Single Nucleotide Polymorphism Genotyping Based On Nucleic Acid-based Signal Amplification And DNAzyme-catalyzed Chemiluminescence Detection

Single nucleotide polymorphism (SNP) is one of the most abundant forms of genetic variations in the human genome. It's found that SNPs in the coding regions of genes is closely related some human diseases. Therefore, sensitive SNP genotyping is crucial in the field of chemical and biological detection, and also has potential applications in clinical diagnosis.Peroxidase-like DNAzymes, which not only can be easily synthesized, but had good thermal stability and catalytic performance as well, are generally becoming ideal sensing elements in biochemistry analysis. Signal amplification technology, on the other hand, can achieve a detection model of one molecular target input and multi molecular reporter output, which greatly improves the detection sensitivity. DNAzyme-based sensor combined with nucleic acid signal amplification technology has a broad application prospect in biochemical analysis.The thesis is mainly composed of three parts:Firstly, recent studies on SNP genotyping are reviewed in Chapter One. The classification and function of nuclease, especially the properties and applications of peroxidase-like G-quadruplex DNAzyme are introduced as well. Moreover, the systematical summary of the nucleic acid signal amplification technology has been presented.Secondly in Chapter Two, by taking rs242557, an Alzheimer's disease related gene fragment-as the target model, we carried out a SNP genotyping research based on target-catalyzed hairpin assembly DNAzyme assay. By using the strategy of amplification assays based on toehold-mediated catalyzed hairpin assembly while coupling with the advantages of DNAzyme-catalyzed microfluidic chemiluminometry, an enzyme-free and non-lable SNP genotyping method has been developed.The applied DNAs in the experiment include one target DNA and two fuel DNA hairpins H1 and H2, of which the sequences were initially fixed based on the result of NUPACK software predicted. Without the triggering of the target DNA, the two fuel DNA hairpins with partially complementary nucleotide sequences, could not open the stem-loop structures to hybridize each other due to the thermodynamic barriers. But once triggered by the target DNA, those two hairpins could open consecutively to hybridize each other in facilitating the formation of G-quadruplex DNAzyme for chemiluminescence determination. The result to the work showed the absolute detection limit of developed sensor was 0.3 fmol, and the differentiating factor (DF) for the SNP genotyping reached as high as 20, which indicated a great potential for clinical diagnose in the future.Last in Chapter Three, the summary and prospect are stated.