Research On New Technology Of Detecting Nucleic Acid Based On Self-assembly Of Hairpin Dna And One-step Nano-gold Colorimetric Method

Nucleic acid as an important genetic material of life,its detection is particularly important to achieve rapid,simple,visualization of nucleic acid detection in the field of biochemical analysis of great significance.Whether it is temperature-dependent nucleic acid amplification reaction,or isothermal nucleic acid amplification reaction,both of them are enzymatic reaction.The high requirements of the experimental conditions for this type of reaction limit the scope of nucleic acid detection.Therefore,the non-enzymatic DNA self-assembly reaction is not only in the field of nucleic acid detection,but also has certain application value in the field of DNA nanostructures and in vivo detection.First,the first chapter of the paper was based on the non-enzyme exponential hairpin assembly(EHA),combined with the pyrene,and the validated fluorescence detection experiment of the scheme was carried out.But,the scheme could not meet the requirements of nucleic acid detection.Therefore,we adjusted the scheme,using EHA reaction combined with Cy3/Cy5 which was fluorescence resonance energy transfer(FRET)pairs for single-stranded nucleic acid detection.In order to obtain the best energy transfer efficiency,the experimental conditions were optimized.The results showed that the detection limit of the method was 10 pM.The anti-interference ability of the method was tested by using the cell culture medium supplemented with 10% fetal bovine serum.The results showed that the method had good anti-interference ability.Experiments with different mismatched targets,proved that the method had good specificity.Second,our study group found that PEG 200 could significantly improve the rate of strand displacement,and DNA self-assembly reaction is a spontaneous replacement and hybridization process.Therefore,we hypothesized that PEG 200 may also promote DNA self-assembly reaction.In the third chapter of the paper,six small molecular substances which commonly used for nucleic acid amplification were selected to be used in the hybridization chain reaction(HCR)and compared the effects of small molecular substances on the HCR reaction.The results showed that PEG 200 was the best to promote the HCR reaction,and then the PEG 200 was used in the EHA reaction.The results showed that the PEG 200 could also contribute to the EHA reaction.With the promotion of 20% PEG 200,the rate of HCR reaction was improved by nearly 100 times,and the EHA reaction was improved by about 10 times.Finally,two kinds of nano-gold modification methods for colorimetric technology were investigated.The two kinds of nano-gold modification methods,fully modified and asymmetric modified,were compared from the salt-resistant ability,the time and the extent of color change after adding the target.According to the experimental results,it was confirmed that the modified nano-gold was suitable for nucleic acid amplification reaction.Then,the modified nano-gold was used for polymerase chain reaction(PCR),loop-mediated isothermal amplification(LAMP).The result showed that the modified nano-gold inhibited the amplification reaction.However,the addition of BSA could reduce the inhibitory effect of the modified nano-gold.Unfortunately,the colorimetric result was not obvious under experimental conditions.We think that the colorimetric technology will be improved by optimizing the experimental conditions and is successfully used to nucleic acid amplification reaction realizing one-step and visual detection of nucleic acids.