| DNA amplification reaction is essentially a DNA replication behavior with DNA polymerase.DNA amplification reactions generally require constant addition of primers,especially for DNA strand displacement amplification reactions under isothermal conditions.However,some special DNAs can undergo conformational changes under certain circumstances and form a hairpin structure to provide primers for DNA amplification reactions.We call this reaction a self-primer strand displacement reaction.This article explores two forms of self-primer strand displacement reactions based on the LAMP reaction,establishes a T4 PNK detection method combined with the transcription reaction initiated by T7 RNA polymerase and constructs a self-strand displacement medium based on the characteristics of d Spacer.1.Construction of T4 PNK activity detection method based on phosphonothioated self-primer strand displacement reaction.Phosphonothioated-modification of DNA double strands can reduce its stability.The phosphonothioated-modification of the dumbbell-like DNA molecule generated in the first step of the LAMP reaction can promote the self-primer strand displacement reaction,so that DNA amplification can be achieved without the addition of additional primers.By combining the phosphonothioated self-primer strand displacement reaction and transcription reaction,we established a highly sensitive and specific detection method for T4polynucleotide kinase（T4 PNK）.This method showed a good linear relationship with the concentration of T4 PNK under the best experimental conditions,with a dynamic range of at least 4 orders of magnitude and could detect T4 PNK as low as 7.5×10-6U/m L.This article also evaluated the potential of this method in screening T4 PNK inhibitors and in complex biological environments.2.The construction of multiple strand displacement reactions mediated by self-strand displacement reactions.First,by introducing a d Spacer into the double-stranded structure of the intermediate product of the LAMP reaction,we developed a new self-strand displacement reactions principle,and the optimal structure of the hairpin primer for self-strand displacement reaction was explored.Secondly,by modifying the 5’end of the primers of multiple strand displacement reaction into the hairpin end proposed in principle of self-strand displacement,a self-strand displacement-mediated multiple strand displacement reaction was proposed.The real-time fluorescence method verified the excellent amplification performance of the multiple-strand displacement reaction mediated by the self-strand displacement reaction.Finally,we explored the reasons for the side reactions of the reaction. |
