Quantification of DNA Using the Luminescent Oxygen Channeling Assay

Abstract

Background: Simplified and cost-effective methods for the detection and quantification of nucleic acid targets are still a challenge in molecular diagnostics. Methods: Luminescent oxygen channeling assay (LOCITM) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types. Results: In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets. Conclusions: This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.

Aptamers Recognizing Glycosylated Hemagglutinin Expressed on the Surface of Vaccinia Virus-Infected Cells

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Assays and Technologies for Developing Proteolysis Targeting Chimera Degraders

“…Meanwhile, the high signal-to-background ratio, high dynamic range, high sensitivity and wash-free procedure associated with the ALPHA makes this technology suitable for HTS application, allowing for the discovery of hits from a big library of compounds [39][40][41]. In addition to the application of screening enzyme or protein inhibitors [42,43], the ALPHA has also been utilized to quantify the concentrations of proteins [44,45], DNAs [46] or small molecules [47], and to capture PPI [48,49], as well as to characterize ternary complexes formed between a target protein, a PROTAC degrader and an E3 ligase [25,29,34,50,51]. Detecting ternary complex formation is one of the most ueful applications of the ALPHA for PROTAC degraders.…”

Fluorescence-Based Cell Viability Screening Assays Using Water-Soluble Oxygen Probes

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