light for the hyperbranched rolling circle amplification reaction of one kettle way that Fig. 6 is various concentration DNA-7a in the embodiment of the present invention
Spectrum.
Fig. 7 is a concentration of 5 pmol/L-200 pmol/L linear relationship curves of DNA-7a in the embodiment of the present invention.
Specific implementation mode
It elaborates to the present invention below with reference to Figure of description and specific embodiment.
Embodiment：
（1）By the padlock probe of a concentration of 1 μm of ol/L of 1 μ L, the dNTPs of 1 a concentration of 10mmol/L of μ L, a concentration of 4 μ of 1 μ L
The A premixs of the 10 μ L of distilled water (DEPC water) mixing composition of the pyrocarbonic acid diethyl ester of the 2nd primer of mol/L and 7 μ L processing
Liquid, then 2 μ L 10 × Taq DNA ligase buffer solutions, 2 μ L 10 × phi29 DNA polymerase buffers, 1 μ L is dense
Degree is the phi29 of the target sequence to be measured of 10 nmol/L, the Taq DNA ligases of 0.5 μ L 40U/ μ L, 0.5 μ L 10U/ μ L
The B premixed liquids of 10 μ L of archaeal dna polymerase and 4 μ L DEPC water mixing composition, then mix A premixed liquids and B premixed liquids equal
It is even, it is blown and beaten with liquid-transfering gun uniform, contains 70 mmol/L Tris-HCl (pH 7.6) in mixed 20 μ L solution, 25
Mmol/L KAc, 10 mmol/L Mg (Ac)2, 1 mmol/L nicotinamide adenine dinucleotide (NAD), 10 mmol/L
MgCl2, 10 mmol/L (NH4)2SO4, 14 mmol/L dithiothreitol (DTT)s (DTT), mass percent concentration is 0.2%
Triton X-100, the dNTPs of 500 μm of ol/L, 0.2 μm of ol/L 2nd primer, 20 U Taq DNA ligases, 5 U
Phi29 archaeal dna polymerases and 0.5 nmol/L target nucleic acids, the above operation carries out in ice face, is subsequently placed at 30 DEG C anti-
It answers 6 hours, makes within 10 minutes at 65 DEG C enzyme inactivation to terminate amplified reaction.
（2）The detection of single nucleotide polymorphism fluorescence：
It takes 2 μ L of amplified production in step 1,2 μ L Syber Green, I (20 ×) solution is added, with 10 mmol/L phosphoric acid
Salt buffer solution（pH7.4）Solution is diluted to 100 μ L, is kept for 10 minutes at room temperature, is measured with the microcolorimetric ware of 350 μ L,
Selective exitation wavelength is 495 nm when fluorescence measurement, and spectrum recording interval measures it from 505 nm to 700 nm at 534 nm
Fluorescence signal intensity.
（3）The parting of single nucleotide polymorphism measures：
It takes 5 μ L of amplified production in step 1,1 μ 6 × Loading of L Buffer and 0.6 μ L Syber Green I is added
Working solution（100×）, 5 minutes are placed at room temperature for, in the Ago-Gel that point sample is 1% to mass percent concentration after encapsulating, runs glue
When with 1 × tbe buffer liquid（0.04 mol/L Tris- boric acid, 0.001 mol/L EDTA, pH=8.0）, run under 100V voltages
Glue 40 minutes, is imaged under gel imager, preserves picture.
Pass through the principle map analysis of the hyperbranched one kettle way rolling circle amplification reaction detection single nucleotide polymorphism of application, such as Fig. 1
Shown, it is one that the reaction of conventional rolling circle amplification is closed three by one kettle way, and connection reaction and amplified reaction are carried out at the same time, padlock probe and mesh
DNA-7a complete complementaries are marked, connect cyclization by template of DNA-7a under the action of Taq DNA ligases, while DNA-7a makees again
Cause rolling circle amplification reaction under the action of phi29 archaeal dna polymerases using the padlock probe of cyclisation as template for primer；In addition,
The second primer being added can cause the repetitive sequence products thereof of the rolling circle amplification product of connecting generated simultaneously with target dna -7a
It carries out replacing reversed amplification, the DNA sequence dna that displacement downstream generates causes hyperbranched amplified reaction, produces during the reaction
The double-stranded DNA and single-stranded DNA product of different length are added I dyestuffs of Syber Green and carry out fluorescence measurement, lead to fluorescence signal
It significantly increases.Comparing, there are two the bases with padlock probe mispairing by DNA-7d, cannot be connected enzyme connection cyclization, line only occurs
Property amplification, produce very weak fluorescence background after Syber Green I are added.Branched rolling circle amplification is introduced, further
Improve the sensitivity of the hyperbranched rolling circle amplification reaction of one kettle way.
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.
As seen from Figure 3, long chain DNA productions of the target sequence DNA-7a after the hyperbranched rolling circle amplification reaction of one kettle way
Object, because its product cannot enter gel very much by electrophoresis greatly, compares in swimming lane 2, and limited amplification only occurs for DNA-7d,
The amplified production of generation is less, so without display in swimming lane 1 does not have.The result shows that target sequence DNA-7a is overspend by one kettle way
Change rolling circle amplification reaction and produces special, efficient amplification.
Single nucleotide polymorphism is detected by comparing one kettle way and hyperbranched one kettle way rolling circle amplification in the embodiment of the present invention
Fluorescence spectrum, as shown in figure 4, DNA-7a products（2）Fluorescence intensity be apparently higher than the intensity of DNA-7d（1）, fluorescence ratio
About 4.8：1, the two is distinguishing the reason is that DNA-7a initiations are rolling circle amplifications, and what DNA-7d caused is limited amplification,
So the two amplification efficiency is different；When the branched amplification of generation of the second primer is added, the fluorescence signal of DNA-7a significantly increases, table
The fluorescence intensity that bright branched amplified reaction generates single double-stranded products is significantly more than linear amplification；DNA-7d only has occurred by comparison
Reversed amplification cannot occur with the second primer for limited amplification, and fluorescence intensity change is smaller after the second primer is added, DNA-7a
It is 5.4 to carry out the fluorescence signal of hyperbranched rolling circle amplification reaction and DNA-7d reaction product fluorescence signal ratios：1, as a result illustrate
The sensitive of detection single nucleotide polymorphism can be improved by introducing the hyperbranched rolling circle amplification reaction of the one kettle way generated after the second primer
Degree and selectivity.
By comparing single nucleotide polymorphism frequency of mutation detection figure in the embodiment of the present invention, as shown in figure 5, we will
DNA-7a and 7d is mixed according to different ratios is used as DNA sample, and the total amount of DNA-7a and 7d is 300 pmol/L in sample, when
When the amount of substance concentration of DNA-7a accounts for 1%, 5%, 10%, 50%, the 100% of total amount, fluorescence intensity and mutation of the sample at 534 nm
Good linear relationship is presented in frequency, and linear equation is the C+57.15 of I=22.95, and linearly dependent coefficient is r=0.9968, as a result
Showing this method can succeed the frequency of mutation of single nucleotide polymorphism in determination sample.
The fluorescence of the hyperbranched rolling circle amplification reaction of one kettle way by comparing various concentration DNA-7a in the embodiment of the present invention
Spectrum, as shown in fig. 6, within the scope of 5 pmol/L-200 pmol/L, the fluorescence signal of DNA-7a with the increase of concentration and
It gradually increases, the logarithm of fluorescence signal and concentration shows good linear relationship.
By comparing a concentration of 5 pmol/L-200 pmol/L linear relationship curves of DNA-7a in the embodiment of the present invention, such as
Shown in Fig. 7, linear equation is the lgC+144.1 of I=124.9, and linearly dependent coefficient is r=0.9955, and detection is limited to 2.9
pmol/L（3 times of signal-to-noise ratio）, the precision of parallel laboratory test evaluation method is carried out, the DNA- of 25 pmol/L and 200 pmol/L are selected
7a carries out 6 repetitions and tests, and relative standard deviation is respectively 3.73% and 4.51%.
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 various process programs of the present inventive concept without substantial differences.
Claims (2)
1. a kind of method of hyperbranched one kettle way rolling circle amplification detection single nucleotide polymorphism, it is characterised in that the specific steps are：
（1）By the padlock probe of a concentration of 1 μm of ol/L of 1 μ L, four kinds of dezyribonucleosides three of 1 a concentration of 10mmol/L of μ L
The coke acid diethyl of the second primer, that is, 2nd primer and 7 μ L of a concentration of 4 μm of ol/L of mixture, that is, dNTPs of phosphoric acid, 1 μ L
The A premixed liquids of the distilled water of ester processing, that is, 10 μ L of DEPC water mixing composition, then by 2 μ 10 × Taq of L DNA connection enzyme buffers
Liquid, 2 μ L 10 × phi29 DNA polymerase buffers, the target sequence to be measured of a concentration of 10 nmol/L of 1 μ L, 0.5 μ L
The Taq DNA ligases of 40U/ μ L, the phi29 archaeal dna polymerases of 0.5 μ L 10U/ μ L and 4 μ L DEPC water mixing composition
A premixed liquids and B premixed liquids, are then uniformly mixed by the B premixed liquids of 10 μ L, are blown and beaten uniformly with liquid-transfering gun, the above operation exists
It is carried out in ice face, is subsequently placed at 30 DEG C and reacts 6 hours, make within 10 minutes at 65 DEG C enzyme inactivation to terminate amplified reaction；
（2）The parting of single nucleotide polymorphism measures：Take step（1）In 5 μ L of amplified production, 16 × Loading of μ L
I working solution of Buffer and 0.6 μ 100 × Syber of L Green, is placed at room temperature for 5 minutes, point sample is dense to mass percent after encapsulating
With 1 × tbe buffer liquid, 0.04 mol/L Tris- boric acid, 0.001 mol/L when degree is in 1% Ago-Gel, runs glue
EDTA, the solution of pH=8.0 run glue 40 minutes under 100V voltages, are imaged under gel imager, preserve picture；
（3）Single nucleotide polymorphism fluoroscopic examination：Take step（1）In 2 μ L of amplified production, 2 μ 20 × Syber of L Green I
Solution is diluted to 100 μ L with 10 mmol/L phosphate buffer solution solution of pH7.4, is kept for 10-15 minutes at room temperature, uses
The microcolorimetric ware of 350 μ L measures, and selective exitation wavelength is 495 nm when fluorescence measurement, spectrum recording interval from 505 nm to
700 nm measure its fluorescence signal intensity at 534 nm.
2. the method for hyperbranched one kettle way rolling circle amplification detection single nucleotide polymorphism as described in claim 1, feature exist
In the step（1）Contain pH 7.6 70 mmol/L Tris-HCl, 25 mmol/L KAc in mixed 20 μ L solution,
10 mmol/L Mg(Ac)2, 1 mmol/L nicotinamide adenine dinucleotide, that is, NAD, 10 mmol/L MgCl2, 10 mmol/
L (NH4)2SO4, 14 mmol/L dithiothreitol (DTT)s, that is, DTT, the Triton X-100 that mass percent concentration is 0.2%, 500
The dNTPs of μm ol/L, 0.2 μm of ol/L 2nd primer, 20 U Taq DNA ligases, 5 U phi29 archaeal dna polymerases and
0.5 nmol/L target nucleic acids.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463108A (en) * | 2016-01-08 | 2016-04-06 | 华侨大学 | SNP (Single nucleotide polymorphism) typing detection method by utilizing padlock probe rolling circle amplification |
| 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 |
-
2018
- 2018-07-18 CN CN201810792602.3A patent/CN108707649A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463108A (en) * | 2016-01-08 | 2016-04-06 | 华侨大学 | SNP (Single nucleotide polymorphism) typing detection method by utilizing padlock probe rolling circle amplification |
| 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 |
|---|
| 周国华 主编: "《SNP检测技术与个体化药物治疗》", 28 February 2015 * |
| 肖乐义 等: ""滚环DNA扩增的原理、应用和展望"", 《中国生物工程杂志》 * |
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Application publication date: 20181026 |