Melting Curve Analysis of Aptachains: Adenosine Detection with Internal Calibration

Abstract

Small molecules are ubiquitous in nature and their detection is relevant in various domains. However, due to their size, sensitive and selective probes are difficult to select and the detection methods are generally indirect. In this study, we introduced the use of melting curve analysis of aptachains based on split-aptamers for the detection of adenosine. Aptamers, short oligonucleotides, are known to be particularly efficient probes compared to antibodies thanks to their advantageous probe/target size ratio. Aptachains are formed from dimers with dangling ends followed by the split-aptamer binding triggered by the presence of the target. The high melting temperature of the dimers served as a calibration for the detection/quantification of the target based on the height and/or temperature shift of the aptachain melting peak.

Development and Application of a High-Resolution Melting Analysis with Unlabeled Probes for the Screening of Short-Tailed Sheep TBXT Heterozygotes

“…Furthermore, we discovered that the concentration of the DNA template in the HRM system should be investigated. According to previous studies [54], the DNA template concentration is an important determinant for the accuracy of genotyping results; however, when the DNA concentration was less than 5 ng/µL in our tests, the genotyping results were inaccurate because multiple nonspecific amplifications occurred during the HRM reaction, reducing the PCR amplification efficiency. The application of molecular identification methods can facilitate a more rapid and accurate determination than a phenotypic identification; therefore, it is important to establish a rapid detection technology for economically important traits in sheep.…”

Rapid on-site nucleic acid testing: On-chip sample preparation, amplification, and detection, and their integration into all-in-one systems

“…Multifunctional gold nanoparticles were used as a Raman reporter and Mg 2+ chelating agent, enabling indirect detection of insoluble LAMP byproducts. Surface plasmon resonance (SPR) is a phenomenon caused by the evanescent wave formed when monochromatic light reflects off the surface of a thin metal film (Lu et al, 2021). An SPR sensor measures the refractive index, angle of refraction, or energy of the reflected light to deduce mass change on the thin metal film.…”

Programmable DNA barcode-encoded exponential amplification reaction for the multiplex detection of miRNAs

“…Then, all the generated ssDNA strands bind with universal probes labeled with uorophores and quench the uorescence signal. Finally, a melting curve analysis (MCA) [32][33][34] is performed following the reaction. The concentration of miRNAs can thus be quantied by calculating the rate of uorescence change at the corresponding T m value.…”