CN101153308B - Optimization method of nucleic acid polymerase chain reaction amplification based on nano metal alloy - Google Patents
Optimization method of nucleic acid polymerase chain reaction amplification based on nano metal alloy Download PDFInfo
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- CN101153308B CN101153308B CN2006100160398A CN200610016039A CN101153308B CN 101153308 B CN101153308 B CN 101153308B CN 2006100160398 A CN2006100160398 A CN 2006100160398A CN 200610016039 A CN200610016039 A CN 200610016039A CN 101153308 B CN101153308 B CN 101153308B
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Abstract
The invention relates to an amplification method for polymerase chain reaction (PCR) in the biotechnological field, which is an optimized amplification method for nucleic acid polymerase chain reaction based on the technology of nanometer alloy, accomplished through adding a certain amount of suspension of nanometer alloy to the system based on the amplification method of PCR in the prior art, provided with significant optimized effect, easy preparation, low cost, and wide applicable fields, and applicable to various PCR amplification, extension of the polymerase, and the other biological methods based on PCR, particularly to the PCR methods with difficult amplification, such as the selective amplification of DNA with extremely low copies from the highly complicated genomic template, amplification of DNA rich in Gs and Cs, amplification of single molecular DNA, amplification of excessive long DNA segment, multiple PCR amplification, and re-amplification for the former unsatisfactory amplification, 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 nano metal alloy.
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 was described as cell-free molecular cloning, and its contriver obtained the Nobel prize in 1992.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, and 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 side reaction product in following situation amplification then can not be ignored, as from the genomic templates of high complexity optionally the utmost point low copy number DNA of specific amplification, amplification unique DNA, amplifying high GC content DNA, amplification overlength dna fragmentation, multiplex PCR amplification, once increase imperfect and amplification etc. again.These light side-reaction then cause non-specific amplification (showing as disperse hangover band and non-specific band 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, and 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.Nano metal alloy is to obtain the microcrystallizing alloy that diameter is 10～20 nanometers more after treatment with non-crystaline amorphous metal, also claims ultramicro-crystal alloy.Because this special construction makes non-crystaline amorphous metal have some unique character, the present invention attempts nano metal alloy is applied to polymerase chain reaction (PCR) amplification, and has obtained good expanding effect.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of optimization method of the amplification of chain reaction of nucleic acid polymerase based on nano metal alloy is provided.This method is by adding the certain amount of nano metal alloy to reach the effective amplification to dna fragmentation in the PCR reaction system, this method optimization effect is remarkable, preparation is easy, with low cost.
The present invention is achieved through the following technical solutions:
A kind of optimization method of the amplification of chain reaction of nucleic acid polymerase based on nano metal alloy, this method may further comprise the steps:
(1). the preparation of nano metal alloy 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 nano metal alloy suspension in the PCR system.
And the preparation method of described nano metal alloy suspension is:
(1). accurately take by weighing sterilized nano metal alloy earlier and place sterilized centrifuge tube;
(2). slowly add aqua sterilisa with pipettor;
(3). carry out promptly making nano metal alloy suspension after ultrasonic wave suspends in 40KHz.
And described nano metal alloy suspension concentration is 10mg/mL, and the addition in the PCR system of its place and the volume percent of PCR system are 0.02～10%.
And described nano metal alloy is nanometer silver platinum alloy, nano silver copper alloy.
And described pcr amplification comprises: the optionally utmost point low copy number DNA of specific amplification, amplifying high GC content PCR, long segment PCR and overlength segment pcr amplification, multiplex PCR amplification, single-molecule PCR amplification and once amplification amplification more afterwards in the genomic templates of conventional pcr amplification, high complexity.
Beneficial effect of the present invention and advantage are:
1. this optimization method optimization effect based on the amplification of chain reaction of nucleic acid polymerase of nano metal alloy is remarkable, prepare easy, with low cost, has wider range of application, can be applied to various pcr amplifications, other biochemical methods on polymerase extension and PCR-based method basis, the PCR method that is particularly useful for some difficult amplification types such as the utmost point low copy number DNA of specific amplification optionally in the genomic templates of high complexity, amplifying high GC content PCR, long segment PCR and overlength segment pcr amplification, the multiplex PCR amplification, the single-molecule PCR amplification, and once increase imperfect or enough and need situation such as amplification again.
2. nano material-the nano metal alloy of the optimization pcr amplification that uses among the present invention is easy to preserve, can prolonged preservation at 4 ℃, and can not lose activity; And nano metal alloy 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 nano metal alloy 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 silver platinum alloy centering lengthy motion picture;
Fig. 2 nano silver copper alloy is to the optimization expanding effect electrophorogram of overlength segment PCR reaction;
Fig. 3 nanometer silver platinum alloy 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 nano metal alloy in the PCR system its concrete operation method is as follows:
1. the preparation of nano metal alloy suspension:
Nano metal alloy is bought the company in Sigma, is example with configuration concentration for the nano metal alloy aaerosol solution of (W/V) 10mg/mL, accurately take by weighing earlier the little centrifuge tube that the sterilized nano metal alloy of 10mg places sterilized 1.5mL, slowly adding aqua sterilisa to volume with pipettor is 1mL, carrying out ultrasonic wave in 40KHz suspends, ultrasonic time is decided according to the suspension situation, should guarantee that generally uniform suspended state keeps more than the 10min.
The nano metal alloy that adopts comprises nanometer silver platinum alloy, nano silver copper alloy and similar alloy.
2.PCR the configuration of system:
The PCR system is according to existing technology, because of the difference of dna segment to be amplified has nothing in common with each other.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 nano metal alloy suspension adds in the system, promptly should deduct the volume of the nano metal alloy suspension of corresponding interpolation.
3.PCR the optimization of system:
To carry out pcr amplification in the middle of an amount of nano metal alloy 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: nano metal alloy distilled water suspension (10mg/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 (containing the staining agent ethidium bromide) of preparation 0.7%;
2) draw electrophoresis chamber point sample on the PCR product of different samples, with the time point molecular weight marker as reference;
3) voltage of 4～5V/cm in addition, electrophoresis;
4) gel imaging system ultraviolet detection analytical results.
Above-mentioned nano metal alloy suspension concentration is 10mg/mL, and the addition in the PCR system of its place and the volume percent of PCR system are 0.02～10%.
Above-mentioned pcr amplification comprises: the optionally utmost point low copy number DNA of specific amplification, amplifying high GC content PCR, long segment PCR and overlength segment pcr amplification, multiplex PCR amplification in the genomic templates of conventional pcr amplification, high complexity, single-molecule PCR amplification and the once amplification again after the amplification, the amplification again after the described once amplification are meant once increases imperfect or enough and needs the situation of amplification again.
The nanometer silver platinum alloy 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 silver platinum alloy (silver-platinum) suspension:
Silver-platinum buys the company in Sigma, configuration concentration is the nanometer silver platinum alloy suspension of (W/V) 10mg/mL, accurately take by weighing earlier the little centrifuge tube that the sterilized nanometer silver platinum alloy 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 (2 μ M) | 3.75μL |
| Primer 2 (2 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 silver platinum alloy suspension 0 μ L, 2.0 μ L, 6.0 μ L, 8.0 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 for common response system result, 3,4,5 results for common response system adding silver-platinum suspension.Non-specific amplification is few among the system pcr amplification result of adding optimization material silver-platinum as can be seen, and the corresponding serious follow-up hangover of conventional PCR system amplification demonstration that does not add silver-platinum shows that non-specific amplification is serious.This pcr amplification result has shown that optimization method effect of the present invention is remarkable.
Nano silver copper alloy (silver-copper) 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 nano silver copper alloy (silver-copper) suspension:
Silver-copper buys the company in Sigma, configuration concentration is the nano silver copper alloy suspended liquid of (W/V) 10mg/mL, accurately take by weighing earlier the little centrifuge tube that the sterilized nano silver copper alloy 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 (2 μ M) | 3.75μL |
| Primer 2 (2 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 2 pipes altogether, and from 2 to 3 numberings.
3.PCR the optimization of system:
Add nano metal alloy suspension 0 μ L, 3.0 μ L respectively successively from the 2nd pipe to the 3 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 results for common response system adding silver-copper.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 disperses obviously alleviate, the remarkable improvement of amplification quality.
Silver-platinum 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 silver platinum alloy (silver-platinum) 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 (2 μ M) | 3.75μL |
| Primer 2 (2 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 silver platinum alloy suspension 0 μ L, 3.0 μ L, 4.0 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 3.2 is conventional PCR system amplification from left to right; 3,4 is to add the present invention to optimize the PCR system amplification of material nano silver-platinum alloy suspension 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 (2)
1. optimization method based on the amplification of chain reaction of nucleic acid polymerase of nano metal alloy, this method may further comprise the steps:
(1). the preparation of nano metal alloy 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 nano metal alloy suspension in the PCR system, described nano metal alloy is nanometer silver platinum alloy, nano silver copper alloy, described nano metal alloy suspension concentration is 10mg/mL, the addition in the PCR system of its place and the volume percent of PCR system are 0.02～10%
The preparation method of described nano metal alloy suspension is:
(1). accurately take by weighing sterilized nano metal alloy earlier and place sterilized centrifuge tube;
(2). slowly add aqua sterilisa with pipettor;
(3). carry out promptly making nano metal alloy suspension after ultrasonic wave suspends in 40KHz.
2. the optimization method of the amplification of chain reaction of nucleic acid polymerase based on nano metal alloy according to claim 1, it is characterized in that: described pcr amplification comprises: the optionally utmost point low copy number DNA of specific amplification, amplifying high GC content PCR, long segment PCR and overlength segment pcr amplification, multiplex PCR amplification in the genomic templates of conventional pcr amplification, high complexity, single-molecule PCR amplification and the once amplification again after the amplification.
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| 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 |
| CN102888428B (en) * | 2011-07-21 | 2013-12-11 | 中国科学院过程工程研究所 | Method for synthesizing nano silver by utilizing Bacillus amyloliquefaciensBacillus amyloliquefaciens LSSE-62 |
| 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 |
| CN113430113B (en) * | 2021-06-30 | 2024-03-15 | 东南大学 | Ultrasonic suspension polymerase chain reaction device and detection method |
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| Min Li et al..Enhancing the efficiency of a PCR using gold nanoparticles.《Nucleic Acids Research》.2005,第33卷(第21期),1-10. * |
| 汪名春 等.纳米材料对长片段基因序列扩增的优化研究.《全国海洋生物技术与海洋药物学术会议论文集》.2006,659-663. * |
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