Homogeneous Detection Of Nucleic Acids And Single Nucleotide Polymorphism With Cation Conjugated Polymers

Detection of nucleic acids and single-nucleotide polymorphism (SNP) is very important in the field of biochemical research and clinical diagnosis. Developing simple, rapid, highly sensitive and specific methods for detecting nucleic acids and SNP have great potential applications, especially, in homogeneous and label-free fashion. This dissertation presents a series of studies for homogeneous and label-free detection of nucleic acids and SNP with Cation Conjugated Polymers (CCPs) by using resonance light scattering, spectrophotometer and molecular fluorescence. The main contents are as follows:1. A label-free detection method for DNA hybridization in homogeneous solution with CCP by using a light scattering technique was established. This method is based on the observation of greatly enhanced light scattering resulted from the hybridization between the ssDNA combined with CCP and its complementary DNA target. The enhancement of light scattering is linearly depended on the concentration of the DNA target in the range of 0.5?10 nmol/L. Moreover, it was also found that the enhanced light scattering intensity has well selectivity against one-base mismatch at the DNA duplex terminal.2. Based on the conformational changes of polythiophene derivative (PMNT) resulted from the formation of interpolyeletrolyte complex with DNA strands, a novel methodology for SNP genotyping assay of DNA and RNA targets has been developed. With the proposed method, all three kinds of SNP genotypes can be colorimetrically identified with one primer extension reaction in homogeneous solution. This procedure could provide simple operation without requirement of any separation steps and inexpensive instrumentation for reliable and robust SNP detection.3. Based on the conformational changes of the conjugate backbone of PMNT, we have developed a rapid and convenient method for detection of miRNA and RNase H activity in a label-free and homogeneous manner. The conformational and colorimetric changes of PMNT in the DNA/PMNT duplex and DNA/miRNA/PMNT triplex are used to conveniently detect the sequence-specific miRNA. The absorbance ratio linearly depend on the concentration of the miRNA in the range of 0.05-1.0?mol/L. This assay can be accomplished with common spectrophotometer, even with visualization by "naked eye", which would significantly reduce the analysis cost and increase the analysis throughput. Combined with the RNase H to digest the miRNA of the DNA/miRNA hybrids, a colorimetric method for RNase H activity has also been developed.4. Combining the miRNA-primed extension reaction and the optical amplification of cationic conjugated polymer, the sensitive, selective and homogeneous method for miRNA determination has been developed based on fluorescence resonance energy transfer (FRET) mechanism. By controlling the amount of DNA polymerase, the proposed method can discriminate one-base difference between miRNA molecules. The homogeneous platform for miRNA assay eliminates the use of labeling probes and the requirement of isolation steps, which greatly simplifies the assay procedure and results in rapid and low-cost detection.