| Rolling circle amplification(RCA)is an isothermal nucleic acid amplification technology.Compared to the traditional method of DNA amplification,RCA technology with high sensitivity,high specificity,easy operation and other characteristics has attracted much attention recently.Based on the principle of RCA,we designed the template DNA and the partially complementary primer DNA.The primer was used to link the ends of the template DNA by the base pairing,in the presence of DNA ligase enzyme.Under room temperature conditions,long chain DNA consisting of repeating units is synthesized by rolling circle amplification under the action of phi29 enzyme.And the influencing factors of RCA products were explored.Also,the self-assembly of gold nanoparticles was researched by RCA product as template.The rheological properties of RCA products were also studied.The main researches are as follows:(1)The synthesis of circle DNA template and the influence factors of synthesis on RCA products were researchedFirstly,the synthesis of cyclic template DNA was explored.The product was characterized by polyacrylamide gel electrophoresis.The template DNA was designed with 63 bases,and the corresponding position on the chromatogram showed that there was a signal,which demonstrated the generating of circular DNA products.The effects of reaction time,primer,enzyme and dNTP on products were investigated.The product was characterized by UV spectrophotometer and agarose gel.The UV spectrophotometer showed that the sample's characteristic absorption peaks was at 260 nm.It indicates that the product is nucleic acid.And the absorption peak 260/280 is about 1.8,which proves that the product has good purity.The gel-imaging has a strong optical density in the agarose gum lane,which proves the existence of product.(2)Preliminary exploration of the rheological properties of the productRCA products are composed of many repeating units of DNA chain segment,similar to the properties of macromolecules.In this paper,the rheological properties of long-chain DNA including the viscosity curve,modulus curve and shear properties of long-chain DNA were studied.Compared with assembly,RCA products in solution were more prone to intertwining and cross-linking.The solution properties were damaged when the solution concentration was high.However,the effect of shear resistance on the twe solutions was diffenrent.Due to high interaction force,RCA products could recover rapidly after high shear rate,with viscosity basically unchanged.Because of internal main structure composed of hydrogen bonds,assembly was prone to be damaged and could not recover within a short period of time.(3)Controlled self-assembly of gold nanoparticles induced by RCA products as templatesIn this paper,three kinds of different loading modes were constructed by using the RCA products as template to bind the AuNPs.A total of seven complementary sequences were designed.The loading of AuNPs under different sequences of addition of reactants,reaction times and reaction temperatures was studied,and characterization was conducted using UV and gel-imaging and transmission electron microscopy.Experimental results demonstrated that the hybridization of short-chain DNA was superior,and AuNPs presented uniformly spaced linear arrays after the loading.RCA products were successfully applied in the loading of AuNPs.the UV curve also shows that the long-chain DNA loaded with gold nanoparticles still have higher purity and strength. |
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