Research Of Nucleic Acid Hybridization Assay Based On Magnetic Mircospheres

With the coming of the genome and post-genomic era,nucleic acid hybridization technology is widely used in sample screening for single nucleotide polymorphisms(SNPs),gene expression studies,medical diagnosis,and food and environmental analysis etc.In particular,various solid phase nucleic acid hybridization techniques with high specificity and sensitivity for detection performance have been rapidly developed because it can facilitate removal of non-target DNA/RNA molecules to greatly reduce the probability of non-specific adsorption.Compared with planar substrates carriers,magnetic microspheres are promising supports because they provide faster reaction kinetics as a result of their uniform dispersion in solution and greatly pseudo liquid phase specific reaction surface.In magnetic microsphere-based molecular diagnostic techniques,the most critical factor is the hybridization efficiency of the immobilized oligonucleotide probe and the target nucleic acid molecule.The hybridization efficiency is the key to determine the performance of molecular diagnostic techniques.Many parameters affect the efficiency of hybridization between the target DNA/RNA and the immobilized oligonucleotide probe,including the hybridization environment,the length of the target DNA/RNA,the length of the oligonucleotide probe,the surface density of the oligonucleotide probe coupling on magnetic microspheres and the distance between the hybridization site and the fluorescent molecular label.However,most of the research are based on the hybridization efficiency of the oligonucleotide probe immobilized on the plate carrier and the target molecule and explore some factors affecting the solid phase nucleic acids hybridization efficiency in order to solve specific application requirements.Few work systematically study the universal regular of solid phase nucleic acid hybridization based on magnetic microspheres.Therefore,this paper focuses on this research,hoping to obtain systematic and universal influence regulars,and provide basic theory and data analysis support for efficient molecular detection methods.Firstly,this paper uses a fragment on the L858 R sequence of exon 21 of the EGFR gene of non-small cell lung cancer as a model nucleic acid molecule for hybridization detection.The oligonucleotide-coupled magnetic microspheres were prepared by covalent coupling and complementary pairing with the Cy5 fluorescent molecule-modified target DNA to construct a nucleic acid hybridization system based on magnetic microspheres.The hybridization performance of nucleic acid based on magnetic microspheres was detected and analyzed by flow cytometry.Secondly,the hybridization environment,such as the composition of the hybridization solution,the length of spacers,the hybridization time,the hybridization temperature and the concentration of the target molecule were studied.The optimal results were obtained under a series of influencing factors.Next,in order to better design oligonucleotide probes to provide higher hybridization efficiency and specificity in nucleic acid hybridization reactions,the effects of different oligonucleotide probes lengths and the different surface density of oligonucleotide probes on the efficient nucleic acids hybridization based on magnetic microspheres were investigated.Then the effects of different target lengths and distances between different hybridization sites and fluorescent molecular lable on the efficient nucleic acids hybridization based on magnetic microspheres were investigated.The results showed that among the three different lengths of 111-bp,210-216-bp,and 320-326-bp target molecules,the 111-bp length DNA target molecules produced higher fluorescence intensity and the 5 end-modified Cy5 fluorescent molecule of DNA target molecules is closer to the magnetic microsphere,the higher the fluorescence intensity produced.The detection limits of nucleic acids hybridization based on magnetic microspheres under optimized conditions were explored and detection results showed that the average number of target molecules bound by each magnetic mircosphere was the least 208,which can be detected.At the same time,in order to elucidate the influence mechanism of the length and density of oligonucleotide probes of magnetic microspheres on the hybridization efficiency,the method of binding constant between immobilized probe and target DNA was successfully established by double reciprocal method.The method test obtained the binding constants of different probe states and target DNA,and gave a quantitative theoretical analysis on the hybridization mechanism based on the immobilized oligonucleotide probes on the surface of magnetic microspheres.In summary,systematic study of the design of the capture probe coupling on magnetic microspheres and the structure of the target molecule,this paper can not only provide guidance and theoretical basis for the design of probes and the selection of target molecules in nucleic acid hybridization technology,thereby improving the efficiency of nucleic acid hybridization reaction and the sensitivity of detection of nucleic acid molecules,and also providing strategies for automated high-throughput bioassay platforms and theoretical and experimental references for the preparation of high performance biocarriers for molecular diagnostics.