Homogeneous And Label-free Detection Of Nucleic Acids Using Rolling Circle Amplification And Nanoparticles

Detection of nucleic acids and single-nucleotide polymorphism (SNP) are very important in the field of biochemical analysis and clinical diagnosis. Developing simple, rapid, highly sensitive and specific methods for detecting nucleic acids and SNP have extensive applications and prospects in homogeneous and label-free fashion. This dissertation presents a series of new methods for homogeneous and label-free detection of nucleic acids and SNP by using rolling circle amplification (RCA) and nanoparticle probe technology integrated with bioluminescence, molecular fluorescence and resonance light scattering detection. The main contents are as follows:1. Integration of simple and efficient RCA and highly sensitive luciferin bioluminescent reaction, a new assay has been developed for homogeneous and lable-free SNP detection. It is based on the mutant DNA can specifically prime the RCA reaction and the large amounts of RCA products, pyrophosphate (PPi), are detected using luciferin bioluminescent reaction. In contrast, the wild DNA is not able to prime RCA reaction. Based on the remarkably differences of bioluminescent signals between wild DNA and mutant DNA, the method is highly sensitive and specific enough to detect 0.45 pmol/L mutant DNA. A mutant frequency as low as 1.0%can be accurately determined.2. Based on the principle of RCA, introduction of a reverse primer complementary to the target-primed RCA products in RCA process leads to the branched RCA (BRCA) and eventually generates the various lengths of ssDNA and double-stranded DNA products, which are sensitively detected using fluorescence dye, SYBR Green I. So a new fluorescent method has been developed using BRCA reaction for SNP detection in homogeneous and label-free format. As the fluorescent signal produced by the mutant DNA was distinctively differentiated from that of the wild DNA, the assay was possible to accurately determine the mutant frequency as low as 1.0%. It was sensitive and specific enough to detect 8.6 finol/L mutant DNA strands.3. MicroRNA (miRNA) has been determined with RCA reaction in homogeneous and label-free process. Designed padlock probe is perfectly complementary to miRNA so that the padlock probe can specifically be ligated and circularized with miRNA in the presence of ligase. Afterward, the miRNA can directly act as primer to initiate RCA reaction. At the same time, introduction of a reverse primer complementary to every tandem sequence on the RCA product can result in the branched RCA reaction. The branched RCA process generates large amounts of various length ssDNA and dsDNA products, which can be detected by fluorescence dyes binding to DNA specifically. The method is simple, sensitive and specific to distinguish miRNAs with a single-base difference without the procedure of reverse transcription.4. Ferric nanoparticles, which are easily prepared with good photostability and biocompatibility by using a simple procedure, can conjugate to DNA through electrostatic interaction and form large aggregates. As a result of aggregation of ferric nanoparticles, the greatly enhanced resonance light scattering (RLS) is determined using highly sensitive RLS technology. Thus, a homogeneous and label-free method is developed for quantitation of DNA with ferric nanoparticles. The assay is simple, rapid, sensitive and inexpensive. Detection limits is 1.9 ng/mL for E. coli K12 genomic DNA. The method may provide a foundation for further study of SNP, RNA and proteins with ferric nanoparticles.