Point Mutation Identification Based On Hairpin Probe And DNA-Modified Detection Of Small Molecules

Single nucleotide polymorphisms (SNPs) are the most common form of geneticvariation．In Human’s3billion bases, single-base mutation (SBM) occurs once every100–300bases. Realizing early, accurate, simple and rapid identification to thesesingle-base mutations is of great importance for the pathogeny and early therapy ofmany corresponding diseases. Accordingly, developing some simple, cost-effective,accurate and easy to be clinically popularized DNA sensing technology hasconsiderable significance.DNA hairpins have been widely used in the preparation of biosensors for theirbetter selectivity and higher specificity. In recent years, by changing the structure ofclassic molecular beacon, scientists have developed many novel detection methods. Inthis paper, We proposed two new DNA sensors by redesigning hairpin probes. Besides,we validate a proof-of-principle for sensitive detection of small molecules employingDNA modification. The detailed content is described as follows:(1) By introducing a hairpin structure and a recognition site for endonuclease intoan oligonucleotide sequence, we developed a simple and effective piezoelectricmethod for DNA detection. In this sensing system, the background value issubstantially suppressed by restriction endonuclease ECoR I while the targethybridization event is amplified by nano-gold-labeled detection probe. The programare as follows: first, a DNA hairpin probe was fixed to the quartz crystal surface whichcan be specifically recognized and cut by endonuclease ECoR I, if the system undertest contains a target DNA, the hairpin probe is opened and the subsequent enzyme isnot their role, and can be combined with nano-gold-labeled detection probe makingsignal amplification. Otherwise, in the absence of target DNA, the hairpin DNA probecan be digested, and nano-Au labeled probes role can not produce any signal. Theexperimental results show that this method is simple and practical, high sensitivityanalytics adapting the detection of DNA. Besides, by sharing ECoR I and the detectionprobe, we designed an electrochemical method using methylene blue as hybridizationindicator, further verified the practicability of our experimental system, and provideda new way for point mutation analysis.(Chapter2)(2) A one-step and reusable electrochemical molecular switch for detection ofsingle-base mutation is proposed in the present work. When the hybridization reaction takes place in the presence of target DNA, the Fc-labeled terminal of the open switchmolecule can be captured by the probe through the predesigned complementary basesof both sequences. By this method, a signal-on sensor is built on. The presentapproach has been demonstrated with the identification of a single-base mutation inHb Constant Spring codon142(terminating codon TAAâ†'CAA) that is one of themajor types of α-thalassemia point mutations for clinical diagnosis. The resultsshowed that the current intensity was linear to the logarithm of the targetconcentration in the range from0.01to100pM with a detection limit0.01pM. Allthese features revealed that the system is a promising candidate for single-basemutation discrimination, owing the advantages of both generalizability and simplicitytoward reagentless detection of DNA with sensitivity and selectivity.(Chapter3)(3) Developed a method for quantifying small target molecules, the screeningscheme is based on the design of target molecule-linked DNA hybrid, when addingtarget molecule sample, the free IAA can bind to the anti-IAA antibody protein by pre-incubating in a solution, inhibiting the formation of antibody/IAA-labeled probe/assembling probe complex in the subsequence step. In this case, the thiol ofassembling probe can randomly run into the QCM surface, thus, the recognition probeis easily captured onto the surface. As a result, HRP is capable of being adsorbed ontothe resultant QCM via the biotin-avidin bridge, and catalyzes the precipitation forming,generating an enhanced mass signal. Utilizing the transducer, not only can the efficientinvestigation of IAA/antibody interaction and sensitive quantification of IAA beaccomplished compared with the conventional assays, but also paves the way todevelop the various sensing platforms for the expansion of IAA detection methods toother small targets and analytes.(Chapter4)...