Digital Ligation-Enabled Fluorescence-Coding PCR (dLiNC PCR) for High-Dimensional Multiplexed Nucleic Acid Detection

Polymerase chain reaction (PCR) is the most commonly used method for nucleic acid amplification due to its ability to detect trace levels of target molecules. However, the development of multiplexed PCR assays has been limited by the complexity of the assay derived from the use of multiple primer/probe sequences as well as the limited number of fluorescence colors. Herein, we describe a digital Ligation-eNabled fluorescence-Coding PCR (dLiNC PCR) that achieves high-dimensional multiplex nucleic acid detection by employing standard digital TaqMan PCR with a pair of universal primers/probes. The proposed assay relies upon a simple preliminary ligation reaction that encodes each target molecule with a distinct fluorescence signature. Target-specific end-point fluorescence signals generated by subsequent digital TaqMan PCR can be readily used to identify multiple targets of interest. We demonstrate the dLiNC PCR by detecting 10 ovarian cancer epigenetic biomarkers at analytical sensitivity as low as 1 aM using only two fluorescence colors. We foresee that the multiplexing capability of the dLiNC PCR can be further enhanced by expanding the number of fluorescence colors as well as a simple modification of oligonucleotide design.