CN101139620A - Nucleic acid polymerase chain reaction optimized expanding method based on nano metal silver - Google Patents
Nucleic acid polymerase chain reaction optimized expanding method based on nano metal silver Download PDFInfo
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- CN101139620A CN101139620A CNA2006100156123A CN200610015612A CN101139620A CN 101139620 A CN101139620 A CN 101139620A CN A2006100156123 A CNA2006100156123 A CN A2006100156123A CN 200610015612 A CN200610015612 A CN 200610015612A CN 101139620 A CN101139620 A CN 101139620A
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Abstract
The invention relates to a method for polymerase chain reaction (PCR) expansion in the technical field of biology, and is an optimal method for nuclein PCR based on silver nanopowder. The method is realized by adding certain amount of silver nanopowder suspension fluid in a system based on a prior PCR expansion method. The method is of obvious optimizing effect, is easy to prepare, of low cost and wide application, can be used for various PCR expanding, polymerase extensions and other biochemical methods based on PCR method, especially suitable for PCR method that is difficult to expand, for example, to expand DNA with high GC content and super-long sectioned PCR, etc.
Description
Technical field
The present invention relates to the method for polymerase chain reaction (PCR) amplification of biological technical field, especially based on the optimization method of the amplification of chain reaction of nucleic acid polymerase of nanometer metallic silver (silver nanopowder).
Background technology
Chain reaction of nucleic acid polymerase (Polymerase Chain Reaction, PCR) be by a kind of DNA amplification in vitro technology of K.Mullis in invention in 1985, this technology can be outside organism in the several hrs with millions of times of amplifications of goal gene of denier, and can any goal gene of specific amplification or dna segment.Round pcr is the revolution on the methodology, and with its significant three big advantage: specificity, high-level efficiency and fidelity have produced tremendous influence to the life science field.PCR is described as cell-free molecular cloning, and its contriver obtained Nobel in 1992 and will encourage.Although the PCR reaction has developed into a mature technology, fault rate is less in the normal experiment, in the actually operating, carries out pcr amplification and has the improved problem of some needs all the time.Distinct issues are exactly that pcr amplification exists in various degree non-specific, PCR is as a kind of in-vitro simulated biochemical reaction, mechanism is very complicated, and interference side reaction to a certain degree always can take place in the chain reaction process, as the primer template may mispairing, primer is in conjunction with forming dimer etc.These side reactions influence to the result when conventional pcr amplification is not clearly, and this is because the template number of conventional pcr amplification is bigger, surpasses 10 usually
3More than, the template amplification number is far longer than the amplification number of side reaction product in the process of exponential amplification, and the non-specific amplification ratio is very low.But the amplification of the side reaction product in following situation then can not be ignored, as amplifying high GC content DNA, overlength dna fragmentation etc.These light side-reaction then cause non-specific amplification (the disperse hangover band and the non-specific band that show as) in follow-up electrophoresis detection, cause amplification efficiency not high; Heavy then may cause the failure of normal amplified reaction.And the specificity that improves pcr amplification not only depends on and the optimization design of primer sequence also depends on the optimization of reaction system and program to a great extent.For many years people have done a large amount of research to the optimization of PCR reactive component, as by in reaction system, adding methane amide, glycerine, dimethyl sulfoxide (DMSO) etc., can improve the non-specific amplification problem to a certain extent, but at actual experiment with in using, some effect and not really remarkable, some adds component (such as dimethyl sulfoxide (DMSO)) too much even suppress the activity of polysaccharase.
Find through retrieval existing scientific and technical literature, United States Patent (USP) (US PATENT) 5,646,019 discloses " a kind of preparation method who causes the amplification of nucleic acid template that is beneficial to ", this method is to have added heat-stable single strand binding protein (SSB in the PCR system, single-stranded nucleic acid binding protein), SSB albumen is only in conjunction with single stranded DNA, but debond double-stranded DNA, contain the non-specific segmental amplification of strand by combination and inhibition, thereby realized the optimization of pcr amplification.Because extract purifying SSB albumen technical sophistication, reagent purity also requires very high, causes preparation cost very high, commercial kit is very expensive, and price is 6～7 times of conventional PCR reagent; Simultaneously in order to keep the biological activity of single strand binding protein, strict being stored in-20 ℃, and its biological activity has short time bar.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of optimization method of the amplification of chain reaction of nucleic acid polymerase based on nanometer metallic silver (silver nanopowder) is provided.This method reaches effective amplification to dna fragmentation by add the certain amount of nano argent in the PCR reaction system, this method optimize effect significantly, preparation is easy, with low cost.
The present invention is achieved through the following technical solutions:
Be somebody's turn to do the optimization method based on the amplification of chain reaction of nucleic acid polymerase of nanometer metallic silver, this method may further comprise the steps:
(1). the preparation of nanometer metallic silver suspension;
(2) configuration of .PCR system;
(3) optimization of .PCR system;
(4) setting of .PCR condition and operation;
(5) the .PCR product detects;
It is characterized in that: the optimization of described PCR system is meant adds nanometer metallic silver suspension in the PCR system.
And described nanometer metallic silver suspension concentration is 10mg/mL, and the volume percent of itself and PCR system is 0.02～10%.
And described pcr amplification comprises: optionally amplifying high GC content and long segment PCR and overlength segment PCR in the genomic templates of conventional PCR, high complexity.
And described nanometer metallic silver can separate from the PCR system.
And described nanometer metallic silver separates from the PCR system, and what pass through is that nanometer metallic silver suspension system is done low-speed centrifugal, can be realized separating by drawing supernatant liquor.
Advantage of the present invention and beneficial effect are:
1. this optimization method optimization effect based on the amplification of chain reaction of nucleic acid polymerase of nanometer metallic silver (silver nanopowder) is remarkable, preparation is easy, with low cost, has wider range of application, can be applied to other biochemical methods on various pcr amplifications, polymerase extension and PCR-based method basis, the PCR method that is particularly useful for some difficult amplification types such as the DNA of amplifying high GC content, overlength fragment PCR or the like.
2. the nano material of the optimization pcr amplification that uses among the present invention---nanometer metallic silver is easy to preserve, can prolonged preservation at 4 ℃, can not lose activity; And nanometer metallic silver is easy to separate from the PCR reaction system, handles and uses for subsequent P CR product and bring convenience.
3. this optimization method based on the amplification of chain reaction of nucleic acid polymerase of nanometer metallic silver (silver nanopowder) simply is easy to grasp, can extensively promote, there is potential and huge using value in fields such as gene test and clone, genetic analysis, clinical diagnosis, gene chip and novel material.
Description of drawings
The optimization expanding effect electrophorogram of the disconnected PCR reaction of Fig. 1 nanometer metallic silver centering lengthy motion picture;
Fig. 2 nanometer metallic silver is to the optimization expanding effect electrophorogram of overlength segment PCR reaction;
Fig. 3 nanometer metallic silver is to the optimization expanding effect electrophorogram of high GC content pcr amplification.
Embodiment
The present invention is described in further detail by following examples in conjunction with the accompanying drawings, but is not limited to following embodiment.
The optimization method of round pcr involved in the present invention realizes that by add nanometer metallic silver (silver nanopowder) in the PCR system its concrete operation method is as follows:
1.silver the preparation of nanopowder suspension:
Silver nanopowder buys the company in Sigma, is example with configuration concentration for the nanometer metallic silver aaerosol solution of (W/V) 10mg/mL, accurately take by weighing earlier the little centrifuge tube that the sterilized nanometer metallic silver of 10mg places sterilized 1.5mL, slowly adding aqua sterilisa to volume with pipettor is 1mL, carries out ultrasonic wave suspension 10min in 40KHz.
2.PCR the configuration of system:
The PCR system is had nothing in common with each other by the difference of dna segment to be amplified according to existing technology.Be in particular in: dNTP, template, Mg
2+, primer addition should select with different expanding fragment lengths according to different templates;
H in the PCR reaction system
2O is distilled water and above rank water, and the addition of water should be adjusted according to the volume that the nanometer metallic silver aaerosol solution adds in the system, promptly should deduct the volume of the nanometer metallic silver aaerosol solution of corresponding interpolation.
3.PCR the optimization of system:
To carry out pcr amplification in the middle of an amount of silver nanopowder suspension adding PCR reaction system, the described consumption of the optimization material that adds in the middle of the PCR reaction system of 25 μ L that is meant in right amount is: silver nanopowder distilled water suspension (10 mg/ml) 0.5 μ L～5 μ L.
4.PCR the setting of condition and operation:
Pre-sex change, the sex change in the PCR reaction conditions and the temperature in each stage of annealing and corresponding time should be selected according to different templates and primer melting temp; Elongating temperature is wherein selected according to the suitable temp of used polysaccharase; The extension time is wherein selected according to the length of institute's amplified fragments; Cycle index is wherein selected according to individual test objective and needs.
5.PCR the detection of product:
DNA sample after the amplification is carried out agarose gel electrophoresis, check amplified band, compare with control systems.Generally detect with agarose gel electrophoresis technology, the last sample volume of the concentration of sepharose and PCR product needs to decide as the case may be.
Described agarose gel electrophoresis technology is summarized as follows according to existing method:
1) sepharose of the required concentration of preparation, concrete concentration should be determined according to the size of amplification of DNA fragments.
2) draw electrophoresis chamber point sample on the PCR product of certain volume, with suitable molecular weight mark on the time point as reference.
3) voltage that adds 3～4V/cm carries out electrophoresis.
When 4) treating indicator, take out offset plate and observe down and photographic recording in the gel imaging system to correct position.
The optimization of above-mentioned PCR system is meant in the PCR system nanometer metallic silver suspension that adds 10 mg/ml, and the volume percent of itself and PCR system is 0.02～10%.
Above-mentioned pcr amplification comprises: optionally amplifying high GC content and long segment PCR and overlength segment PCR in the genomic templates of conventional PCR, high complexity.
Above-mentioned nanometer metallic silver can separate from the PCR system, reclaims the PCR product that amplification obtains if desired, then can isolate the optimization material easily from reaction end system.Silvernanopowder suspension system is done low-speed centrifugal, draw supernatant liquor and can reach the separation purpose.
Silver nanopowder improves at some optimizations that form the pcr amplification of non-specific amplification the optimization of PCR reaction, and amplification length is the long segment of 4249bp, comprises the following steps:
1. the preparation of nanometer metallic silver (silver nanopowder) suspension:
Silver nanopowder buys the company in Sigma, configuration concentration is the nanometer metallic silver aaerosol solution of (W/V) 10mg/mL, accurately take by weighing earlier the little centrifuge tube that the sterilized nanometer metallic silver of 10mg places sterilized 1.5mL, slowly adding aqua sterilisa to volume with pipettor is 1mL, carries out ultrasonic wave suspension 10min in 40KHz.
2.PCR the configuration of system, concrete table composed as follows:
| Taq enzyme (5U/ μ L) | 0.45μL |
| Pfu enzyme (2.5U/ μ L) | 0.45μL |
| 10 * PCR damping fluid | 2.5μL |
| DNTP substrate (2.5mM) | 3.5μL |
| Mg 2+(25mM) | 1.75μL |
| Primer 1 (1 μ M) | 3.75μL |
| Primer 2 (1 μ M) | 3.75μL |
| Template | 1μL |
Wherein, template is λ DNA, and available from three rich polygala root bio-engineering corporations, the Taq enzyme is available from three rich polygala root bio-engineering corporations.
Dispose PCR system 4 pipes altogether, and from 2 to 5 numberings.
3.PCR the optimization of system:
Add nanometer metallic silver aaerosol solution 0 μ L, 0 μ L, 0.5 μ L, 2.0 μ L respectively successively from the 2nd pipe to the 5 pipes.Supplying cumulative volume with distilled water at last is 25 μ L.
4.PCR the setting of condition and operation:
Utilize round pcr that template molecule is increased, amplification condition is: 93 ℃ of preheating 2min, and 33 circulations, each circulation comprises: 93 ℃ of sex change 10s, 58.1 ℃ of annealing 30s, 68 ℃ are extended 3min, and last 68 ℃ are extended 10min.
5, the DNA sample after the amplification being carried out agarose gel electrophoresis detects.
The results are shown in Figure 1.The purpose fragment is 4249bp, 1 is molecular weight marker LambdaDNA/HindIII (MBI company from left to right, by dna fragmentation from top to bottom is 23,130bp, 9,416bp, 6,557bp, 4,361bp, 2,322bp, 2,027bp, 564bp and 125bp and constitute) 2,3 for common response system result, 4,5 results for common response system adding silver nano[owder suspension.Non-specific amplification is few among the system pcr amplification result of adding optimization material silver nanopowder as can be seen, and the corresponding serious follow-up hangover of conventional PCR system amplification demonstration that does not add silver nanopowder shows that non-specific amplification is serious.This pcr amplification result has shown that optimization method effect of the present invention is remarkable.
Nanometer metallic silver is to the optimization of overlength fragment PCR amplification, and at the optimization improvement of overlength fragment PCR amplification, amplification length is the long segment of 15000bp, comprises the following steps:
1. the preparation of nanometer metallic silver (silver nanopowder) suspension:
With embodiment 1.
2.PCR the configuration of system, concrete table composed as follows:
| Taq enzyme (5U/ μ L) | 0.45μL |
| Pfu enzyme (2.5U/ μ L) | 0.45μL |
| 10 * PCR damping fluid | 2.5μL |
| DNTP substrate (2.5mM) | 3.5μL |
| Mg 2+(25mM) | 1.75μL |
| Primer 1 (1 μ M) | 3.75μL |
| Primer 2 (1 μ M) | 3.75μL |
| Template | 1μL |
Wherein, template is λ DNA, and available from three rich polygala root bio-engineering corporations, the Taq enzyme is available from three rich polygala root bio-engineering corporations.
Dispose PCR system 3 pipes altogether, and from 2 to 4 numberings.
3.PCR the optimization of system:
Add nanometer metallic silver aaerosol solution 0 μ L, 3.0 μ L, 5.0 μ L respectively successively from the 2nd pipe to the 4 pipes.Supplying cumulative volume with distilled water at last is 25 μ L.
4.PCR the setting of condition and operation:
Utilize round pcr that template molecule is increased, amplification condition is 33 circulations, 93 ℃ of preheating 2min, and 93 ℃ of sex change 10s, 54.8 ℃ of annealing 30s, 72 ℃ are extended 20min, and last 72 ℃ are extended 10min.
5. the DNA sample after the amplification being carried out agarose gel electrophoresis detects.
The results are shown in Figure 2.The purpose fragment is 15kb, 1 is molecular weight marker LambdaDNA/HindIII (MBI company from left to right, by dna fragmentation from top to bottom is 23,130bp, 9,416bp, 6,557bp, 4,361bp, 2,322bp, 2,027bp, 564bp and 125bp and constitute) 2 for common response system result, 3,4 results for common response system adding silver nanopowder.Above reaction conditions is identical, and the non-specific amplification product disperse of broad etc. appears in 2 sample amplified bands as can be seen, and 3,4 disperses obviously alleviate, the remarkable improvement of amplification quality.
Silver nanopowder is to the optimization of high GC content pcr amplification, and at the optimization improvement of high GC content amplification, amplification length is the dna molecular of 2872bp, comprises the following steps:
1. the preparation of nanometer metallic silver (silver nanopowder) suspension:
With embodiment 1.
2.PCR the configuration of system, concrete table composed as follows:
| Taq enzyme (5U/ μ L) | 0.45μL |
| Pfu enzyme (2.5U/ μ L) | 0.45μL |
| 10 * PCR damping fluid | 2.5μL |
| DNTP substrate (2.5mM) | 3.5μL |
| Mg 2+(25mM) | 1.75μL |
| Primer 1 (1 μ M) | 3.75μL |
| Primer 2 (1 μ M) | 3.75μL |
| Template | 1μL |
Wherein, template is λ DNA, and available from three rich polygala root bio-engineering corporations, the Taq enzyme is available from three rich polygala root bio-engineering corporations.
Dispose PCR system 4 pipes altogether, and from 2 to 5 numberings.
3.PCR the optimization of system:
Add nanometer metallic silver aaerosol solution 0 μ L, 1.0 μ L, 4.0 μ L, 5.0 μ L respectively successively from the 2nd pipe to the 5 pipes.Supplying cumulative volume with distilled water at last is 25 μ L.
4.PCR the setting of condition and operation:
Utilize round pcr that template molecule is increased, amplification condition is 33 circulations, 93 ℃ of preheating 2min, and 93 ℃ of sex change 10s, 54.8 ℃ of annealing 30s, 72 ℃ are extended 20min, and last 72 ℃ are extended 10min.
5. the DNA sample after the amplification being carried out agarose gel electrophoresis detects.
The results are shown in Figure 3.2 is conventional PCR system amplification from left to right; 3,4,5 is to add the present invention to optimize the PCR system amplification of material nano argent aaerosol solution to high GC content.1 is molecular weight marker (being 23 from top to bottom, 130bp, 9,416bp, 6,557bp, 4,361bp, 2,322bp, 2,027bp, 564bp and 125bp).Have multi-ribbon non-specific amplification or product hangover etc. to occur as can be seen in the 2 sample amplifications, 3,4 sample bands are clear, obviously optimized.
Claims (5)
1. optimization method based on the amplification of chain reaction of nucleic acid polymerase of nanometer metallic silver, this method may further comprise the steps:
(1). the preparation of nanometer metallic silver suspension;
(2) configuration of .PCR system;
(3) optimization of .PCR system;
(4) setting of .PCR condition and operation;
(5) the .PCR product detects;
It is characterized in that: the optimization of described PCR system is meant adds nanometer metallic silver suspension in the PCR system.
2. the optimization method of the amplification of chain reaction of nucleic acid polymerase based on nanometer metallic silver according to claim 1, it is characterized in that: described nanometer metallic silver suspension concentration is 10mg/mL, the volume percent of itself and PCR system is 0.02～10%.
3. the optimization method of the amplification of chain reaction of nucleic acid polymerase based on nanometer metallic silver according to claim 1, it is characterized in that: described pcr amplification comprises: optionally amplifying high GC content and long segment PCR and overlength segment PCR in the genomic templates of conventional PCR, high complexity.
4. the optimization method of the amplification of chain reaction of nucleic acid polymerase based on nanometer metallic silver according to claim 1, it is characterized in that: described nanometer metallic silver can separate from the PCR system.
5. the optimization method of the amplification of chain reaction of nucleic acid polymerase based on nanometer metallic silver according to claim 4, it is characterized in that: described nanometer metallic silver separates from the PCR system, and what pass through is that nanometer metallic silver suspension system is done low-speed centrifugal, can be realized separating by drawing supernatant liquor.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792787A (en) * | 2010-04-06 | 2010-08-04 | 山东大正医疗器械股份有限公司 | Method for optimizing PCR with composite nano material |
| 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 |
| CN106834550A (en) * | 2017-04-18 | 2017-06-13 | 福建省农业科学院畜牧兽医研究所 | A kind of avian influenza virus nano PCR detection kit and its application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792787A (en) * | 2010-04-06 | 2010-08-04 | 山东大正医疗器械股份有限公司 | Method for optimizing PCR with composite nano material |
| 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 |
| CN106834550A (en) * | 2017-04-18 | 2017-06-13 | 福建省农业科学院畜牧兽医研究所 | A kind of avian influenza virus nano PCR detection kit and its application |
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