CN106591287A - Polymerase chain reaction enhancing method - Google Patents
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Abstract
The invention discloses a polymerase chain reaction enhancing method. The polymerase chain reaction enhancing method includes the steps of adding a PCR (polymerase chain reaction) enhancer into a PCR amplification system so as to obtain an enhanced PCR amplification system, wherein the PCR enhancer refers to a mixture of hexanehexol and trehalose; setting program parameters of the enhanced PCR amplification system, and conducting amplification test so as to obtain an amplified product; testing the amplified product on a fluorescence quantitative PCR amplification instrument. The polymerase chain reaction enhancing method has the advantages that since the mixture of the hexanehexol and the trehalose is used as the PCR enhancer, nucleic acid amplification reaction efficiency and/or yield can be remarkably improved and/or increased, and the nucleic acid sensitivity detected by nucleic acid amplification is improved.
Description
Technical field
The present invention relates to biological technical field technical field, the enhanced method of more particularly to a kind of PCR.
Background technology
PCR (polymerase chain reaction；PCR it is) a kind of specific for amplification amplification
The Protocols in Molecular Biology of nucleic acid fragment (DNA/RNA), it is considered as the special DNA replication dna of in vitro, and the maximum of PCR is special
Point, is to be significantly increased micro nucleic acid.General PCR is predominantly adopted and is combined selected nucleic acid-templated (such as DNA or RNA) extremely
Few upstream, two kinds of downstream Oligonucleolide primers, in the effect of archaeal dna polymerase (such as Taq archaeal dna polymerases, Tth archaeal dna polymerases)
Under purpose nucleic acid template segments are expanded.Purify from restrictive digestion content using conventional method and obtain above two widow
Nucleotide primer, or above-mentioned primer can be produced by synthetic method.The primer preferably efficiency highest in amplification is single-stranded, but
Primer can also be double-strand.Double-chain primer is denatured first (such as make its denaturation using heating means), that is, process to separate each chain.
PCR experiment adopt it is nucleic acid-templated need not purify, can by technology as described in patent CN101665785B from
Nucleic acid is extracted in sample.Nucleic acid is available from various sources, such as plasmid or natural origin, including bacterium, yeast, virus, organelle
Or advanced bio.If nucleic acid-templated is single stranded RNA, need to be made before as pcr template single stranded RNA reverse transcription for DNA,
That is the DNA under RNA is instructed synthesizes.Can be by any suitable clone method, including chemistry or Enzymology method are completed under RNA guidances
DNA synthesis, through heat denatured, (denaturation temperature general range is about 90-105 DEG C to subsequent DNA, and the time typically carries out about 30 seconds
Or more long) become cDNA.If nucleic acid-templated is double-strand, need to open two chain separations before as pcr template.Can lead to
Any suitable denaturation method is crossed, including physics, chemistry or Enzymology method make chain separation.
Since being established from PCR method, in the time of 30 years, have developed rapidly, a series of existing PCR methods are devised
Come, and be widely used in each field such as diagnosis, tumor research, pathogen detection, Genotyping, the legal medical expert of genetic disease, having
Very strong practicality.
PCR amplification procedures are restricted by several factors, such as the performance of archaeal dna polymerase, PCR cushioning liquid composition, primer
The condition such as base sequence and length, the annealing temperature for adopting.Amplification to some specific nucleic acid fragment regions, design of primers position
It is limited；Or in multiple PCR reactions, the various feelings such as the optimization of the condition such as PCR cushioning liquid composition, annealing temperature is limited
Condition, can all cause the imperfect of PCR amplifications.
The content of the invention
The goal of the invention of the present invention is to provide a kind of PCR enhanced method, to improve PCR reactions
Efficiency and amplification yield.
Embodiments in accordance with the present invention, there is provided a kind of enhanced method of PCR, methods described includes：
PCR reinforcing agents are added in PCR amplification system, enhanced PCR amplification system is obtained, the PCR reinforcing agents are
The mixture of hexitol and trehalose；
The program parameter of the enhanced PCR amplification system is set, and carries out amplification experiment, obtain amplified production；
The amplified production is placed on fluorescent quantitative PCR instrument and is tested.
Further, the PCR reinforcing agents are the mixture of D-sorbite and trehalose.
Further, the PCR reinforcing agents are the mixture of mannitol and trehalose.
Further, the PCR reinforcing agents are the mixture of mannitol, D-sorbite and trehalose.
Further, the PCR amplification system, including：PCR cushioning liquid, dNTPs, H-Taq enzyme, upstream primer, downstream is drawn
Thing, label, and base template, the base template is DNA profiling or RNA templates, the label be fluorescence probe or
The dyestuffs of SYBR Green I.
Further, the PCR amplification system also includes：Tth enzymes and manganese acetate.
Further, it is described that the program parameter of enhanced PCR amplification system is set, and amplification experiment is carried out, expanded
The step of product, includes：
If the base template is DNA profiling：
The DNA profiling is pre-processed, 94 degrees Celsius of heating-up temperature, obtains pretreated at 5 minutes heat times
DNA profiling；
Degenerative treatments are carried out to the pretreated DNA profiling, denaturation temperature is 94 degrees Celsius, and denaturation time is 15
Second, obtain the DNA profiling after denaturation；
DNA profiling after the denaturation is annealed and extension is processed, the temperature of annealing is 57 degrees Celsius；Annealing when
Between be 10 seconds, obtain amplified production.
Further, it is described that the program parameter of enhanced PCR amplification system is set, and carry out expanding experiment and obtain amplification and produce
The step of thing, includes：
If the base template is RNA templates：
Activation Taq enzyme, while heating destroys part duplex structure in the RNA templates, obtains single stranded RNA template, plus
Hot temperature is 95 degrees Celsius, and the heat time is 1 minute；
Reverse transcription process is carried out to the single stranded RNA template, cDNA templates are obtained, the reverse transcription temperature is 60 Celsius
Degree, reverse transcription time is 30 minutes；
The cDNA templates are heated, pretreated base template is obtained, the heating-up temperature is taken the photograph for 95
Family name's degree, the heat time is 1 minute；
Degenerative treatments are carried out to the pretreated base template, the base template after denaturation is obtained, at the denaturation
The temperature of reason is 95 degrees Celsius, and the time of the degenerative treatments is 15 seconds；
Base template after the denaturation is annealed and extension is processed, obtained amplified production, the temperature of the annealing
For 60 degrees Celsius, the time of the annealing is 30 seconds.
From above technical scheme, the embodiment of the present invention illustrates a kind of enhanced method of PCR, described
Method comprises the steps：PCR reinforcing agents are added in the PCR amplification system, enhanced PCR amplification system, institute is obtained
PCR reinforcing agents are stated for hexitol and the mixture of trehalose；The program parameter of the enhanced PCR amplification system is set, and
Amplification experiment is carried out, amplified production is obtained；The amplified production is placed on fluorescent quantitative PCR instrument and is tested.This
Invention provides PCR enhanced method, can be shown as PCR reinforcing agents using the mixture of hexitol and trehalose
The efficiency and/or yield for improving nucleic acid amplification reaction is write, so as to improve the sensitivity that nucleic acid is detected using nucleic acid amplification reaction.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to institute in embodiment
The accompanying drawing that needs are used is briefly described, it should be apparent that, drawings in the following description are only some enforcements of the present invention
Example, for those of ordinary skill in the art, on the premise of not paying creative work, can be being obtained according to these accompanying drawings
Obtain other accompanying drawings.
Fig. 1 is the flow chart according to an a kind of enhanced method of PCR for being preferable to carry out exemplifying；
Fig. 2 is the DNA that extracted with the oropharynx swab sample of 8 Respiratory Tract Adenovirus (ADV) the infecteds as treating test sample
This, control group augmentation detection result；
Fig. 3 is the DNA that extracted with the oropharynx swab sample of 8 Respiratory Tract Adenovirus (ADV) the infecteds as treating test sample
This, experimental group augmentation detection result；
Fig. 4 is the RNA that extracted with the plasma sample of 7 human immunodeficiency virus (HIV) the infecteds as treating test sample
This control group augmentation detection result；
Fig. 5 is the RNA that extracted with the plasma sample of 7 human immunodeficiency virus (HIV) the infecteds as treating test sample
This control group augmentation detection result；
Fig. 6 is the α-globin gene for detecting human peripheral sample DNA, by agarose gel electrophoresis analysis result；
Fig. 7 is the type RNA nucleic acid of parainfluenza 3 in detection mouth throat swab, by agarose gel electrophoresis analysis result.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Whole description, it is clear that described embodiment is only a part of embodiment of the invention, rather than the embodiment of whole.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
The embodiment of the present invention illustrates a kind of enhanced method of PCR, as shown in figure 1, methods described include as
Lower step：
S101, adds PCR reinforcing agents in the PCR amplification system, obtains enhanced PCR amplification system, the PCR
Reinforcing agent is the mixture of hexitol and trehalose；
S102, arranges the program parameter of the enhanced PCR amplification system, and carries out amplification experiment, obtains amplification and produces
Thing；
S103, the amplified production is placed on fluorescent quantitative PCR instrument and is tested.
Further, the PCR reinforcing agents are the mixture of D-sorbite and trehalose.
Further, the PCR reinforcing agents are the mixture of mannitol and trehalose.
Further, the PCR reinforcing agents are the mixture of mannitol, D-sorbite and trehalose.
PCR reinforcing agents typically PCR reaction start before be added in PCR amplification system, the concentration that PCR reinforcing agents are used by
The determination of many factors such as composition, the design conditions of primer and/or probe, the PCR primer length of PCR amplification system, this
Bright preferred version is：In template is for the PCR amplification system of DNA, the concentration of D-sorbite and trehalose is respectively
0.2M and 0.25M；In template in the PCR amplification system of RNA, the concentration of D-sorbite and trehalose be respectively 0.1M and
0.25M.By our experimental verification, an isomer mannitol of D-sorbite, it is used simultaneously with trehalose,
The expanding effect of PCR can be improved, in the present invention, it is preferred to D-sorbite and trehalose are enhancing of the blend compositions as PCR
Agent.
Further, PCR cushioning liquid, dNTPs, H-Taq enzyme, upstream primer, downstream primer, label, and base template,
The base template is DNA profiling or RNA templates, and the label is fluorescence probe or the dyestuffs of SYBR Green I.
Add a kind of PCR reinforcing agents in the PCR amplification system of embodiment of the present invention PCR reaction, can significantly improve PCR's
Efficiency and/or yield.PCR amplification system composition at least contains following component：PCR cushioning liquid, archaeal dna polymerase, dNTPs, draw
Thing and/or probe and the metal cation required for catalytic dna polymerase.Common PCR cushioning liquid by Tris-HCl,
MgCl2, the buffer system such as KCl, Triton X-100 constitutes.Cumulative volume is 20-200 μ l, PCR in general single PCR reaction tubes
The component such as archaeal dna polymerase, dNTPs, metal cation of addition suitable concn in cushioning liquid, wherein primer and/or probe
Concentration can be 0.05 μM -5 μM.
For base template is the PCR amplification system of DNA profiling, preferred embodiments of the present invention are：
| Component | Volume/concentration in single reaction |
| 5 × PCR cushioning liquid * | 10μL |
| dNTPs(100mM) | 1.5μL |
| H-Taq enzymes (5U/ μ L) | 1.0μL |
| Upstream primer | 0.5μM |
| Downstream primer | 0.5μM |
| Fluorescence probe | 0.25μM |
| The dyestuffs of 20 × SYBR Green I | 2.5μL |
| DNA profiling | 10ng |
| Sterilizing purified water | Up to 50μL |
Wherein：5 × PCR cushioning liquid by 200mmol/L Tris-HCl, 30mmol/L MgCl2,500mmol/L KCl,
0.2% (V/V) Triton X-100,10% (V/V) formamide (formamide) and the purified water of sterilizing composition, -20 ± 5 DEG C
Preserve.
Further, the PCR amplification system also includes：Tth enzymes and manganese acetate.
For base template is the PCR amplification system of RNA templates, preferred embodiments of the present invention are：
| Component | Volume/concentration in single reaction |
| 5 × PCR cushioning liquid * | 10μL |
| dNTPs(100mM) | 1.5μL |
| Tth enzymes (5U/ μ l) | 1.0μL |
| H-Taq enzymes (5U/ μ l) | 1.0μL |
| Mn(OAc)2(150mM) | 1.0μL |
| Upstream primer | 0.5μM |
| Downstream primer | 0.5μM |
| Fluorescence probe | 0.25μM |
| The dyestuffs of 20 × SYBR Green I | 2.5μL |
| RNA templates | 10ng |
| Sterilizing purified water | Up to 50μL |
Wherein：5 × PCR cushioning liquid by 0.25mol/L N, N- bicine N-s/potassium hydroxide (Bicine/
KOH),pH 8.2；The potassium acetate (Potassium acetate) and 40% (v/v) glycerine (glycerol) group of 575mmol/L
Into.
Further, it is described that the program parameter of enhanced PCR amplification system is set, and carry out being wrapped the step of amplification is tested
Include：
If the base template is DNA profiling：
94 degrees Celsius of the heating-up temperature that the DNA profiling is pre-processed, 5 minutes heat times, after being pre-processed
DNA profiling；
The denaturation temperature that degenerative treatments are carried out to pretreated DNA profiling is 94 degrees Celsius, and denaturation time is 15
Second, obtain the DNA profiling after denaturation；
The DNA profiling to after denaturation is annealed, and the temperature of the annealing that extension and fluorescent collecting are processed is 57 Celsius
Degree；The time of annealing is 10 seconds, obtains amplified production.
Pcr amplification reaction process shown in the embodiment of the present invention is：
1) heat denatured, the condition of denaturation heating depends on the length and core of buffer solution salt ionic concentration and denaturing nucleic acid
Thuja acid is constituted, but general scope is about 90 DEG C -105 DEG C, and time of denaturation depends on response feature and length nucleic acid, but typically
Carry out -4 minutes about 30 seconds.
2) anneal, make on the target sequence of each primer annealing to template nucleic acid.The temperature of annealing is usually 35 degree Celsius -65 and takes the photograph
Family name's degree (such as from about 40 degrees Celsius -60 degrees Celsius), the time of annealing can be 10 seconds to 1 minute (such as from about -40 seconds 30 seconds).
3) extend, that is, be enough to be extended the temperature of the product for producing complementary with template nucleic acid by annealing primer, but in the temperature
Under, (elongating temperature is generally 40 degrees Celsius -80 degrees Celsius to extension products, and extension of time can be about with complementary template consistency
10 seconds to 5 minutes).
For base template be DNA profiling, the program parameter of enhanced PCR amplification system, preferred embodiments of the present invention
For：
Further, it is described that the program parameter of enhanced PCR amplification system is set, and carry out being wrapped the step of amplification is tested
Include：
If the base template is RNA templates：
The activation Taq enzyme, while heating destroys part duplex structure in the RNA templates, obtains single stranded RNA template
Heating-up temperature be 95 degrees Celsius, the heat time be 1 minute, obtain single stranded RNA template；
The reverse transcription temperature that reverse transcription process is carried out to single stranded RNA template is 60 degrees Celsius, and reverse transcription time is 30
Minute；
The heating-up temperature heated to cDNA templates is 95 degrees Celsius, and the heat time is 1 minute；
Described that degenerative treatments are carried out to pretreated base template, the temperature of the degenerative treatments is 95 degrees Celsius, institute
The time for stating degenerative treatments is 15 seconds；
The base template to after denaturation is annealed, and the temperature of the annealing that extension and fluorescent collecting are processed is 60 Celsius
Degree, the temperature of the annealing is 30 seconds.
For base template be RNA templates, the program parameter of enhanced PCR amplification system, preferred embodiments of the present invention
For：
Interpretation of result：
Embodiment one
The DNA extracted using the oropharynx swab sample of 8 Respiratory Tract Adenovirus (ADV) the infecteds as sample to be tested,
With the PCR reaction systems detection of reinforcing agent is not added with as control, another is to be added with D-sorbite and trehalose to portion
PCR reaction systems are detected as experimental group, shown in Fig. 2 and Fig. 3：
Control group, experimental group Ct Data-Statistics：
It can be seen that, the present invention provides PCR enhanced method, extra large using addition 0.2M D-sorbites and 0.25M
The mixture of algae sugar can significantly improve the efficiency of 8 Respiratory Tract Adenovirus (ADV) DNA nucleic acid amplification reactions as PCR reinforcing agents
And/or yield, so as to improve the sensitivity that nucleic acid is detected using nucleic acid amplification reaction.
Embodiment two
The RNA extracted using the plasma sample of 7 human immunodeficiency virus (HIV) the infecteds as sample to be tested,
With the PCR reaction systems detection of reinforcing agent is not added with as control, another is to be added with D-sorbite and trehalose to portion
PCR reaction systems are detected as experimental group, as a result shown in Fig. 4 and Fig. 5：Control group, experimental group Ct Data-Statistics：
It can be seen that, the present invention provides PCR enhanced method, using 0.1M D-sorbites and 0.25M trehaloses
Mixture the efficiency of 7 human immunodeficiency virus (HIV) RNA nucleic acid amplification reactions can be significantly improved as PCR reinforcing agents
And/or yield, so as to improve the sensitivity that nucleic acid is detected using nucleic acid amplification reaction
Embodiment three：
The α-globin gene of detection human peripheral sample DNA, by agarose gel electrophoresis analysis result such as Fig. 6 institutes
Show：
Wherein, α-thalassemia α α/α α wild-type amplification results (1.7kb) 3：DNA Markers；1：Experimental group (adds
Plus 0.2M D-sorbites and 0.25M trehaloses)；
2 control groups (are not added with)；
It can be seen that, the present invention provides PCR enhanced method, using 0.2M D-sorbites and 0.25M trehaloses
Mixture can significantly improve as PCR reinforcing agents human peripheral sample DNA α-globin nucleic acid amplified reaction efficiency
And/or yield, so as to improve the sensitivity that nucleic acid is detected using nucleic acid amplification reaction.
Example IV：
The type RNA nucleic acid of parainfluenza 3 in detection mouth throat swab, by agarose gel electrophoresis analysis result such as Fig. 7；
Wherein, the type target conservative fragments of parainfluenza 3 detection (150bp) 3：DNA Markers；4：Experimental group (addition 0.1M
D-sorbite and 0.25M trehaloses)；
5 control groups (are not added with).
It can be seen that, the present invention provides PCR enhanced method, using 0.2M D-sorbites and 0.25M trehaloses
Mixture as PCR reinforcing agents can significantly improve in oropharynx swab the efficiency of the amplified reaction of the type RNA nucleic acid of parainfluenza 3 and/
Or yield, so as to improve the sensitivity that nucleic acid is detected using nucleic acid amplification reaction.
From above technical scheme, the embodiment of the present invention illustrates a kind of enhanced method of PCR, described
Method comprises the steps：PCR reinforcing agents are added in the PCR amplification system, enhanced PCR amplification system, institute is obtained
PCR reinforcing agents are stated for hexitol and the mixture of trehalose；The program parameter of the enhanced PCR amplification system is set, and
Amplification experiment is carried out, amplified production is obtained；The amplified production is placed on fluorescent quantitative PCR instrument and is tested.This
Invention provides PCR enhanced method, can be shown as PCR reinforcing agents using the mixture of hexitol and trehalose
The efficiency and/or yield for improving nucleic acid amplification reaction is write, so as to improve the sensitivity that nucleic acid is detected using nucleic acid amplification reaction.
Those skilled in the art will readily occur to its of the present invention after considering specification and putting into practice invention disclosed herein
Its embodiment.The application is intended to any modification of the present invention, purposes or adaptations, these modifications, purposes or
Person's adaptations follow the general principle of the present invention and including the undocumented common knowledge in the art of the present invention
Or conventional techniques.Description and embodiments are considered only as exemplary, and true scope and spirit of the invention are by following
Claim is pointed out.
It should be appreciated that the precision architecture for being described above and being shown in the drawings is the invention is not limited in, and
And can without departing from the scope carry out various modifications and changes.The scope of the present invention is only limited by appended claim.
Claims (8)
1. a kind of enhanced method of PCR, it is characterised in that methods described includes：
PCR reinforcing agents are added in PCR amplification system, enhanced PCR amplification system is obtained, the PCR reinforcing agents for oneself six
The mixture of alcohol and trehalose；
The program parameter of the enhanced PCR amplification system is set, and carries out amplification experiment, obtain amplified production；
The amplified production is placed on fluorescent quantitative PCR instrument and is tested.
2. method according to claim 1, it is characterised in that the PCR reinforcing agents are the mixed of D-sorbite and trehalose
Compound.
3. method according to claim 1, it is characterised in that the PCR reinforcing agents are the mixing of mannitol and trehalose
Thing.
4. method according to claim 1, it is characterised in that the PCR reinforcing agents are mannitol, D-sorbite and marine alga
The mixture of sugar.
5. the method according to any one of claim 1-4, it is characterised in that the PCR amplification system, including：PCR is buffered
Solution, dNTPs, H-Taq enzyme, upstream primer, downstream primer, label, and base template, the base template is DNA profiling
Or RNA templates, the label is fluorescence probe or the dyestuffs of SYBR Green I.
6. method according to claim 5, it is characterised in that the PCR amplification system also includes：Tth enzymes and manganese acetate.
7. method according to claim 5, it is characterised in that the program ginseng of the enhanced PCR amplification system of the setting
Number, and carries out amplification experiment, includes the step of obtain amplified production：
If the base template is DNA profiling：
The DNA profiling is pre-processed, 94 degrees Celsius of heating-up temperature, obtains pretreated DNA at 5 minutes heat times
Template；
Degenerative treatments are carried out to the pretreated DNA profiling, denaturation temperature is 94 degrees Celsius, and denaturation time is 15 seconds, is obtained
DNA profiling to after denaturation；
DNA profiling after the denaturation is annealed and extension is processed, the temperature of annealing is 57 degrees Celsius；The time of annealing is
10 seconds, obtain amplified production.
8. method according to claim 6, it is characterised in that the program ginseng of the enhanced PCR amplification system of the setting
Counting, and carry out the step of amplification experiment obtains amplified production includes：
If the base template is RNA templates：
Activation Taq enzyme, while heating destroys part duplex structure in the RNA templates, obtains single stranded RNA template, heating temperature
Spend for 95 degrees Celsius, the heat time is 1 minute；
Reverse transcription process is carried out to the single stranded RNA template, cDNA templates are obtained, the reverse transcription temperature is 60 degrees Celsius, inverse
The transcription time is 30 minutes；
The cDNA templates are heated, pretreated base template is obtained, the heating-up temperature are 95 degrees Celsius,
Heat time is 1 minute；
Degenerative treatments are carried out to the pretreated base template, the base template after denaturation is obtained, the degenerative treatments
Temperature is 95 degrees Celsius, and the time of the degenerative treatments is 15 seconds；
Base template after the denaturation is annealed and extension is processed, obtained amplified production, the temperature of the annealing is 60
Degree Celsius, the time of the annealing is 30 seconds.
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Cited By (5)
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| CN107119137A (en) * | 2017-05-26 | 2017-09-01 | 广州华弘生物科技有限公司 | A kind of detection kit and detection method of quick detection HER 2/neu gene expressions |
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| CN111893216A (en) * | 2020-08-11 | 2020-11-06 | 北京毅新博创生物科技有限公司 | Product for detecting DNA/RNA by nucleic acid mass spectrum and detection method |
| CN116121349A (en) * | 2022-12-20 | 2023-05-16 | 南京诺唯赞生物科技股份有限公司 | Method for amplifying nucleic acid sample containing ethanol |
| CN116121349B (en) * | 2022-12-20 | 2024-04-09 | 南京诺唯赞生物科技股份有限公司 | Method for amplifying nucleic acid sample containing ethanol |
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Application publication date: 20170426 |