Ligation-Enabled Fluorescence-Coding PCR for High-Dimensional Fluorescence-Based Nucleic Acid Detection

Abstract

Polymerase chain reaction (PCR) is by far the most commonly used method of nucleic acid amplification and has likewise been employed for a plethora of diagnostic purposes. Nonetheless, multiplexed PCR-based detection schemes have hitherto been largely limited by technical challenges associated with nonspecific interactions and other limitations inherent to traditional fluorescence-based assays. Here, we describe a novel strategy for multiplexed PCR-based analysis called Ligation-eNabled fluorescence-Coding PCR (LiNC PCR) that exponentially enhances the multiplexing capability of standard fluorescence-based PCR assays. The technique relies upon a simple, preliminary ligation reaction in which target DNA sequences are converted to PCR template molecules with distinct endpoint fluorescence signatures. Universal TaqMan probes are used to create target-specific multicolor fluorescence signals that can be readily decoded to identify amplified targets of interest. We demonstrate the LiNC PCR technique by implementing a two-color-based assay for detection of 10 ovarian cancer epigenetic biomarkers at analytical sensitivities as low as 60 template molecules with no detectable target cross-talk. Overall, LiNC PCR provides a simple and inexpensive method for achieving high-dimensional multiplexing that can be implemented in manifold molecular diagnostic applications.

Micro- and nanosystems for the detection of hemorrhagic fever viruses

“…A recently developed strategy accomplished multiplexed detection by using two-color-based fluorescent probes with varied red and/or green binding sites. 151 With the use of only two fluorescent colors, the number of detectable targets is increased to ten. In the future, this approach may be utilized in specific amplification strategies for the detection of HFV analytes.…”

Configuration-regulated highly luminescent hydrogen–organic frameworks for detection of phenelzine and propofol

“…In this view, fluorescence-based detection methods have emerged to meet the demand for a more rapid detection. Fluorescence sensing, featuring a simple operation, fast response, and high efficiency, [15][16][17] is a promising approach for PZ and PPF detection. The development of fluorescent sensors has made great progress in the past decade and these have become a pillar in the fields of life sciences, electronic information, and environmental monitoring.…”

Ratiometric PCR in a Portable Sample-to-Result Device for Broad-Based Pathogen Identification

“…It demonstrates the identification of extracted DNA from 15 bacterial species with a six-color benchtop real-time PCR instrument . Another strategy to further expand the multiplexed analyses is by first ligating color-coded templates with the target DNA and then amplifying the ligated product with a pair of universal primers and two-color probes, ultimately encoding each target with a specific endpoint fluorescent intensity across two fluorescence channels. , This strategy demonstrates both an elegant method to expand the number of targets using two-color fluorescence detection platforms and the capability to implement the assay on a droplet microfluidic device. However, this method requires an additional step to ligate DNA target and color-coded templates, increasing assay complexity and adding another barrier to work directly from crude samples.…”