CN103773757A - Method for optimizing polymerase chain reaction (PCR) by using graphene quantum dots - Google Patents
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Abstract
The invention discloses a method for optimizing a polymerase chain reaction (PCR) by using graphene quantum dots. The method comprises the following steps: (1) preparing an aqueous solution of the graphene quantum dots; (2) uniformly mixing the aqueous solution of the graphene quantum dots and a PCR amplification reaction system according to an optimizing ratio; (3) carrying out DNA (Deoxyribonucleic Acid) amplification according to procedures by a PCR instrument. The method has the advantages that through optimizing the PCR by the graphene quantum dots, the comprehensive optimization effect is better than that of the other existing nano-materials, so that the sensitivity of a PCR technology can be effectively improved, the yield of amplification products is increased, and the specificity of the amplification products is improved; moreover, the dosage of the graphene quantum dots required for optimization is low; in addition, the graphene quantum dots are simple in preparation step and convenient in storage.
Description
Technical field
The present invention relates to biological technical field, specifically a kind of method of utilizing graphene quantum dot aggregation polymerase chain reaction.
Background technology
Graphene quantum dot (GQDs) is a kind of novel carbon nanomaterial, has the two-dirnentional structure of monatomic thickness, and its size distribution is less than 100 nm.Because its unique structure and chemistry, physical properties, graphene quantum dot is widely used in biological medicine every field.For example: research finds that graphene quantum dot can be used as a kind of core target medicine carrier (Enhancing Cell Nucleus Accumulation and DNA Cleavage Activity of Anti-Cancer Drug via Graphene Quantum Dots. Scientific Reports, 2013,3,2852), graphene quantum dot also can with the DNA molecular effect of ad hoc structure, be expected to expression (the Stabilization and Induction of Oligonucleotide i-Motif Structure via Graphene Quantum Dots. ACS Nano of regulatory gene, 2013, 7, 531-537), graphene quantum dot can modifying DNA structure, (the Photo-Fenton Reaction of Graphene Oxide:A New Strategy to Prepare Graphene Quantum Dots for DNA Cleavage. ACS Nano that is expected to there is certain using value aspect cancer therapy drug, 2012, 6 (8), 6592).In addition, graphene quantum dot also can be used for detection (the Detection of the ovarian cancer biomarker CA-125 using chemiluminescence resonance energy transfer to graphene quantum dots of cancer markers, and cell imaging (Strongly green-photoluminescent graphene quantum dots for bioimaging applicationsw Chem. Commun. DOI:10.1039/c3cc47701k), 2011,47,6858 – 6860.).
Polymerase chain reaction (PCR), as a kind of DNA Amplification Technologies, since 1985 are invented, has been widely used in medical science and field of biology.But, being different from amplification in body, the not accurate regulation and control of PCR system and mismatch repair mechanism, cause pcr amplification efficiency not ideal enough sometimes.In recent years, there is the report of a large amount of optimization and improvement PCR method and technology.For example, utilize various nano materials to improve the performance of round pcr as toughener.As Jenner's grain of rice (Nanoparticle PCR:Nanogold-Assisted PCR with Enhanced Specificity. Angew. Chem. 2005,117,5230-5233); Some complex optimum DeGrain, as carbon nano powder (patent No.: CN101134975A), carbon nanotube (Effects of single-walled carbon nanotubes on the polymerase chain reaction. Nanotechnology, 2004,15,154-157) etc.But some of them nano material raw material ratio is more expensive, and cost is high, so there is certain defective.Recently the method for utilizing graphene oxide optimize PCR of report has overcome the deficiency of some of them optimization methods, the PCR complex optimum effect (patent No.: 01010546630) that is improved.For example, but still have some shortcomings, graphene oxide size is larger, so biocompatibility is poor, and in the method, graphene oxide usage quantity is larger.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing graphene quantum dot aggregation polymerase chain reaction.
Second object of the present invention is to provide the application of graphene quantum dot in aggregation polymerase chain reaction.
For realizing first object of the present invention, the present invention discloses following technical scheme: a kind of method of utilizing graphene quantum dot aggregation polymerase chain reaction, it is characterized in that, and said method comprising the steps of:
(1) prepare the graphene quantum dot aqueous solution;
(2) the graphene quantum dot aqueous solution is mixed by optimized proportion with pcr amplification reaction system;
(3) on PCR instrument, follow procedure carries out DNA cloning;
Optimized proportion described in step (2) refers to that the concentration of graphene quantum dot is to determine by the following method: graphene quantum dot is carried out joining in PCR system and reacting after gradient dilution, and the ratio of the scope that reaches optimization take actual expanding effect in system is the optimized proportion of graphene quantum dot.
As a preferred version, graphene quantum dot water solution preparation method is in step (1): take the synthetic graphite oxide aqueous solution of Hummers method as initiator, utilize Photo-Fenton reaction, with H
2o
2for oxygenant, Fe
3+for catalyzer, under ultraviolet radiation, be prepared from, product is dialysed in ultrapure water, remove unreacted H
2o
2small molecules with reaction produces, obtains the pure graphene quantum dot aqueous solution.
As a preferred version, what step (2) PCR increased comprises simple plasmid and complex genome, and described complex genome comprises calf thymus DNA.
As a preferred version, the described optimized proportion of step (2) is 0.016-0.320 ng/ μ l, quality/reaction system liquor capacity that ng/ μ l is graphene quantum dot.
For realizing second object of the present invention, the application of the open graphene quantum dot of the present invention in aggregation polymerase chain reaction.
The invention has the advantages that: utilize the complex optimum effect of graphene quantum dot optimize PCR better, be better than other existing nano material, can effectively improve the sensitivity of round pcr, improve output and the specificity of amplified production.And it is very low to optimize required graphene quantum dot consumption.In addition, the preparation process of graphene quantum dot is simple, and it is convenient to store.
Accompanying drawing explanation
Fig. 1 is the GQDs of the different concns impact on PCR sensitivity and output.
Fig. 2 is that the GQDs of different concns is on the specific impact of plasmid PCR product.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique using in following embodiment if no special instructions, is ordinary method.Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.
The first step: prepare the graphene quantum dot aqueous solution
The described graphene quantum dot aqueous solution is take the synthetic graphite oxide aqueous solution of Hummers method as initiator, utilizes Photo-Fenton reaction, with H
2o
2for oxygenant, Fe
3+for catalyzer, under UV-irradiation, be prepared from, product is dialysed in ultrapure water 2 days, remove unreacted H
2o
2small molecules with reaction produces, obtains the pure graphene quantum dot aqueous solution.Concrete preparation method can reference literature (Photo-Fenton Reaction of Graphene Oxide:A New Strategy to Prepare Graphene Quantum Dots for DNA Cleavage. ACS Nano, 2012,6 (8), 6592-6599).
Second step: graphene quantum dot solution is mixed by optimized proportion with pcr amplification reaction system
The optimized proportion of the described graphene quantum dot aqueous solution and pcr amplification reaction system is the ratio of concentration in reaction system that has an effect of optimization.In the present invention, graphene quantum dot optimization range is 0.016-0.320 ng/ μ l, quality/reaction system liquor capacity that ng/ μ l is graphene quantum dot.
Archaeal dna polymerase used can be Taq enzyme, can be also other polysaccharases such as Ex Taq; Pcr amplified dna template can be plasmid, can be also other DNA profiling.
Mix and refer to the required all components of amplification is added after PCR pipe, with the continuous pressure-vaccum of liquid-transfering gun 7-8 time.
The 3rd step: follow procedure carries out DNA cloning on PCR instrument
PCR condition and each operation steps all can operate according to a conventional method.In PCR, agents useful for same and raw material all can be bought in market or make by existing method.In the present embodiment, PCR instrument is BIO-RAD(T100
tMthermal Cycler, U.S.A.).
Experiment reagent and material:
Primer 1:5 '-CGCTAACGGATTCACCAC-3 ' (SEQ ID NO.1);
Primer 2: 5 '-CACGGAAACCGAAGACCA-3 ' (SEQ ID NO.2).
The graphene quantum dot aqueous solution (0.1 ng/ μ l and 1 ng/ μ l), ultrasonic 1 min at normal temperatures before use.Other reagent is shown in below in step.
PCR condition: (1) 94 ℃ of preheating 5 min, (2) 95 ℃ of sex change 30 s, (3) 52 ℃ of annealing 30 s, (4) 72 ℃ are extended 30 s, 36 circulations are repeated in step (2)～(4), (5) 72 ℃ are extended 5 min, (6) 12 ℃ of cooling 10 min.
Operation steps:
The first step: the minimum DNA profiling amount of screening PCR.The DNA profiling of difference amount is joined in PCR system and reacted.Template amount is respectively: 1.75 × 10
-2, 1 × 10
-2, 2.5 × 10
-3, 1.75 × 10
-3, 1 × 10
-3, 2.5 × 10
-4, 1.75 × 10
-4, 1 × 10
-4, 2.5 × 10
-5, 1.75 × 10
-5, 1 × 10
-5, 2.5 × 10
-6ng.
Other component of PCR reaction system: (2.5 μ l) for 10 × PCR buffered soln; DNTP(2 mM, 2.5 μ are l); MgCl
2(25 mM, 1.5 μ are l); Primer 1 and 2 (each 5 mM, 1 μ is l); Taq polysaccharase (5 U/ μ l, 0.3 μ is l); Sterilized water is supplied 25 μ l.DNA product after amplification is carried out to agarose gel electrophoresis detection.
Second step: select the minimum template amount 2.5 × 10 of amplified production
-6ng, and add respectively the different GQDs that measure.GQDs amount is respectively: 0,0.4,0.6,0.8,2,4,6,8 ng, and other component is the same, and sterilized water is supplied 25 μ l.DNA product after amplification is carried out to agarose gel electrophoresis, and experimental result as shown in Figure 1.
When template amount is 2.5 × 10
-6when ng, almost there is no amplified production by conventional PCR condition, but add after GQDs, as shown in Figure 1, along with GQDs add-on improves gradually, pcr amplification product increases gradually, PCR output and sensitivity be improved significantly.When GQDs final concentration scope is during at 0.016-0.16 ng/ μ l, effect of optimization is obvious.Add 0.8 ng GQDs(ultimate density be 0.032 ng/ μ l), the output of PCR is not add 1.8 times of GQDs, is equivalent to add 2.5 × 10
-5the output of ng template amount, remolding sensitivity does not add GQDs and has improved an order of magnitude.
The first step: 0.25 ng DNA profiling is joined in PCR system and reacted, and archaeal dna polymerase is used Ex Taq enzyme instead, other components of PCR system are with embodiment 2 step 1, and sterilized water is supplied 25 μ l.
Second step: take the amplified production in step 1 as template, dilute 10 times, get 1 μ l and add PCR system, add the different GQDs that measure to carry out second and take turns PCR reaction.GQDs amount is respectively as 0,1.5,2.5,3.5,4.5,5.5,6.5 ng, the same step 1 of other component, and sterilized water is supplied 25 μ l.
DNA product after amplification is carried out to agarose gel electrophoresis, and experimental result as shown in Figure 2.As can be seen from the figure, along with the amount that adds GQDs increases, in product, non-specific band reduces gradually, effect of optimization the best in the time that graphene quantum dot ultimate density is 0.12-0.14 ng/ μ l.
Experiment reagent and material:
Primer 1:5 '-ATTAAGGACATCTTAGGGGCCCTCT-3 ' (SEQ ID NO.3);
Primer 2: 5 '-GGGTTTGATGTGAGGGGGTGTGTTG-3 ' (SEQ ID NO.4).
The graphene quantum dot aqueous solution (0.1 ng/ μ l and 1 ng/ μ l), ultrasonic 1 min at normal temperatures before use.Other reagent is shown in below in step.
PCR condition: (1) 95 ℃ of preheating 8 min, (2) 95 ℃ of sex change 30 s, (3) 71 ℃ of annealing 30 s, (4) 72 ℃ are extended 25 s, 35 circulations are repeated in step (2)～(4), (5) 72 ℃ are extended 5 min, (6) 12 ℃ of cooling 10 min.
The GQDs of difference amount is joined in PCR system and reacted.GQDs amount is followed successively by: 0,0.4,0.6,0.8,2,4,6,8 ng.Other component of PCR system: calf thymus DNA (0.5 × 10
-3ng/ μ l, 1 μ is l); (2.5 μ l) for 10 × PCR buffered soln; DNTP (2 mM, 2.5 μ are l); MgCl
2(25 mM, 2 μ are l); Primer 1 and 2 (each 5 mM, 1 μ is l); Taq polysaccharase (5 U/ μ l, 0.3 μ is l); Sterilized water is supplied 25 μ l.
DNA product after amplification is carried out to agarose gel electrophoresis.Along with the amount that adds GQDs increases, PCR product output also increases gradually.Adding 0.8 ng GQDs(final concentration is that 0.032 ng/ μ output l) is not add 2.1 times of GQDs.
In sum, utilize the complex optimum effect of graphene quantum dot optimize PCR better, the sensitivity that can effectively improve round pcr, output and the specificity of raising amplified production.And it is very low to optimize required graphene quantum dot amount.What PCR increased can be simple plasmid, can be also the complicated genomes such as calf thymus DNA.The polysaccharase that pcr amplification reaction system is used does not have particular requirement, can be Taq enzyme, can be other archaeal dna polymerases such as Ex Taq yet.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
SEQUENCE LISTING
<110> East China University of Science
Mono-kind of <120> utilizes the method for graphene quantum dot aggregation polymerase chain reaction
<130> 。
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> synthetic
<400> 1
cgctaacgga ttcaccac 18
<210> 2
<211> 18
<212> DNA
<213> synthetic
<400> 2
cacggaaacc gaagacca 18
<210> 3
<211> 25
<212> DNA
<213> synthetic
<400> 3
attaaggaca tcttaggggc cctct 25
<210> 4
<211> 25
<212> DNA
<213> synthetic
<400> 4
gggtttgatg tgagggggtg tgttg 25
Claims (5)
1. a method of utilizing graphene quantum dot aggregation polymerase chain reaction, is characterized in that, said method comprising the steps of:
(1) prepare the graphene quantum dot aqueous solution;
(2) the graphene quantum dot aqueous solution is mixed by optimized proportion with pcr amplification reaction system;
(3) on PCR instrument, follow procedure carries out DNA cloning;
Optimized proportion described in step (2) refers to that the concentration of graphene quantum dot is to determine by the following method: graphene quantum dot is carried out joining in PCR system and reacting after gradient dilution, and the ratio of the scope that reaches optimization take actual expanding effect in system is the optimized proportion of graphene quantum dot.
2. a kind of method of utilizing graphene quantum dot aggregation polymerase chain reaction according to claim 1, it is characterized in that, graphene quantum dot water solution preparation method is in step (1): take the synthetic graphite oxide aqueous solution of Hummers method as initiator, utilize Photo-Fenton reaction, with H
2o
2for oxygenant, Fe
3+for catalyzer, under ultraviolet radiation, be prepared from, product is dialysed in ultrapure water, remove unreacted H
2o
2small molecules with reaction produces, obtains the pure graphene quantum dot aqueous solution.
3. a kind of method of utilizing graphene quantum dot aggregation polymerase chain reaction according to claim 1, is characterized in that, what step (2) PCR increased comprises simple plasmid and complex genome, and described complex genome comprises calf thymus DNA.
4. a kind of method of utilizing graphene quantum dot aggregation polymerase chain reaction according to claim 1, it is characterized in that, the described optimized proportion of step (2) is 0.016-0.320 ng/ μ l, quality/reaction system liquor capacity that ng/ μ l is graphene quantum dot.
5. the application of graphene quantum dot in aggregation polymerase chain reaction.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104150468A (en) * | 2014-07-14 | 2014-11-19 | 华东理工大学 | Method for separating graphene quantum dot |
| CN105647909A (en) * | 2016-03-25 | 2016-06-08 | 厦门大学 | Method for improving PCR specificity |
| CN106244577A (en) * | 2015-06-15 | 2016-12-21 | 中国科学院上海应用物理研究所 | A kind of multiplex polymerase chain re-action method and application thereof |
| CN110358811A (en) * | 2019-08-20 | 2019-10-22 | 上海纳米技术及应用国家工程研究中心有限公司 | A method of optimization loop-mediated isothermal amplification |
| CN110423796A (en) * | 2019-08-19 | 2019-11-08 | 上海纳米技术及应用国家工程研究中心有限公司 | A method of improving nucleic acid in vitro amplified reaction efficiency |
| CN113265451A (en) * | 2020-02-14 | 2021-08-17 | 成都中医药大学 | Method for improving specificity of real-time fluorescent quantitative PCR |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104150468A (en) * | 2014-07-14 | 2014-11-19 | 华东理工大学 | Method for separating graphene quantum dot |
| CN104150468B (en) * | 2014-07-14 | 2016-02-03 | 华东理工大学 | A kind of separation method of graphene quantum dot |
| CN106244577A (en) * | 2015-06-15 | 2016-12-21 | 中国科学院上海应用物理研究所 | A kind of multiplex polymerase chain re-action method and application thereof |
| CN106244577B (en) * | 2015-06-15 | 2021-07-06 | 中国科学院上海应用物理研究所 | Multiple polymerase chain reaction method and application thereof |
| CN105647909A (en) * | 2016-03-25 | 2016-06-08 | 厦门大学 | Method for improving PCR specificity |
| CN105647909B (en) * | 2016-03-25 | 2018-09-21 | 厦门大学 | A method of improving PCR specificity |
| CN110423796A (en) * | 2019-08-19 | 2019-11-08 | 上海纳米技术及应用国家工程研究中心有限公司 | A method of improving nucleic acid in vitro amplified reaction efficiency |
| CN110423796B (en) * | 2019-08-19 | 2024-02-13 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for improving nucleic acid in-vitro amplification reaction efficiency |
| CN110358811A (en) * | 2019-08-20 | 2019-10-22 | 上海纳米技术及应用国家工程研究中心有限公司 | A method of optimization loop-mediated isothermal amplification |
| CN110358811B (en) * | 2019-08-20 | 2024-02-13 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for optimizing loop-mediated isothermal amplification reaction |
| CN113265451A (en) * | 2020-02-14 | 2021-08-17 | 成都中医药大学 | Method for improving specificity of real-time fluorescent quantitative PCR |
| CN113265451B (en) * | 2020-02-14 | 2023-08-29 | 成都中医药大学 | Method for improving real-time fluorescence quantitative PCR specificity |
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