CN101343650B - Polyase chain reaction optimization method based on arborized polymer - Google Patents
Polyase chain reaction optimization method based on arborized polymer Download PDFInfo
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Abstract
The invention discloses a polymerase chain reaction optimizing method based on dendritic polymer, which belongs to the technical field of the biology. A dendritic polymer material is added into a polymerase chain reaction system as an additive agent to optimize the polymerase chain reaction, the dendritic polymer comprises multivalent nucleus combined with at least two dendritic branching covalences and a branch structure which can ensure the polymerization degree to achieve at least 2.0 through at least two generations of expansion. The dendritic polymer is diluted or concentrated and added into the polymerase chain reaction system in a gradient way, so as to achieve the range of the optimization purpose through actual amplification, and the range is the effective use amount range of the required dendritic polymer. The invention has the advantages that the optimization and amplification effects are remarkable, the cost of the additive agent is low, the amplified product is easy to be preserved, the application of the amplified product is wide, the specificity of the amplified product is obviously improved, and the output of the product in certain reactions is obviously enhanced.
Description
Technical field
What the present invention relates to is the reaction method in a kind of biological technical field, particularly a kind of polyase chain reaction optimization method based on branch-shape polymer.
Background technology
Polymerase chain reaction (PCR) is a kind of nucleic acid amplification technologies at in-vitro simulated natural dna replication dna.Round pcr is similar to the natural reproduction process of DNA, and its specificity depends on and target sequence two ends complementary Oligonucleolide primers.PCR mainly extends three step thermal cyclings repeatedly by high-temperature denatured, low-temperature annealing and thermophilic and constitutes; The DNA that circulation is each time produced all can become round-robin template next time; The special district of the DNA copy number that circulates each time between the primer that all makes two synthetic increases one times, and the PCR product is able to 2
nExponential form increase rapidly, after 25～30 circulations, can make gene amplification 10 in theory
9Doubly.
But round pcr is high, highly sensitive because of its specificity, simple fast, to advantages such as the low quantitative templates numbers of the purity requirement of sample, extensive day by day in the application of bio-science various fields.But in the actually operating, carry out pcr amplification and still have the improved problem of some needs, like problems such as specificity, sensitivity, speed of response.Distinct issues are exactly that pcr amplification exists in various degree non-specific.PCR is as a kind of in-vitro simulated DNA chain type amplified reaction, and mechanism is very complicated, and interference side reaction to a certain degree always can take place in the Kettenreaktion process, as the primer template maybe mispairing, primer combines to form amplification that dimer etc. causes or the like.Scientists attempts addressing this problem from different angles, as from template, primer, Mg
2+Concentration, annealing temperature equal angles are improved its specificity, and perhaps with synergistic agent such as methane amide, glycerine, DMSO 99.8MIN., trimethyl-glycine, Tetramethylammonium chloride etc. are introduced the PCR system.But in actual application, some effect is not fully up to expectations, even can suppress the amplification procedure of PCR.
U.S. Pat PATENT5,646 are found in retrieval through to the prior art document; 019 discloses " a kind of preparation method who causes the amplification of nucleic acid template that is beneficial to ", and this method is in the PCR system, to have added heat-stable single strand binding protein (SSB, single-stranded nucleic acid binding protein); SSB albumen only combines single stranded DNA; But the debond double-stranded DNA contains the non-specific segmental amplification of strand through combining and suppressing, thereby has 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 a 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 keeps time limit shorter.
Summary of the invention
The present invention is directed to the deficiency of prior art, a kind of polyase chain reaction optimization method based on branch-shape polymer is provided, make its specific amplification that improves the PCR reaction and the output of target dna, do not cause obviously increasing of cost simultaneously.The present invention adds to through the branch-shape polymer material with significant quantity and carries out the PCR reaction in the system of polymerase chain reaction, reaches the specificity that increases the PCR reaction, improves the effect of the output of PCR title product.Simultaneously,, be easy to preserve, reduce greatly than costs such as other biological molecule interpolation material SSB because branch-shape polymer is with low cost.
The present invention realizes that through following technical scheme the present invention adds the branch-shape polymer material and optimizes the polymerase chain reaction as additive in the system of polymerase chain reaction.
Described branch-shape polymer comprises and two covalently bound multivalence nuclears of dendroid branch, and through the expansion of at least two generations, make the polymerization degree reach 2.0 (>=G at least at least
2) branched structure.
In principle, all branch-shape polymers all are applicable to the present invention.Branch-shape polymer (generally acknowledge in the industry and be referred to as dendrimer) is represented the three-dimensional macromole of one type of dendritic structure, and it has the geometrical shape and the chemical structure of clearly definition.The branch of this quasi-molecule mind-set from a core or has the peripheral radiation of functional group, and inside has wide cavity, has very high surface density and ordering degree, is easy to carry out functionalized.The branch-shape polymer instance that is fit to is seen Angew.Chem.Int.Ed.Engl, and 1990,29 (2) the 138-175 pages said.
Suitable branch-shape polymer comprise have the branch-shape polymer that improves branched structure and in branched structure defective branch-shape polymer, the branch-shape polymer of symmetrical branching preferably among the present invention.
Described branch-shape polymer preferably contains the branch-shape polymer of N-terminal end group group.According to the increase of the polymerization degree, the terminal amino group group of polymkeric substance is Exponential growth, i.e. terminal amino group group number=2
(polymerization degree+2)
On the N-terminal group of said branch-shape polymer, can partly or entirely connect various modification functional groups, preferably from the modification group of following groups: ethanoyl, carboxylic acid group, hydroxyl.The modifying method of end group is known, can obtain through all or part of and suitable reaction reaction of amino end group that makes last step.
Described branch-shape polymer is polymeric amide-amine type or Vestolen PP 7052 imines type dendrimer.Wherein polymeric amide-amine type polymkeric substance is nucleus growth with quadrol or tetramethylenediamine.Polymeric amide-amine type (PAMAM) dendrimer that the present invention preferably examines based on quadrol.
Said polymeric amide-amine type branch-shape polymer based on quadrol nuclear is the aqueous solution, can make the polymerization degree by oneself and be 1.0 to 10.0 sample, and its preparation method is known at present, and concrete method for making can be referring to document (Macromolecules, 1986,19,2466; Angew.Chem.Int.Ed.Engl, 1993,32 (9), 1306), the modifying method of end group is known.For example, the reaction of ethanoyl modification dendrimer terminal amino group is:
The reaction that the carboxylic acid group modifies is:
The amino reaction of hydroxyl modified is:
The polymerization degree of said polymeric amide-amine type branch-shape polymer based on quadrol nuclear is with 3.0 to 8.0 (G
3～G
8) be good, preferred degree of polymerization 4.0 or 5.0 (G
4Or G
5) polymeric amide-amine type dendrimer.Following chemical formula shows that the polymerization degree is polymeric amide-amine type dendrimer of 4.0.
In practical application, the branch-shape polymer material of significant quantity joined as additive carry out the PCR reaction in the system of polymerase chain reaction.Said significant quantity is meant the effective level scope that produces optimization function.Different to different, the terminal end groups of the branch-shape polymer material polymerization degree, the polymerase chain reaction system is different, and this branch-shape polymer effective level scope that produces the required interpolation of optimization function is different.Those skilled in the art can test through following method easily and draw effective level:
To various polymerization PCR system; The branch-shape polymer of the different end group and the polymerization degree produces the effective level scope of optimization function and confirms through following method: through this kind branch-shape polymer being carried out gradient dilution or concentrating; Join in this polymerase chain reaction system with the concentration gradient mode, reaching the scope of optimizing purpose with the reality amplification is the effective level scope of required this branch-shape polymer.
The amplification system that said polymerase chain reaction system can be those systems that are easy to generate non-specific amplification, GC content is high, template number copy (less than 10 for low
3) system, complicated genetic background system or comprise the system of above-mentioned several factors.
For example, the PCR that produces non-specific amplification amplification system again can be used as a typical system.PCR amplification system again is meant that the product with amplification for the first time dilutes the template of back as the amplification second time, carries out the amplification second time.When 1000 times of templates of product dilution as the amplification second time with amplification for the first time; And when under conventional pcr amplification system, increasing; Can produce a large amount of non-specific amplification products; And if in this PCR system, add the branch-shape polymer material of an amount of particular end end group, then can eliminate these non-specific amplifications, on agarose gel electrophoresis figure, obtain single title product;
Said polymerase chain reaction system also can be to adopt the amplification system of pfu archaeal dna polymerase.
The general composition of described PCR system is shown in various biology tool books commonly used or various commercial polysaccharase specification sheets.With Takara company Ex Taq enzyme is example, the consisting of an of pcr amplification reaction:
| Ex Taq enzyme (5U/ μ L) | 0.125μL |
| 10 * PCR damping fluid (does not contain Mg 2+) | 2.5μL |
| DNTP substrate (2.5mM) | 2μL |
| Mg 2+(25mM) | 1.5μL |
| Primer 1 (10 μ M) | 0.5μL |
| Primer 2 (10 μ M) | 0.5μL |
| Template | 0.5μL |
| H 2O | Adding water, to supply TV be 25 μ L |
The general process of described PCR reaction is:
(1) preparation PCR reaction system comprises the archaeal dna polymerase, primer, template, dNTP, PCR damping fluid of suitable amounts etc.;
(2) on the PCR appearance, carry out the PCR reaction with the program of setting;
(3) product after amplification is accomplished detects.
When this PCR reaction needed is optimized, can in preparation PCR reaction system process, add the additive of optimization function, in the present invention, additive is the branch-shape polymer material of significant quantity.
The PCR product detects can adopt multiple detection method; As being carried out agarose gel electrophoresis, the DNA sample after the amplification detects; Or the back sample that increases is carried out capillary electrophoresis detect, or on the real-time quantitative PCR appearance, carry out the real-time amplification procedure that real-time quantitative detects PCR.
The present invention compares with existing method, and it is remarkable to have the effect of optimizing amplification, and additive branch-shape polymer lower cost for material is easy to preserve widely used advantage.The specificity of amplified production is significantly improved, and the output of product also is significantly improved in some reaction.The aqueous solution of branch-shape polymer can prolonged preservation at 4 ℃, and do not have deactivated worry.Commercialization branch-shape polymer price (generally be lower than 0.05 yuan/reaction) is also cheap than single strand binding protein (0.48 yuan/reaction), and reagent was all once good, is easy to preserve and accurate application of sample.PCR reaction system after the optimization of the present invention is not only applicable to the accurate PCR appearance of temperature control, is applicable to the PCR appearance that temperature control performance is relatively poor yet.The method of in the PCR reaction, adding branch-shape polymer of the present invention development 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: once amplification is unsuccessful and needs increases again, the amplification that copies template is hanged down in the amplification of high GC content, the amplification of human genome GAP, high background, extinct plants and animal DNA cloning or the like.The present invention also is applicable to the real-time quantitative PCR reaction.The present invention has potential and huge using value to fields such as gene test and clone, genetic analysis, clinical diagnosis, gene chip and novel materials.
Description of drawings
Fig. 1 adds the polymeric amide-amine type dendrimer (G of the polymerization degree 4.0
4-dendrimer) the serious PCR that increases again of non-specific amplification is reacted the design sketch that is optimized;
Fig. 2 adds the polymeric amide-amine type dendrimer (G of the polymerization degree 5.0
5-dendrimer) the serious PCR that increases again of non-specific amplification is reacted the design sketch that is optimized;
Fig. 3 adds the polymeric amide-amine type dendrimer (G of the acetylizad polymerization degree 5.0 of 25% terminal amino group
5-dendrimer-25%Ac) the serious PCR that increases again of non-specific amplification is reacted the design sketch that is optimized;
Fig. 4 adds the polymeric amide-amine type dendrimer (G of the acetylizad polymerization degree 5.0 of 50% terminal amino group
5-dendrimer-50%Ac) PCR to amplification rat Thy-1 gene reacts the design sketch that is optimized;
Fig. 5 adds the polymeric amide-amine type dendrimer (G of the terminal amino group 75% acetylizad polymerization degree 5.0
5-dendrimer-75%Ac) the serious PCR that increases again of non-specific amplification is reacted the design sketch that is optimized;
Fig. 6 adds the polymeric amide-amine type dendrimer (G of the terminal amino group 50% carboxylic acidization 50% acetylizad polymerization degree 5.0
5-dendrimer-50%COOH-50%Ac) PCR to amplification rat Thy-1 gene reacts the design sketch that is optimized;
Fig. 7 adds the polymeric amide-amine type dendrimer (G of the polymerization degree 5.0
5-dendrimer) PCR that increases again to low temperature thermal oxidation reacts the design sketch that is optimized.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
Carrying out some To Templates seldom or during the extremely rare amplification experiment of sample; Usually can finding once increases can't obtain target stripe; At this moment just need increase again; Promptly proceed secondary amplification as template, improve the output of target stripe in the product with the product that obtains that once increases.But also can form some nonspecific amplifications when improving output with increasing again.
To the PCR of this formation non-specific amplification situation that increases again, realize optimization to it through the branch-shape polymer that adds significant quantity to PCR again in the amplification system.
Particularly, comprise the following steps:
1, preparation PCR system is carried out the amplification first time.
System is formed like following table:
| Takara Ex Taq enzyme (5U/ μ L) | 0.125μL |
| 10 * PCR damping fluid (does not contain Mg 2+) | 2.5μL |
| DNTP substrate (2.5mM) | 2μL |
| Mg 2+(25mM) | 1.5μL |
| Primer p1 (10 μ M) | 0.5μL |
| Primer p2 (10 μ M) | 0.5μL |
| Template | 0.5μL |
| H 2O | Supply TV to 25 μ L |
Wherein, template is 1ambdaDNA, and dilution is 10
7Copy/ μ L, Ex Taq enzyme are available from Takara company, and expanding fragment length is 283bp.Primer sequence is:
p1：5’-GGCTTCGGTCCCTTCTGT-3’；
p2：5’-CACCACCTGTTCAAACTCTGC-3’。
Amplification condition is: 95 ℃ of preheatings 3 minutes; 30 circulations, each circulation comprises: 94 ℃ of sex change 20 seconds, 55 ℃ of annealing 30 minutes, 72 ℃ were extended 30 seconds; Last 72 ℃ are extended 5min.
DNA sample after the amplification is carried out agarose gel electrophoresis detect, find bright simple target band, explain that amplification is good for the first time.
2, prepare PCR amplification system again.
System is formed as above table, but different be that template is above-mentioned first time of the amplified production of 1000 times of dilutions, primer sequence is still:
p1：5’-GGCTTCGGTCCCTTCTGT-3’；
p2：5’-CACCACCTGTT?CAAACTCTGC-3’。
Simultaneously, adding that water supplies is before the 25 μ L, needs to add a certain amount of G
4-dendrimer solution.
3, branch-shape polymer is optimized material prepn
The polymeric amide of the polymerization degree 4.0-amine type dendrimer is expressed as G available from Sigma company (article number 412449-2.5G)
4-dendrimer, concentration is 1 μ g/ μ L, and is subsequent use after need in super clean bench, diluting 1000 times before using.
4, the G that in above PCR system, adds different volumes
4-dendrimer makes that the quality of dendrimer is respectively 0.5ng in per 25 μ L PCR reaction systems, 0.7ng, and 0.9ng, 1.0ng does simultaneously and does not add the corresponding contrast of optimizing medium.
5, utilize round pcr that template molecule is increased, amplification program is with amplification condition is identical for the first time.
6, the DNA sample after the amplification is carried out agarose gel electrophoresis and detect, the inspection amplified band compares with control systems.
Amplification is as shown in Figure 1.Be followed successively by M from left to right: molecular weight marker (DL2000 of Takara company is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp); 1 for not adding G
4The amplification system again of-dendrimer aqueous solution; 2,3,4,5 for adding 0.5ng respectively, 0.7ng, 0.9ng, 1.0ngG
4The amplification system again of-dendrimer; 6 for not adding the blank of template.Can find out and add G
4-dendrimer is that non-specific amplification obviously reduces among the system pcr amplification result of 0.5ng～0.9ng, particularly G
4The simple target band that amplification obtained becoming clear when-dendrimer add-on was the 0.7ng～0.9ng left and right sides, and do not add G accordingly
4The conventional PCR system amplification of-dendrimer shows serious follow-up hangover, shows that non-specific amplification is serious.This pcr amplification result has shown that the present invention has significant optimization effect for pcr amplification, particularly can improve the output of specific amplification and amplified production.
Reaction system and detailed process are with embodiment 1, and different is that the optimization agent of adding is homemade G
5-dendrimer, concentration is 3.24 μ g/ μ L, and is subsequent use after need in super clean bench, diluting 1000 times before using.
Amplification is as shown in Figure 2.Be followed successively by M from left to right: molecular weight marker (DL2000 of Takara company is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp); 1 for not adding G
5The amplification system again of-dendrimer aqueous solution; 2,3,4,5 for adding 0.65ng respectively, 0.81ng, 0.88ng, 0.97ngG
5The amplification system again of-dendrimer; 6 for not adding the blank of template.Can find out and add G
5-dendrimer is that non-specific amplification obviously reduces among the system pcr amplification result of 0.65ng～0.88ng, particularly G
5The simple target band that amplification obtained becoming clear when-dendrimer add-on was 0.81ng～0.88ng, and do not add G accordingly
5The conventional PCR system amplification of-dendrimer shows serious follow-up hangover, shows that non-specific amplification is serious.This pcr amplification result has shown that the present invention has significant optimization effect for pcr amplification, particularly can improve the output of specific amplification and amplified production.
Reaction system and detailed process are with embodiment 1, and different is that the optimization agent of adding is homemade G
5-dendrimer-25%Ac, concentration is 2.84 μ g/ μ L, and is subsequent use after need in super clean bench, diluting 1000 times before using.
Amplification is as shown in Figure 3.Be followed successively by M from left to right: molecular weight marker (DL2000 of Takara company is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp); 1 for not adding G
5The amplification system again of-dendrimer-25%Ac aqueous solution; 2,3,4,5 for adding 0.28ng respectively, 0.99ng, 1.14ng, 1.28ng G
5The amplification system again of-dendrimer-25%Ac; 6 for not adding the blank of template.Can find out and add G
5-dendrimer-25%Ac is that non-specific amplification obviously reduces among the system pcr amplification result of 0.28ng～1.14ng, particularly G
5The simple target band that amplification obtained becoming clear when-dendrimer-25%Ac add-on was 0.99ng～1.14ng, and do not add G accordingly
5The conventional PCR system amplification of-dendrimer-25%Ac shows serious follow-up hangover, shows that non-specific amplification is serious.This pcr amplification result has shown that the present invention has significant optimization effect for pcr amplification, particularly can improve the output of specific amplification and amplified production.
Reaction system and detailed process are with embodiment 1, and different is that the optimization agent of adding is homemade G
5-dendrimer-50%Ac, concentration is 4.92 μ g/ μ L, and is subsequent use after need in super clean bench, diluting 1000 times before using.
Amplification shows G
5-dendrimer-50%Ac is 0.25ng～2.0ng for increase the again effective level of PCR reaction of 25 μ L, and wherein optimizing consumption is 0.74ng～2.0ng.
Can obtain the very significantly band of several non-specific amplifications when being template amplification Thy-1 gene,, realize optimization it through the branch-shape polymer that in the PCR system, adds significant quantity to this formation non-specific amplification situation with the rat gene group.
Particularly, comprise the following steps:
1, preparation PCR system.
System is formed like following table:
| Takara Ex Taq enzyme (5U/ μ L) | 0.25μL |
| 10 * PCR damping fluid (does not contain Mg 2+) | 2.5μL |
| DNTP substrate (2.5mM) | 2μL |
| Mg 2+(25mM) | 1.5μL |
| Primer p3 (10 μ M) | 0.5μL |
| Primer p4 (10 μ M) | 0.5μL |
| Template | 0.5μL |
| H 2O | Supply TV to 25 μ L |
Wherein, template is the rat gene group, and concentration is 100ng/ μ L, and Ex Taq enzyme is available from Takara company, and expanding fragment length is 1026bp.Primer sequence is p3:5 '-ATGAACCCAGTCATCAGCA-3 '; P4:5 '-ATAGTTTTATTGGAGCTTGT-3 '.
2, branch-shape polymer is optimized material prepn
The optimization agent of adding is homemade G
5-dendrimer-50%Ac, concentration is 4.92 μ g/ μ L, and is subsequent use after need in super clean bench, diluting 1000 times before using.
3, the G that in above PCR system, adds different volumes
5-dendrimer-50%Ac makes that the quality of dendrimer is respectively 24.6ng in per 25 μ L PCR reaction systems, 39.4ng, and 49.2ng, 59.0ng, 68.9ng does simultaneously and does not add the corresponding contrast of optimizing medium.
5, utilize round pcr that template molecule is increased, amplification condition is: 94 ℃ of preheatings 3 minutes; 30 circulations, each circulation comprises: 94 ℃ of sex change 15 seconds, 49 ℃ of annealing 15 seconds, 72 ℃ were extended 1 minute; Last 72 ℃ are extended 3min.
6, the DNA sample after the amplification is carried out agarose gel electrophoresis and detect, the inspection amplified band compares with control systems.
Amplification is as shown in Figure 4.Be followed successively by M from left to right: molecular weight marker (DL2000 of Takara company is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp); 1 for not adding G
5The amplification system again of-dendrimer-50%Ac aqueous solution; 2,3,4,5,6 for adding 24.6ng respectively, 39.4ng, 49.2ng, 59.0ng, 68.9ng G
5The amplification system again of-dendrimer-50%Ac.Can find out and add G
5-dendrimer-50%Ac is that non-specific amplification obviously reduces among the system pcr amplification result of 24.6ng～68.9ng, particularly G
5The simple target band that amplification obtained becoming clear when-dendrimer-50%Ac add-on was the 49.2ng～68.9ng left and right sides, and do not add G accordingly
5The conventional PCR system amplification of-dendrimer-50%Ac shows serious follow-up hangover, shows that non-specific amplification is serious.This pcr amplification result has shown that the present invention has significant optimization effect for pcr amplification, particularly can improve the output of specific amplification and amplified production.
Reaction system and detailed process are with embodiment 1, and different is that the optimization agent of adding is homemade G
5-dendrimer-75%Ac, concentration is 2 μ g/ μ L, and is subsequent use after need in super clean bench, diluting 1000 times before using.
Amplification is as shown in Figure 5.Be followed successively by M from left to right: molecular weight marker (DL2000 of Takara company is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp); 1 for not adding G
5The amplification system again of-dendrimer-75%Ac aqueous solution; 2,3,4,5,6 for adding 2.0ng respectively, 3.0ng, 4.0ng, 5.0ng, 6.0ng G
5The amplification system again of-dendrimer-75%Ac; 7 for not adding the blank of template.Can find out and add G
5-dendrimer-75%Ac is that non-specific amplification obviously reduces among the system pcr amplification result of 2.0ng～4.0ng, particularly G
5The simple target band that amplification obtained becoming clear when-dendrimer-75%Ac add-on was 3.0ng～4.0ng, and do not add G accordingly
5The conventional PCR system amplification of-dendrimer-75%Ac shows serious follow-up hangover, shows that non-specific amplification is serious.This pcr amplification result has shown that the present invention has significant optimization effect for pcr amplification, particularly can improve the output of specific amplification and amplified production.
Reaction system and detailed process are with embodiment 1, and different is that the optimization agent of adding is homemade G
5-dendrimer-100%Ac, particle diameter is about 5nm, and concentration is 8 μ g/ μ L.
Amplification shows G
5-dendrimer-100%Ac is 20 μ g～24 μ g for increase the again effective level of PCR reaction of 25 μ L, and wherein optimizing consumption is 20 μ g～22 μ g.
Embodiment 8 adds the polymeric amide-amine type dendrimer (G of the terminal amino group 25% carboxylic acidization 75% acetylizad polymerization degree 5.0
5-dendrimer-25%COOH-75%Ac) the serious PCR of amplification again reaction is optimized to non-specific amplification
Reaction system and detailed process are with embodiment 1, and different is that the optimization agent of adding is G
5-dendrimer-25%COOH-75%Ac, concentration is 4 μ g/ μ L.
Amplification shows G
5-dendrimer-25%COOH-75%Ac is 28 μ g～50 μ g for increase the again effective level of PCR reaction of 25 μ L, and wherein optimizing consumption is 28 μ g～40 μ g.
Embodiment 9 adds the polymeric amide-amine type dendrimer (G of the terminal amino group 50% carboxylic acidization 50% acetylizad polymerization degree 5.0
5-dendrimer-50%COOH-50%Ac) the serious PCR of amplification again reaction is optimized to non-specific amplification
Reaction system and detailed process are with embodiment 1, and different is that the optimization agent of adding is homemade G
5-dendrimer-50%COOH-50%Ac, concentration is 5.6 μ g/ μ L.
Can know G according to amplification
5-dendr imer-50%COOH-50%Ac is 5.6 μ g～80.0 μ g for increase the again effective level of PCR reaction of 25 μ L, and wherein optimizing consumption is 5.6 μ g～61.6 μ g.
Embodiment 10 adds the polymeric amide-amine type dendrimer (G of the terminal amino group 50% carboxylic acidization 50% acetylizad polymerization degree 5.0
5-dendrimer-50%COOH-50%Ac) the PCR reaction to amplification rat Thy-1 gene is optimized
Reaction system and detailed process are with embodiment 5, and different is that the optimization agent of adding is homemade G
5-dendrimer-50%COOH-50%Ac, concentration is 5.6 μ g/ μ L, and is subsequent use after need in super clean bench, diluting 1000 times before using.
Amplification is as shown in Figure 6.Be followed successively by M from left to right: molecular weight marker (DL2000 of Takara company is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp); 1 for not adding G
5The amplification system again of-dendrimer-50%COOH-50%Ac aqueous solution; 2,3,4,5,6 for adding 28g respectively, 39.2g, 50.4g, 61.6g, 72.8g G
5The amplification system again of-dendrimer-50%COOH-50%Ac.Can find out and add G
5-dendrimer-50%COOH-50%Ac is that non-specific amplification obviously reduces among the system pcr amplification result of 39.2g～72.8g, particularly G
5The simple target band that amplification obtained becoming clear when-dendrimer-50%COOH-50%Ac add-on was 61.6g～72.8g, and do not add G accordingly
5The conventional PCR system amplification of-dendrimer-50%COOH-50%Ac shows serious follow-up hangover, shows that non-specific amplification is serious.This pcr amplification result has shown that the present invention has significant optimization effect for pcr amplification, particularly can improve the output of specific amplification and amplified production.
Embodiment 11 adds the polymeric amide-amine type dendrimer (G of the terminal amino group 75% carboxylic acidization 25% acetylizad polymerization degree 5.0
5-dendrimer-75%COOH-25%Ac) the serious PCR of amplification again reaction is optimized to non-specific amplification
Reaction system and detailed process are with embodiment 1, and different is that the optimization agent of adding is homemade G
5-dendrimer-75%COOH-25%Ac, concentration is 7.12 μ g/ μ L.
Can know G according to amplification
5-dendrimer-75%COOH-25%Ac is 10.6 μ g～80.0 μ g for increase the again effective level of PCR reaction of 25 μ L, and wherein optimizing consumption is 10.6 μ g～40.1 μ g.
Embodiment 12 adds the polymeric amide-amine type dendrimer (G of the 100% terminal amino group carboxylic acidifying polymerization degree 5.0
5-dendrimer-100%COOH) the serious PCR of amplification again reaction is optimized to non-specific amplification
Reaction system and detailed process are with embodiment 1, and different is that the optimization agent of adding is homemade G
5-dendrimer-100%COOH, concentration is 12.5 μ g/ μ L.
Can know G according to amplification
5-dendrimer-100%COOH is 2.5 μ g～90.0 μ g for increase the again effective level of PCR reaction of 25 μ L, and wherein optimizing consumption is 37.5 μ g～62.5 μ g.
Embodiment 13 adds the polymeric amide-amine type dendrimer (G of the polymerization degree 5.0
5-dendrimer) PCR of the amplification again reaction to low temperature thermal oxidation is optimized
Annealing temperature is to influence one of specific important factor of PCR.The selection of annealing temperature and time depends primarily on length, based composition and the concentration thereof of primer, also has the length of template sequence.Though select high degenerate temperature can improve the specificity of reaction, output can reduce greatly, this is very unfavorable of the extremely precious sample of considerably less or template for template; But reduce annealing temperature and be easy to introduce nonspecific amplification again.
To owing to reduce the PCR situation that non-specific amplification that annealing temperature forms increases the weight of, realize optimization to it through the branch-shape polymer that adds significant quantity to the PCR of different annealing temperature again in the amplification system.
Reaction system and detailed process be with embodiment 2, and in the program that the PCR that different is adopts increases again, annealing temperature descends successively, is respectively 55 ℃, and 50 ℃, 45 ℃, 40 ℃.In addition, the G that in the PCR of above 4 different annealing temperature system, adds equal volume
5-dendrimer makes G in per 25 μ LPCR reaction systems
5The quality of-dendrimer is 0.9ng, does simultaneously not add the corresponding contrast of optimizing medium.
Amplification is as shown in Figure 7.Be followed successively by M from left to right: molecular weight marker (DL2000 of Takara company is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp); 1,3,5,7 for not adding G
5The amplification system again of-dendrimer aqueous solution, its annealing temperature are respectively 55 ℃, and 50 ℃, 45 ℃, 40 ℃; 2,4,6,8 for to add 0.9ng G respectively under corresponding annealing temperature
5The amplification system again of-dendrimer; 9 for not adding the blank of template, and annealing temperature is 40 ℃.Can find out and add G
5The simple target band that the system pcr amplification of-dendrimer can increase and obtain becoming clear, and do not add G accordingly
5The conventional PCR system amplification of-dendrimer shows serious follow-up hangover, shows that non-specific amplification is serious.This pcr amplification result has shown that the present invention has significant optimization effect for the pcr amplification of low temperature thermal oxidation, particularly can improve the output of specific amplification and amplified production.
Claims (5)
1. polyase chain reaction optimization method based on branch-shape polymer; It is characterized in that; In the system of polymerase chain reaction, add the branch-shape polymer material and optimize the polymerase chain reaction as additive, said branch-shape polymer is based on the polyamide-amide type dendrimer of quadrol nuclear, wherein said quadrol nuclear and at least two dendroid branch covalent attachment; Said branch-shape polymer makes the polymerization degree from 3.0 to 8.0 through the expansion of at least two generations.
2. the polyase chain reaction optimization method based on branch-shape polymer according to claim 1 is characterized in that, described branch-shape polymer comprises N-terminal end group group.
3. the polyase chain reaction optimization method based on branch-shape polymer according to claim 2; It is characterized in that; On the N-terminal group of said branch-shape polymer, partly or entirely be connected with the modification group that is selected from following groups: ethanoyl, carboxylic acid group, hydroxyl.
4. the polyase chain reaction optimization method based on branch-shape polymer according to claim 1 is characterized in that, the said branch-shape polymer polymerization degree is 4.0 or 5.0.
5. according to each described polyase chain reaction optimization method of claim 1 to 3 based on branch-shape polymer; It is characterized in that; Said branch-shape polymer; Its effective level scope is confirmed through following method: through branch-shape polymer being carried out gradient dilution or concentrating, join in this polymerase chain reaction system with the concentration gradient mode, reaching the scope of optimizing purpose with the reality amplification is the effective level scope of required this branch-shape polymer.
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| CN1631936A (en) * | 2003-12-24 | 2005-06-29 | 上海市血液中心 | Polyamide-amine type branch-shape polymer nano materials, synthesis method and use thereof |
| CN1680573A (en) * | 2005-02-03 | 2005-10-12 | 上海交通大学 | Optimizing method of amplifying nucleic acid polymerase chain reaction |
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| CN1631936A (en) * | 2003-12-24 | 2005-06-29 | 上海市血液中心 | Polyamide-amine type branch-shape polymer nano materials, synthesis method and use thereof |
| CN1680573A (en) * | 2005-02-03 | 2005-10-12 | 上海交通大学 | Optimizing method of amplifying nucleic acid polymerase chain reaction |
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