Programmable multiple temperature curves for barcoding massive and versatile bioassays

Biomarkers are measurable indicators of biological processes, conditions, or diseases in the human body. They can be classified into genetic, proteomic, and metabolomic biomarkers based on their origin and function. DNA and proteins are two primary types of biomarkers that play vital roles in healthcare and medical research. They enable early diagnosis of diseases and help physicians identify appropriate treatment courses. Additionally, they can be applied to assess the safety and efficacy of new drugs, accelerating drug discovery, and reducing costs.

There are two main methods for detecting these biomarkers. One is the low-throughput specific method, such as qPCR, which can specifically quantify target genes individually. However, the number of detected signals is limited by the color channel of the readout platform. The other method is the high-throughput non-specific approach, including next-generation sequencing. While these methods have no limit to the detection number, they require significant infrastructure, complex data analysis, and are time-consuming and expensive. Simpler, faster alternative readout methods are desirable.

In this study, we utilize the melting temperature of DNA as the readout signal, representing the temperature at which half of the double-stranded DNA (dsDNA) becomes single-stranded (ssDNA). By adding a melting temperature barcode to target molecules, we can generate a unique melting curve pattern for each target molecule. These distinct melting curve patterns can be used as fingerprints for the specific identification of each targeted molecule. Moreover, by combining this approach with digital PCR, we can absolutely quantify the targeted molecules.