Isothermal Amplification of Nucleic Acids

Abstract

Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

Signal Amplification for Highly Sensitive Immunosensing

“…T4 DNA ligase helps circularization of DNA template, and Phi29 DNA polymerase initiates an RCA polymerization from the prime-template hybrids, which generate long ssDNA with many tandem copies of the complement to the circularized molecule in a few minutes [66]. Since Ward et al firstly described an adaptation of the RCA for the detection of protein, termed ''immuno-RCA'' in 2000 [67], RCA has been explored as important strategies for signal enhancement in immunoassay due to the sensitivity, simplicity, and versatility of the RCA technique [68]. For example, Cheng et al [58] introduced the biotin-streptavidin system to bind primers to antibody and combined the RCA technique with oligonucleotide functionalized quantum dots (QDs), and anodic stripping voltammetric test, realizing detection of protein target at ultralow concentration (Fig.…”

<strong>Electrochemical Detection of <em>Salmonella</em> via On-surface Isothermal Amplification of its Genetic Material onto Highly Stable and Reproducible Indium Tin Oxide Platforms</strong>

“…Its high thermal and storage stability combined with its good reproducibility opens up a range of applications to develop integrated platforms for both electrochemical and optical detection of isothermal nucleic acid amplification products. There are several isothermal nucleic acid amplification methods 9 that are more easily adapted to simple instrumentation than PCR. Among them, helicase-dependent amplification (HDA) follows a reaction scheme similar to PCR but taking advantage of the helicases' ability to unwind dsDNA at a constant temperature.…”

“…In these technologies the amplification reaction takes place at a constant temperature, usually higher than 37 1C, by adapting enzymatic mechanisms from natural biological processes. 8,9 However, to truly integrate isothermal amplification and electrochemical detection there still exist important hurdles to overcome, mainly reproducibility and thermal stability of the sensing phase. Gold surfaces functionalized with DNA oligonucleotides through the formation of thiol-based self-assembled monolayers (SAMs) are a common approach for electrochemical genosensing, even though the poor stability under dry storage conditions and thermal stability in aqueous solutions limit their widespread commercial application.…”

Aptasensors Based on Stripping Voltammetry

“…Multiple amplification strategies include PCR, SDA, HCR, RCA, etc. [43][44][45]79]. RCA is a simple and powerful isothermal enzymatic process.…”