Detection Methods Of Single Nucleotide Polymorphisms And Protein

Disorders within gene fragment have close relationship to particular phenotypes, of which single nucleotide polymorphisms (SNPs) is the widely distributed genetic variation. Accurate, sensitive and specific identification of these point mutations has become socially concerned and holds great promise in DNA diagnostics. Protein is a type of important biomarkers, whose analysis plays an essentially important role in life sciences, medical diagnostics.This research work forcuses on development and innovation of detection methods for single nucleotide polymorphisms (SNPs) and protein, and achieves the goal of quick, sensitive, and accurate detection of the target. The details are as follows:1. Horseradish peroxidase (HRP) can catalyze the oxidation reaction between luminol and hydrogen peroxide (H2O2) followed by the generation of chemiluminescence. Moreover, the complex formed by hemin and its aptamer is able to mimic the biocatalytic activity of horseradish peroxidase, using such properties, we first reported a SNPs detection method based on aptamer-hemin catalyzed chemiluminescence (in Chapter 2). During our strategy, two linear amplification approaches are employed, ligase detection reaction (LDR) and an isothermal amplification (cooperation by DNA polymerase and nicking enzyme). For the mutant target, LDR can generate primers ligated fragments, while for the wild-type target, the the junction can't be linked. The isothermal amplification step depends not only on the replication of DNA polymerase, but also on the scission of nicking enzyme, which can generate a great number of aptamer. The aptamer is a guanine-rich oligonucleotide which can form a G-quadruplex intramolecular structure, hemin could be specifically intercalated in such structure and bound tightly to it, contributing to make that complex function as a superior catalyst analogous to HRP, chemiluminescence generated between luminal and H2O2 is proportionate to the concentration of the target DNA in a certain range, and based on such relationship, we can realize the quantificational detection of target with a detection limit of 1pM. In order to demonstrate the feasibility and sensitivity of the present approach, we have applied it to identify a single base mutation in CYP2D6, which is a drug metabolizing enzyme playing a significant role in the metabolism of many drugs ,and the wild type and mutant type could be perfectly distinguished.2. Based on the principle analogous to part 1, we have improved the method, making the analysis more sensitive and more rapid (in Chapter 3). In the whole analysis procedure, there are two cooperation-consecutive isothermal amplification circles. The nucleic acid generated in the first circle can act as a primer, initiating the second circle of autonomous synthesis and displacement of aptamer.3. Combining aptasensing technologies and proximity-dependent DNA hybridization assays, we constitute a new strategy for protein fluorescence detection (in Chapter 4). In our assay, two aptamer-based proximity probes are employed in which there is certain tail sequence respectively to hybridize together. In the absence of the target (thrombin), because of the predesigned low melting tempreture, the complementary fragment can't promote effective annealing, while in the presence of thrombin, the aptamer-based probes can simultaneously recognize the target and bind to it with high sensitivity and selectivity, making their local concentration increased substantially and the hybridization can be achieved, then the replication of the oligonucleotide by the polymerase could be initiated, generating the promoter sequence. In the presence of T7 RNA Polymerase , aptamer for Malachite green(MG) can be efficiently produced, and the adaptive binding of aptamer to MG is able to trigger greatly enhanced fluorescence signal.