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

被引:69
作者
Yeh, HC
Ho, YP
Shih, IM
Wang, TH [1 ]
机构
[1] Johns Hopkins Univ, Dept Mech Engn, Baltimore, MD 21218 USA
[2] Johns Hopkins Univ, Whitehead Biomed Engn Inst, Baltimore, MD 21218 USA
[3] Johns Hopkins Univ, Dept Pathol, Baltimore, MD 21218 USA
[4] Johns Hopkins Univ, Dept Oncol, Baltimore, MD 21218 USA
[5] Johns Hopkins Univ, Dept Gynecol & Obstet, Baltimore, MD 21218 USA
基金
美国国家科学基金会;
关键词
D O I
10.1093/nar/gkl021
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 > 10(5).
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页数:8
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