Construction Of Structural Stability Nucleic Acid Probes For Application In Plant Molecular Detection

Nucleic acid is a biological molecule that carries most of the genetic information.Its importance is unquestionable.The detection of nucleic acid has become a basic prerequisite for many applications of molecular genetics.Common nucleic acid detection techniques include DNA microarray,Northern blotting and q PCR,etc.,but the operation is relatively complicated and the detection cost is high,and Northern blotting involves the use of radioisotopes.In recent years,a series of signal amplification techniques such as strand displacement amplification,rolling circle amplification,and catalytic hairpin assembly have been applied to nucleic acid molecule detection.These methods have the advantages of rapid,sensitive,and strong specificity.In this paper,based on the combination of molecular beacons,catalyzed hairpin assembly and enzyme isothermal amplification,three structurally stable probes were constructed respectively,and their corresponding new methods for detecting nucleic acids were established.The main results are as follows:1.Detection of miR160 by catalytic hairpin assembly based on molecular beacons.Four hairpin probes WA,WB,WC and WD were designed using molecular beacon and catalytic hairpin assembly technology.Experiments such as gel electrophoresis and fluorescence signal detection have verified that the hairpin loop structure of these probes can be successfully induced to open in the presence of miR160 to form a nucleic acid tetramer,which can specifically recognize single-base mutation miR160.This method can achieve highly sensitive detection of target miR160 as low as 10 p M,and can detect targets in complex matrices.2.DNA tetrahedron detection of miR21 based on molecular beacons.A DNA tetrahedron was designed based on DNA self-assembly technology.The molecular beacon HP2 and complementary hairpin probe HP1 were designed using the principle of catalytic hairpin assembly,and they were connected to different tetrahedron vertices.The assembly of the tetrahedral structure was verified by gel electrophoresis and atomic force microscopy,which proved that miR21 can induce the hairpin loop structure of the probe to open and form a double tetrahedral structure,and specifically recognize the single-base mutant miR21.This method can enhance the anti-degradation ability of the probe,and realize the detection of miR21 as low as 10 p M.At the same time,the confocal microscopy analysis proves that the method can be applied to the detection of live cells.3.3D probe based on molecular beacon to detect KRAS gene.A new type of 3D probe was constructed by modifying the cholesterol at the 5’end of the molecular beacon HP2-Chol.The molecular beacon can form a spherical 3D probe through the aggregation of cholesterol to identify the target KRAS gene and develop a clip ring structure.The 3D probe is connected by the palindrome at the end,and the amplification of the Klenow enzyme is used to initiate a strand displacement reaction to achieve signal amplification.This method can achieve high-sensitivity detection of target KRAS mutant genes as low as 10 p M,and can detect KRAS mutant genes in complex biological substrates.In this paper,a molecular detection method based on structure-stable nucleic acid probes is established,which has the advantages of simple and quick operation,good specificity,low detection cost and stable structure,and has high application value in plant nucleic acid detection.