Nucleic Acid Detection Based On Enzymatic Amplification

The development in research of gene structure and gene function, especially the rapid progress of "Human Genome Project" makes the study of DNA a hot pot in the field of life science. Living organisms, bacteria and viruses have unique nucleic acid sequence. The detection of these specific sequences as well as their mutation hasfar-reaching significance on early diagnosis of epidemic, infectious diseases, cancer and genetic diseases. Various techniques have been developed for DNA detection including blot hybridization, flow cytometry, and fluorescence in situ hybridization. Although these methods have been utilized in clinical diagnosis, they still have some shortcomings such as complicated pretreatment, time-consuming operation, poor precision and expensiveness. Electrochemical sensor has been applied in this thesis due to their simplicity, fast response, low fabrication cost. Coupling with enzymatic catalysis cycle, new methods analyzing nucleic acid have been explored. The main results were summarized as follows:1. Ultrasensitive electrochemical detection of BCR/ABL fusion gene fragment based on circular strand replacement polymerization coupling with cascade hybridization reactionAfter the molecular beacons (MB) was immobilized on the electrode through the Au-thiol interaction, target sequence was added to open up the loop, enabling assist-DNA1 to attach on the stem part. In the presence of Klenow Fragment(3’â†'5’ exo"), DNA polymerization occurred. The lengthening new strand could release the target sequence, which would trigger new polymerization cycle, resulting in the multiplication of assist-DNA1 on the electrode. Then assist-DNA2 probes hybridized with complementary regions on each of two assist-DNA1 probe to create long concatamers structure containing abundant amino-group. Via the classic EDC coupling reaction, QDs could be tagged easily. Dissolved by HNO3, an anodic stripping voltammetry signal of Cd2+was obtained. The dynamic range of the sensor was from 10-14 mol ·L-1 to 10-10 mol·L-1, and the detection limit was 2×10-15 mol·-1. The biosensor could discriminate perfectly matched target DNA from single-base mismatch DNA and thus might have a promising future in early diagnosis and prognosis of chronic myelogenous leukaemia. Additionally, the assay could be used for detecting other DNA chain by changing bases of the capture MB probe conveniently.2. Dual channel sensitive detection of hsa-miR-21 based on rolling circle amplificationThe capture probe was immobilized on the surface of Au electrode. In the absence of target miRNA, the rolling circle amplification (RCA) could be initiated by introducing RCA template, dNTPs and Phi29 DNA polymerase into the system. The obtained RCA product was a micrometer long single-strand DNA containing thousands of repeated sequences for linear periodic hybridization of the QD-modified detection probes. Subsequently, the Cd2+ in the QD-attached RCA product was released by HNO3 solution and could be quantified by anodic stripping voltammetry (ASV) or fluorescence spectra with Rhod-5N. When the target miRNA existed, it competed with RCA template to hybridize with the capture probe leading to the inhibition of the RCA reaction. The biosensor exhibited novel dual amplification method based on the integration of RCA reaction and QDs tagging, providing reliable experiment data. The dynamic range of miR-21 was from 10-16 mol · L-1 to 3×10-13 mol · L-1 with a detection down to 3.3×10-17 mol · L-1. The desired strategy proposed a simple method for miRNA detection. In addition, the proposed method could be easily modified for other miRNA detection systems through changing the design of capture DNA, thus providing a promising detection method for prevention and early diagnosis of cancer.