Studies On Recognition Of Single-nucleotide Polymorphism Based On Fluorescence Enhancement Of Inorganic Materials

Single nucleotide polymorphism (SNP), the third genetic markers, accounts for high density in the genetic polymorphism, having intimate relation with the occurrence and development of diseases. Recently, more and more interests have been focused on SNP, which in turn facilitates rapid development of the detection methods for SNP. In this thesis, the basic properties of SNP and the methods for SNP detection are briefly summarized. Among these methods, fluorescence recognition of SNP based on DNA abasic site (AP site) has attracted much attention due to the merits of simplicity, rapidity, and effectiveness. On the basis of these, the DNA sequences containing AP site or GAP site (produced by eliminating one nucleotide from a DNA duplex) from fragments of cancer supression gene p53were choosen as models, and we have successfully developed the method for recognition of single-nucleotide polymorphism based on fluorescence enhancement of inorganic materials. The main contents are as follows:1. Study on recognition of single-nucleotide polymorphism based on fluorescence enhancement of terbium ion upon binding at an abasic site in DNARecognition of single-nucleotide polymorphism was proposed in this work on the basis of fluorescence enhancement of an inorganic ligand, terbium ion (Tb3+), upon binding at an abasic site in DNA. The AP site was deliberately introduced in a probe strand just opposite the base of interest in a target strand. Selective binding of Tb3+at the AP site was found by observations of its characteristic fluorescence enhancement for AP site-containing DNA by comparison with fully matched DNA without the AP site. The enhancement is very dependent on the target base and the bases flanking the AP site. When the target base is G, the maximum enhancement of Tb3+fluorescence can be obtained especially for Gs flanking the AP site. This method was successfully applied in detection of P177R mutation of codon177in cancer repression gene p53, manifesting its potential applications in SNP detection with signal-on fashion.2. Gap site-specific formation of fluorescent silver nanoclusters and in situ selective DNA biosensingWe exploited the potential of a gap site in DNA duplexes as a new scaffold for the synthesis of silver nanoclusters (Ag NCs). We found that fluorescent Ag NCs were highly selectively formed when cytosine faced toward the gap site in DNA duplexes, and they could be utilized as readout with signal-on signaling for certain DNA mutations. Compared to the commonly used single-stranded DNA templates for the synthesis of Ag NCs, the gap site in DNA duplexes was found to facilitate rapid formation of fluorescent Ag NCs without sacrifice of bright emission and excellent stability of the nanoclusters. This base-selective growth of the fluorescent Ag NCs at the gap site would find promising applications in practical detection of single nucleotide polymorphism and potential DNA-based functional sensors by label-free and cost-effective fashions.3. Upconversion emisisons of fluorescent silver nanoclusters and in situ selective DNA biosensingWe investigated the upconversion emissions of Ag NCs templated by single-and double-stranded DNAs. The DNA-templated Ag NCs exhibit upconversion emissions at the identical wavelengths as those observed for the corresponding Stokes emissions. Thus the Ag NCs’upconversion behaviors can be easily tuned by the examined DNA sequences. In addition, relative to the Stokes mode, the Ag NCs become more stable at the upconversion emission conditions. As a proof-of-concept application, DNA biosensing in nucleobase recognition with the in situ formed Ag NCs is realized by the Ag NCs’upconversion emissions. We expect that the Ag NCs’upconversion emissions should be more advantageous than the previously used rare-earth materials at least in the aspect of easy modulation of the emission energies by DNA sequences and could find wide applications in sensor designs.