Hairpin DNA-mediated isothermal amplification techniques for nucleic acid testing

Abstract

Nucleic acid detection is of great importance in a variety of areas, from life science and clinical diagnosis to environmental monitoring and food safety. Unfortunately, nucleic acid targets are always found in trace amounts and their response signals are difficult to be detected. Amplification mechanisms are then practically needed to either duplicate nucleic acid targets or enhance the detection signals. Polymerase chain reaction (PCR) is one of the most popular and powerful techniques for nucleic acid analysis, serving as a gold standard for detection. But the requirement of costly devices for precise thermo-cycling procedures in PCR has severely hampered the wide applications of PCR. Fortunately, isothermal molecular reactions have emerged as promising alternatives. The past decade has witnessed significant progress in the research of isothermal molecular reactions utilizing hairpin DNA probes (HDPs). Based on the nucleic acid strand interaction mechanisms, the hairpin DNA-mediated isothermal amplification (HDMIA) techniques can be mainly divided into three categories: strand assembly reactions, strand decomposition reactions, and strand creation reactions. This thesis introduces the HDMIA methods with ultra-high performances. Their sensing principles and advanced designs are evaluated, along with their wide applications, especially those benefiting from the utilization of G-quadruplexes and nanomaterials. The thesis also discusses the current challenges encountered, highlights the potential solutions, and points out the possible future directions. The first project in Chapter 2 addresses ultrasensitive fluorescent strategy for DNA detection. The method discussed utilizes a molecular beacon, hairpin DNA probe, and a nicking enzyme to trigger dual-cycling reactions, showing ultra-sensitivity, and very high selectivity over mismatched and random DNA sequences. Chapter 3 focuses attention on the utilization of advanced material to trigger dual-signal amplification for the determination of low DNA concentration. The method involves the use of graphene oxide (GO), exonuclease enzyme, and two specially designed fluorophore-labelled hairpin probes. The combination of GO-induced quenching and exonuclease enzyme-mediated dual regeneration of analytes lead to extremely low detection limit. In Chapter 4, DNAzyme technique is utilized to circumvent the requirements of bulky devices and costly reagents, while maintaining the sensing method with dual-stage signal amplification ability based on the coupling use of catalytic hairpin assembly (CHA) and Mg²⁺-dependent DNAzyme. The experimental data shows a great improvement, and the results from spiked fetal bovine serum samples further verify the reliability for practical applications. All the proposed methods are simple and relatively resolved the problems of false amplification. They are enzyme-assisted recycling methods and immobilization-free strategy. The elimination of protein based enzyme was also explored (DNAzyme), whose performance is similar to protein-based enzymes. Future work has also been proposed to utilize CRISPR/Cas techniques with HDMIA, which is expected to improve the detection limit by two to three orders of magnitude. This is a promising pathway to the fabrication of simple but sensitive point-of-care testing devices for low-cost detection.

Date of Award	2021
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Zhigang LI (Supervisor)