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Nucleic Acids Research 2006 34(5):e35; doi:10.1093/nar/gkl021
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Published online 3 March 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Methods Online

Homogeneous point mutation detection by quantum dot-mediated two-color fluorescence coincidence analysis

Hsin-Chih Yeh1, Yi-Ping Ho1, Ie-Ming Shih3,4,5 and Tza-Huei Wang1,2,*

1Department of Mechanical Engineering, The Johns Hopkins University Baltimore, MD 21218, USA 2Whitaker Biomedical Engineering Institute, The Johns Hopkins University Baltimore, MD 21218, USA 3Department of Pathology, The Johns Hopkins University Baltimore, MD 21218, USA 4Department of Oncology, The Johns Hopkins University Baltimore, MD 21218, USA 5Department of Gynecology and Obstetrics, The Johns Hopkins University Baltimore, MD 21218, USA

*To whom correspondence should be addressed. Tel: +1 410 516 7086; Fax: 1 410 516 7254; Email: thwang{at}jhu.edu

Received April 6, 2005. Revised July 5, 2005. Accepted February 14, 2006.

This report describes a new genotyping method capable of detecting low-abundant point mutations in a homogeneous, separation-free format. The method is based on integration of oligonucleotide ligation with a semiconductor quantum dot (QD)-mediated two-color fluorescence coincidence detection scheme. Surface-functionalized QDs are used to capture fluorophore-labeled ligation products, forming QD-oligonucleotide nanoassemblies. The presence of such nanoassemblies and thereby the genotype of the sample is determined by detecting the simultaneous emissions of QDs and fluorophores that occurs whenever a single nanoassembly flows through the femtoliter measurement volume of a confocal fluorescence detection system. The ability of this method to detect single events enables analysis of target signals with a multiple-parameter (intensities and count rates of the digitized target signals) approach to enhance assay sensitivity and specificity. We demonstrate that this new method is capable of detecting zeptomoles of targets and achieve an allele discrimination selectivity factor >105.


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