Quantitative analysis of specific nucleic acid sequences by two-color single-molecule fluorescence detection

被引:5
作者
Goodwin, PM [1 ]
Nolan, RL [1 ]
Cai, H [1 ]
机构
[1] Los Alamos Natl Lab, Biosci Div, Los Alamos, NM 87545 USA
来源
MANIPULATION AND ANALYSIS OF BIOMOLECULES, CELLS AND TISSUES | 2003年 / 4962卷
关键词
single-molecule; fluorescence; detection; two-color; burst cross-correlation; FCS; quencher; sequence; DNA; mRNA;
D O I
10.1117/12.488210
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Single-molecule fluorescence techniques are used to detect a specific nucleic acid sequence in a mixture of unrelated sequences. Co-hybridization of a pair oligonucleotide hybridization probes, each labeled with a spectrally-distinct fluorophore and complementary to a specific sub-sequence of the target nucleic acid, forms a fluorescent adduct containing both fluorophores. The presence of the specific sequence is signaled by the simultaneous detection of both fluorophore labels on a single target fragment. We demonstrate quantitative detection of target nucleic acid sequences at fragment concentrations as low as 100 fM with a simple instrument that uses low-power, continuous-wave laser excitation.. Furthermore, we show that a cross-correlation analysis of the arrival times of individual single-molecule fluorescence photon bursts detected in spectrally separate channels permits quantitative detection of the dual-color labeled species at concentrations approximately 1000x lower than can be quantitatively detected using the photon cross-correlation between the two detection channels. We also demonstrate that a pair of quencher-labeled oligonucleotides each complementary to the fluorescent hybridization probes can be used to reduce unbound probe fluorescence, substantially improving the sensitivity of the assay. We use this approach to detect beta-actin messenger RNA (mRNA) transcripts.
引用
收藏
页码:78 / 88
页数:11
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