Clinical Chemistry
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Clinical Chemistry 46: 1471-1477, 2000;
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(Clinical Chemistry. 2000;46:1471-1477.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Quantification of DNA Using the Luminescent Oxygen Channeling Assay

Rajesh Patel1, Reinhold Pollner1,1, Steve de Keczer1, John Pease1, Marcel Pirio1, Neal DeChene1, Alan Dafforn1 and Sam Rosea,1

1 Advanced Diagnostics Group, Dade Behring Inc., PO Box 49013, San Jose, CA 95161-9013.
a Author for correspondence. Fax 408-239-2707; e-mail samuel_rose{at}dadebehring.com

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.




The following articles in journals at HighWire Press have cited this article:


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J. Hynes, S. Floyd, A. E. Soini, R. O'Connor, and D. B. Papkovsky
Fluorescence-Based Cell Viability Screening Assays Using Water-Soluble Oxygen Probes
J Biomol Screen, June 1, 2003; 8(3): 264 - 272.
[Abstract] [PDF]

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 Genome ResHome page
L. Beaudet, J. Bédard, B. Breton, R. J. Mercuri, and M. L. Budarf
Homogeneous Assays for Single-Nucleotide Polymorphism Typing Using AlphaScreen
Genome Res., April 1, 2001; 11(4): 600 - 608.
[Abstract] [Full Text]

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 Clin. Chem.Home page
A. Dafforn, H. Kirakossian, and K. Lao
Miniaturization of the Luminescent Oxygen Channeling Immunoassay (LOCITM) for Use in Multiplex Array Formats and Other Biochips
Clin. Chem., September 1, 2000; 46(9): 1495 - 1497.
[Full Text] [PDF]


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