Research On Technology For Detecting DNA Single-nucleotide Variants

Single-nucleotide variants(SNV)are important molecular marker for biomedical research and clinical applications.Therefore,the development of SNV detection technology with superior performance has been the focus of nucleic acid biotechnology.Watson-crick hybridization rule is the basic principle of nucleic acid detection technology.Currently,many methods for SNV identification based on hybridization specificity have been developed.For example,methods based on single hybridization probes,molecular beacons,and binary DNA probes.However,the above hybridization-based SNV identification methods still have the following problems:(1)These methods can only determine whether the whole strand is wildtype or mutation,but it is difficult to locate the mutation to a smaller region.As a result,in accurate SNV identification,high cost and long time are spent on the detection of irrelevant areas,which limits the improvement of SNV identification efficiency.(2)The unexpected secondary structure of target DNA formed by intermolecular base pairs creates a higher energy barrier for intermolecular hybridization,and it significantly slowed DNA hybridization,resulting in the SNV identification in unit time being affected and sometimes even erroneous.(3)In low-abundance SNV identification,it is difficult to further block a large number of existing non-targets indiscriminately,which limits the improvement of specificity and selectivity.Here,we conduct following researches to solve the problems above:1.This paper proposes a strategy to identify domain-level SNV based on the elimination of an unexpected secondary structure of target.The target DNA is assembled into rigid double strand by hybridization.In the rigid structure,the self-folding target is stretched to eliminate the unexpected secondary structure of the target.Since SNV site at different locations on target may affect double-stranded stability or branch migration during hybridization,locating the SNV site to the domain level can be achieved by using probes to inquire about the properties of the double strand.The results show that the strategy successfully eliminates the unexpected secondary structure of the target,and reports more domain-level information of the target through probes,thus providing a domain-level SNV identification platform that can run at room temperature.This strategy not only successfully distinguished the wild-type from the mutant-type,but also accurately identified the BRAF-V600 E mutation domain from the three mutation possibilities.2.A strategy based on indiscriminately blocking interfering sequences to achieve high specificity SNV identification was developed.In a reversible reaction,the thermodynamics of the reaction is easily regulated by the concentration of reactants or products.Inspired by this,based on the reversible nature of the toehold exchange reaction,the forward reaction was inhibited by adding excessive by-products to the reaction.Ideally,the unexpected hybridization between all interference sequences and the probes can be further inhibited,so as to block all interference sequences indiscriminately.The results show that compared with the system without concentration inhibition,this strategy can greatly improve the specificity of SNV identification.Moreover,the concentration inhibition enables non-targets mutated at different positions to be inhibited indiscriminately.Based on the above advantages,the strategy finally identified the target with abundance of 1%.3.A strategy is proposed to integrate concentration inhibition with entropy-driven amplification to achieve highly selective low-abundance SNV identification.In the concentration inhibition,additional by-products were added to inhibit forward reaction,thereby blocking the non-target to improve the specificity of SNV identification.However,this improvement will sacrifice the target yield.Therefore,in order to improve the yield of the target on the basis of ensuring high specificity,concentration inhibition and entropy-driven catalytic amplification were combined.In theory,the target can hybridize with the toehold exchange probe to activate the subsequent entropy-driven catalytic amplification network,while a large number of non-targets are blocked by excessive concentration inhibition and difficult to hybridize with the probe.The results show that the SNV identification specificity of this strategy is superior to any single concentration inhibition or entropy-driven catalytic amplification module.This strategy can detect the target SNV with 0.1% abundance.In addition,even if the mixture contains three 1000-fold interference sequences,the proposed strategy can still identify the target SNV.