Novel Methods Of Optical Analysis For The DNA And Protein Biological Markers

Living organisms are utterly dependent on all kinds of biological macromolecules. There are four basic classes of biological macromolecules, such as carbohydrates, lipids, proteins and nucleic acids. Among them, nucleic acids and proteins are the most important species for life science. Single nucleotide polymorphisms (SNPs) are the most frequently occurring and stable forms of genetic variations in human genome, and relative to various diseases of human being. While the changes of protein abundance can indicate the situation of metabolism in vivo and be used for estimation of health risks associated with various diseases. Therefore, investigations about proteins and DNA are crucial in the fields of life science research, biomedical research and clinical application. Most of the earlier investigation have been carried out through the measurement of fluorescence spectrometry, fluorescence anisotropy, fluorescence resonant energy transfer and surface plasmon resonance. Notwithstanding the importance of these techniques, it’s been a topic of intensive interest in analytical chemistry to develop highly selective and high sensitive methods and techniques with desirable operational attributes. Because techniques based on spectroscopy or luminescence are high sensitive and selective, cost-effective, easy to curry out without expensive equipment, and liable to realize miniaturization, these techniques have excellent prospects in biological assay in the future. Consequently, several new optical techniques or strategies for genotyping, DNA detection or detection of protein have been developed in the presented paper and described as follows:(1) We reported a homogeneous label-free assay for genotyping of single nucleotide polymorphism, using ligation-mediated strand displacement amplification with dnazyme-based chemiluminescence detection. In this assay, genotyping of SNPs was accomplished by DNA ligase, which only joins the probes perfectly matching with target gene. Consecutive SDA reactions were initiated by the ligated product, via self-primed extension reaction of hairpin-structure3’end in the presence of vent exo-DNA polymerase and dNTPs. Hence, a cycle of nickase cleavage, polymerase extension, and subsequent replicated strand release is created, which renders a SDA of the ligated product. The first SDA reaction produce a great amount of replicated strand which can launch the second SDA for the ligated product to produce abundant anti-hemin aptamers, which can catalyze oxidation reaction of luminal-H2O2and give readout of chemiluminescence. According to this, we can realize genotyping for target gene. Results reveal that the developed technique possesses superb selectivity in discriminating single-base mismatches, displays very low detection limit (0.1fM), and has a wide dynamic range (1fM to1nM) and a high signal-to-background ratio (-150). Because of its label-free, homogeneous, and chemiluminescence-based detection format, this proposed technique should be rather robust, cost-efficient, easily automated, and scalable up to parallel assays of hundreds of samples.(2) We developed a label-free method for genotyping of cytochrome P4502D6*10using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence. In this method, genotyping of single nucleotide polymorphisms was accomplished by thermostable DNA ligase in repeated thermal cycles of denaturation at hing temperature and ligation at low temperature, which ongly covalently joins common probe with discriminating probe,3’end perfectly matching with the target, and resultes in a lot of ligation product. Single SDA reactions were initiated by the self-primed extension of the hairpin-structure3’end of the ligated product, and to produce a large number of anti-hemin aptamer, which can catalyze chemiluminescence reaction of luminal-H2O2and realize the detection of target genotype. Thermolstable ligase furnished this method with high specificity in identification of SNPs, and the using of ligase chain reactiong and constant temperature-linear SDA allowed highly efficient amplification with a limit of detection at0.1pM. Tht employment of chemiluminescence in this technique displayed a wide dynamic range from1pM to1nM and a high signal-to-background ratio of～57. Because it is label-free, homogeneous, robust, cost-efficient, readily automated and scalable for parallel assays of hundreds of samples, the proposed genotyping method might provide a highly sensitive and specific genotyping platform for pharmacogenomics research and clinical diagnostics.(3) We designed a novel assay strategy for simultaneous genotyping of several targets based on LCR-mediated common PCR and biological barcode-HPLC technique. We designed a pair of allele-specific discriminant probe and a common probe according to target gene. Discrimination of single-base mismatches is first accomplished using DNA ligase in repeated thermal cycles to generate a ligation product between a discriminant probe and a common probe. The ligated products as templates could be duplicated and amplified through PCR reaction using common primers, one being labeled with fluorophore. The amplified products with fluorophore were digested by restriction endonuclease into two parts, a random sequence and a fluorophore labeled barcode sequence with specific length. The digested products were separated and analyzed through HPLC equipped with fluorescence detector. The fluorophore labeled barcode sequence with specific length is relative to special genotype of target gene. According to this, we can realize the goal of genotyping for a given target gene. Furthermore, HPLC is a high-through technique and can be performed analysis on a great deal of analytes one time. The developed assay was successfully demonstrated by simultaneous analyzing two targets, with a detection limit of0.1fM.(4) A general homogeneous method on the basis of exonuclease aided target recycling strategy was developed in Chapter5for amplified fluorescence DNA detection. In this chapter, a probe labeled with a fluorophore at its3’end and a quencher near the fluorophore was designed according to the given target DNA. The two-labeled probe holds the fluorophore in close proximity to the quencher, resulting in very weak fluorescence because of FRET between fluorophore and quencher. After challenge with a perfectly matched target, the labeled probe formed a dsDNA that contains exonulcease Ⅲ-active3’recessed end. Using the recessed-end cleavage activity of exonulcease Ⅲ, the labeled probe was hydrolyzed via the stepwise removal of monoclueotieds from its3’terminus, while the fluorophore and the target were liberated. After that, the released target could hybridize with another labeled probe, whence the cycle of exonuclease aided hydrolyzation started anew, realizing amplified DNA detection. It was demonstrated via detecting a model DNA target that the developed exonuclease-amplified assay is sensitive and specific and can reach the detection limit of0.1pM.(5) A assay based on biological barcode technique and HPLC for simultaneous detection of several target was developed in Chapter6. We designed a pair of target-specific discriminant primer according to target gene. Target-specific amplification was first finished using a few cycles of first-stage PCR to generate a certain amount of amplified product. The amplified products as templates could be duplicated and amplified through second-stage PCR amplification using common primers, one being labeled with fluorophore. The second-amplified products with fluorophore were digested by restriction endonuclease into two parts, a random sequence and a fluorophore labeled barcode sequence with specific length. The digested products were separated and analyzed through HPLC equipped with fluorescence detector. The fluorophore labeled barcode sequence with specific length is relative to corresponding target. Based on this, we can realize special detection for a given target. Moreover, HPLC is a high-through technique and can be used to analyze a great amount of analytes at one-time assay. The developed assay was successfully proved via simultaneous detecting four interesting targets, with a detection limit of0.01fM.(6) A novel aptamer sensor strategy based on fluorescence protection assay was proposed for homogeneous detection of protein targets in Chapter7. The fluorophore-labeled aptamer was used as sensitive element for specific recognition of protein targets. The formation of aptamer-protein complex dramatically increase steric hindrance, which protects the fluorophore labeled near the binding site from interacting with and quenching by anti-fluorophore antibody. This assay strategy of aptamer was demonstrated via performing detection of model protein IgE, an important biomarker relative to atopic allergic diseases. The results prove that the aptamer method is highly sensitive (>6-fold in fluorescence enhancement), and highly specific to other co-existing interference proteins. The dynamic detection range of the method is0.1nM to50nM, and the detection limit at0.1nM. Moreover, other proteins could be investigated directly by using fluorophore-labeled corresponding aptamers. Furthermore, all kinds of anti-fluorophore antibody being available, the aptamer strategy could be carried out for multiplex assay of different proteins, via utilizing various fluorophore labels with non-overlapping emission peaks. With the characters of robustness, generality, simple operation, and multiplexing, this proposed homogeneous fluorescence protection aptamer strategy contributes a new methodology to design aptamer biosensors for protein detection.