Resonance Light Scattering Spectrum Probe In Dna Hybridization And Polymorphism Detection And Its Research And Application

Detection of DNA hybridization and polymorphism widely used in the diagnosis of viral and genetic diseases, has caused the attention of molecular biology, pharmacology, biochemistry and analytical chemistry and other workers. On the basis of the accumulation of years of research and the investigation and research a large number of references, we synthesis the fluorescent polymer poly [5- methoxy -2-(3-sulfonated propoxy) -1,4- phenylene vinylene (MPS-PPV) as a resonance light scattering probe, and use of triphenylmethane dyes bromocresol green (BG), surfactant cetyltrimethylammonium bromide (CTAB), polycyclic aromatic hydrocarbons naphthalene (NAP) and metal Cu (Ⅱ)meso-(4-alkoxyphenyl) porphyrin (Cu(Ⅱ)-TAOPP) as probes, and used various of spectra means and electron microscopic technology to study the mechanism of the reactions. established 5 new methods of accurate, simple and quickly identify fully complementary and mismatched DNA base pairs and 2 new analytical methods of nucleic acids and protein.1) Synthesized a water soluble fluorescent polymer poly [5-methoxy-2-(3-sulfonyl isopropoxide)-1, 4-PPV (MPS-PPV), used 1H-NMR, IR to characterize the molecular structure, researched the UV absorption, fluorescence properties of polymer and utilized scanning electron microscope to study on surface morphology of the polymer. The results showed that the maximum UV-vis spectra of polymer was at 450nm with a wide absorption peak, fluorescence peak located at 550 nm and its surface has a microporous honeycomb structure with an irregular appearance which closed to a ball shape.We applied MPS-PPV into the determination of DNA hybridization,inspected the best conditions of the reaction, studied the properties of RLS spectra, fluorescence spectra and electrochemical reaction, discussed the reaction mechanism, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of polymer/ssDNA and polymer/dsDNA, and established a new non-labeled methods for the determination of DNA hybridization. In pH7.2 Tris-HCl buffer, at 40 oC, hybridization reaction for half an hour, RLS of polymer-DNA system with the maximum scattering peak located at 460 nm. Under the optimum conditions,the enhanced RLS intensity was proportional to the concentration of target DNA over the range of 0.3×10-7~1.0×10-7mol/L. Mechanism research indicated that MPS-PPV can interact with negatively charged double-stranded DNA (dsDNA, P1≈T1) by electrostatic binding in the presence of CTAB. The binding effect weakened the negative charge of P1≈T1 skeleton, enhanced its skeleton's hydrophobicity, and led the aggregation of MPS-PPV-CTAB and P1≈T1, resulting in the amplification of RLS signal. By measuring the amplification of RLS signal, complete complementary and bases mismatched DNA sequences can easily be detected and identified. This method does not need to lable the target DNA and probe DNA sequence. The novel method is simple and fast, and has potential application value in disease diagnosis.2) We applied MPS-PPV into the determination of DNA, studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM, discussed the reaction mechanism, and proposed a simple and speedy assay for DNA determination. In pH5.0 BR buffer, MPS-PPV has synergistic effect in the RLS signal of CTAB-DNA with the maximum scattering peak located at 342 nm. Under the optimum conditions , the enhanced RLS intensity was proportional to the concentration of fsDNA, the related coefficient is 0.9996, and the limit of detection is 3.10 ng/mL. Mechanism research indicated that electrostatic force is the main force among MPS-PPV, CTAB and DNA, together with hydrophobic effect and intercalation.3) We applied MPS-PPV into the determination of protein, studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM, discussed the reaction mechanism, and proposed a simple and speedy assay for protein determination. In pH 3.22 BR buffer, MPS-PPV interacted with BSA (Bovine Serum Albumin) by electrostatic force and hydrophobic effect with the maximum scattering peak located at 342 nm. The enhanced RLS intensity was proportional to the concentration of BSA, the related coefficient is 0.9991, and the limit of detection is 3.99 ng/mL.4) We applied triphenylmethane dye Bromocresol Green (BG) into the determination of DNA hybridization, discussed the properties of RLS spectra of other different kinds of triphenylmethane dyes methyl violet, chromazurine, bright green, xylenol orange and alkaline magenta with double strand DNA (dsDNA) and single strand DNA (ssDNA) and utilized Gaussian03 to calculate the molecular volume of those dyes and the effects on the interaction with DNA. We studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM of BG-dsDNA, discussed the reaction mechanism, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of BG/ssDNA and BG/dsDNA , and established a new non-labeled methods for the determination of DNA hybridization. Mechanism research indicated that BG can interact with negatively charged double-stranded DNA (dsDNA, P1≈T1) by groove binding. The binding effect weakened the negative charge of P1≈T1 skeleton, enhanced its skeleton's hydrophobicity, and led the aggregation of BG and P1≈T1, resulting in the amplification of RLS signal.5) We applied cationic surfactant CTAB into the determination of DNA hybridization, discussed the properties of RLS spectra of other different kinds of surfactants CPB, SDBS, SDS, TX-100 and T-80 with dsDNA and ssDNA. We studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM of CTAB-dsDNA, discussed the reaction mechanism, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of CTAB/ssDNA and CTAB/dsDNA,and established a new non-labeled methods for the determination of DNA hybridization. Mechanism research indicated that CTAB can interact with dsDNA (P1≈T1) by electrostatic force and hydrophobic effect leading the aggregation of CTAB and P1≈T1,resulting in the amplification of RLS signal.6) We applied PAH (polycyclic aromatic hydrocarbon) naphthalene into the determination of DNA hybridization, discussed the properties of RLS spectra of other different kinds of PAHs anthracene, fluoranthene, pyrene, phenanthrene with dsDNA and ssDNA. We studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM of naphthalene-dsDNA, discussed the reaction mechanism, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of naphthalene/ssDNA and naphthalene/dsDNA, and established a new non-labeled method for the determination of DNA hybridization. Mechanism research indicated that naphthalene can interact with dsDNA (P1≈T1) by groove binding which depends on G-C sequenees of dsDNA and the volume of naphthalene. The binding effect weakened the negative charge of P1≈T1 skeleton, enhanced its skeleton's hydrophobicity, and led the aggregation of naphthalene and P1≈T1, resulting in the amplification of RLS signal. By measuring the amplification of the RLS signal, entirely complementary and bases mismatched DNA sequences can easily be detected and recognized. The method has potential application value in disease diagnosis.7) We applied Cu (Ⅱ)meso-(4- alkoxyphenyl) porphyrin(Cu(Ⅱ)-TAOPP) into the determination of DNA hybridization, discussed the properties of RLS spectra of other different kinds of metalloporphyrin Co(Ⅱ)-TAOPP, Cr(Ⅱ)-TAOPP, Mg(Ⅱ)-TAOPP, Zn(Ⅱ)-TAOPP and Ni(Ⅱ)-TAOPP with dsDNA and ssDNA. We studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM of Cu(Ⅱ)-TAOPP-dsDNA, discussed the reaction mechanism, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of Cu(Ⅱ)-TAOPP/ssDNA and Cu(Ⅱ)-TAOPP/dsDNA, and established a new non-labeled methods for the determination of DNA hybridization. Mechanism research indicated that hydrophobic Cu(Ⅱ)-TAOPP can interact with dsDNA(P1≈T1) leading the aggregation of Cu(Ⅱ)-TAOPP and P1≈T1. The large aggregates showed strong RLS signal amplification and fluorescence quenching. By measuring the amplification of the RLS signal, entirely complementary and bases mismatched DNA sequences can easily be detected and recognized. The method has potential application value in disease diagnosis.