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

Goodwin, Peter M ; Nolan, Rhiannon L ; Cai, Hong

SPIE 2003

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  • Título:
    Quantitative analysis of specific nucleic acid sequences by two-color single-molecule fluorescence detection
  • Autor: Goodwin, Peter M ; Nolan, Rhiannon L ; Cai, Hong
  • Notas: Conference Location: San Jose, CA, United States
    Conference Date: 2003-01-25|2003-01-31
  • Descrição: 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 β-actin messenger RNA (mRNA) transcripts.
  • Editor: SPIE
  • Data de criação/publicação: 2003
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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