The Research On The Recognition And Enrichment Methods For Nucleic Acid Based On Strand Displacement Reaction

With the rapid development of biotechnology,DNA is not only a target for medical diagnosis,but also a carrier of data information,which plays an important role in the field of synthetic biology.Due to the advantages of flexible design,simple operation,low cost and so on,the strand displacement reaction has a very good application prospect in the field of medical diagnosis and synthetic biology,such as Point-of-Care Testing(POCT),DNA computing and DNA data storage.However,applying strand displacement reaction in biosensing and the amplification of DNA pool still remain great gaps for improving.For example,using strand displacement reaction,there is a lack of systematic studies of detecting cell-free DNA and the amplification of DNA,such as the specificity and sensitivity of detection,as well as the amplification preference and error rate.By analyzing the biochemical molecule mechanism of strand displacement reaction,we designed a strand displacement probe and constructed an isothermal amplification reaction suitable for a large-scale DNA oligo pool,promoting the better application of DNA materials in biosensing and DNA data information.By introducing the toehold-mediated strand displacement reaction into the padlock probe,we designed and constructed a novel biosensor,termed toehold assisted padlock probe.The new biosensor contains a padlock probe and a short oligonucleotide perfectly complementary to target,named toehold blocker.Based on toehold-mediated strand displacement,the toehold-assisted padlock probe and toehold blocker were demonstrated with new controllability in significantly suppressing 85% undesired cross-reaction,promoting target recycling and low concentration of variant alleles(0.1%)detection only spanning a narrow sequence window of 29 nucleotides on average by tuning the thermodynamic properties of the toehold between the blocker and padlock.For the real cf DNA sample,a unique asymmetric PCR technique was developed for the highly fragmented DNA by bringing dd NTPs in asymmetric PCR.A combination of broad-spectrum enrichment method and high-resolution padlock probe technique improved the accurate genotyping,which obtained the observed mutation frequency was two times than the traditional method.Meanwhile,an energy-efficient,low-bias isothermal amplification reaction was constructed using enzyme-mediated strand displacement amplification,which realized the practical DNA molecular storage of MB-level digital information.This is the first time that the isothermal amplification reaction has been applied to DNA data storage.Results demonstrated that i DR could produce single-stranded DNA(ss DNA)or doublestranded DNA(ds DNA)in a controlled manner.And the amplified product carried a 5’terminal phosphate group.In contrast to the conventional method,the thermal reaction could save 98% of the electrical consumption.i DR could achieve successful decoding requiring 70-fold less than the traditional method.In addition,a cost-effective and scalable DNA oligo pool normalizing(OPN)method was developed based on barcode strategy.Combination of OPN with i DR achieved a perfect manipulation of large-scale DNA oligo pool,which could save hundreds of sequencing resources.In summary,based on the strand displacement reaction,this study successfully developed a biosensor with high specificity and a method for amplifying the large-scale DNA oligo pool,promoting the application of DNA in biosensing,nanomaterials,and DNA data storage.