Fluorescently cationic conjugated polymer as an indicator of ligase chain reaction for sensitive and homogeneous detection of single nucleotide polymorphism

Ligase chain reaction (LCR) offers a simple and robust alternative platform for nucleic acid amplification, but its application has been limited because the LCR products are mostly detected by gel electrophoresis separation or heterogeneous analysis. In this paper, we report a novel homogeneous LCR assay by using cationic conjugated polymers (CCPs) as an indicator for detection of single-nucleotide polymorphism (SNP). For LCR, we design two pairs of unique target-complement probes. Each pair of probes contains two adjacent probes, in which one probe is designed with phosphorothioate modification at its 3'-end, and the other probe is labeled with fluorescein at its 5'-end. After the LCR, the two adjacent probes are ligated to form one DNA strand with a fluorescein label at its 5'-end and phosphorothioate modification at its 3'-end, which is resistant to the exonuclease I and exonuclease III degradation. When the CCP is added, because of the strong electrostatic interactions between CCP and DNA, effective fluorescence resonance energy transfer (FRET) from the CCP to the fluorescein-labeled DNA can be observed. In contrast, the unligated fluorescein-labeled probes are degraded to the mononucleotides by exonuclease I and exonuclease III. Introduction of CCP leads to inefficient FRET results because much weaker electrostatic interactions between the fluorescein-labeled mononucleotides and CCP keep the fluorescein far away from CCP. Accordingly, homogeneous LCR for SNP detection is performed successfully. The method is sensitive and specific enough to detect 1 fM (600 zmol) DNA molecules. It is possible to quantify SNP and accurately determine the allele frequency as low as 1.0%. This proposed assay strategy extends the application of LCR and provides a new platform for homogeneous detection of SNP.