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Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes

Nature Biotechnology volume 23, pages 885–889 (2005)Cite this article

Abstract

Rapid, multiplexed, sensitive and specific molecular detection is of great demand in gene profiling, drug screening, clinical diagnostics and environmental analysis1,2,3. One of the major challenges in multiplexed analysis is to identify each specific reaction with a distinct label or 'code'4. Two encoding strategies are currently used: positional encoding, in which every potential reaction is preassigned a particular position on a solid-phase support such as a DNA microarray5,6,7,8, and reaction encoding, where every possible reaction is uniquely tagged with a code that is most often optical or particle based4,9,10,11,12,13. The micrometer size, polydispersity, complex fabrication process and nonbiocompatibility of current codes limit their usability1,4,12. Here we demonstrate the synthesis of dendrimer-like DNA-based, fluorescence-intensity-coded nanobarcodes, which contain a built-in code and a probe for molecular recognition. Their application to multiplexed detection of the DNA of several pathogens is first shown using fluorescence microscopy and dot blotting, and further demonstrated using flow cytometry that resulted in detection that was sensitive (attomole) and rapid.

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Figure 1: Synthesis of nanobarcodes.
Figure 2: Microbead-based DNA detection using fluorescence microscopy.
Figure 3: DNA blotting assay with nanobarcodes.
Figure 4: Multiplexed DNA detection using flow cytometry.

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Acknowledgements

We wish to acknowledge the Cornell Center for Advanced Technology and the Cornell Center for Vertebrate Genomics for financial support. This material is based upon work supported in part by the Science and Technology Center Program of the National Science Foundation under agreement no. ECS-9876771. We thank Yung-Fu Chang for providing pathogen genomic DNA (Mycobacterium avium subsp. paratuberculosis) and Carol Bayles for technical support on microscopy.

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  1. Department of Biological and Environmental Engineering, Cornell University, Ithaca, 14853-5701, New York, USA

    Yougen Li, Yen Thi Hong Cu & Dan Luo

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  1. Yougen Li
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  2. Yen Thi Hong Cu
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Correspondence to Dan Luo.

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A patent for similar technology is being filed, the value of which may be increased by this publication.

Supplementary information

Supplementary Fig. 1

Evaluation of nanobarcodes with agarose gel electrophoresis. (PDF 745 kb)

Supplementary Fig. 2

Schematic drawing of nanobarcode denaturation (without showing fluorescence dyes). (PDF 96 kb)

Supplementary Fig. 3

DNA nanobarcode quantitative decoding based on microbead populations. (PDF 42 kb)

Supplementary Fig. 4

Statistics multiplexed DNA detection using flow cytometry. (PDF 55 kb)

Supplementary Table 1

Building oligonucleotides (PDF 47 kb)

Supplementary Table 2

Capture probes, report probes and target DNA (PDF 47 kb)

Supplementary Table 3

Y-DNA building blocks (PDF 43 kb)

Supplementary Table 4

DNA nanobarcodes (PDF 40 kb)

Supplementary Table 5

Code library (PDF 45 kb)

Supplementary Note 1 (PDF 66 kb)

Supplementary Note 2 (PDF 42 kb)

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Li, Y., Cu, Y. & Luo, D. Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes. Nat Biotechnol 23, 885–889 (2005). https://doi.org/10.1038/nbt1106

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