A double-stranded molecular probe for homogeneous nucleic acid analysis

Daniel Meserve, Zhaohui Wang, Donna Zhang, Pak Kin Wong

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

This paper reports the design and optimization of a double-stranded molecular probe for homogeneous detection of specific nucleotide sequences. The probes are labeled with either a fluorophore or a quencher such that the probe hybridization brings the two labels into close proximity, and this diminishes the fluorescence signal in the absence of a target. In the presence of a target, the fluorophore probe is thermodynamically driven to unzip from its hybridized form and bind with the target. An equilibrium analysis, which successfully describes all the major features of the assay without any fitting parameter, is performed to generalize the design of the probe. Several key parameters affecting the performance of the assay are examined. We show that the dynamic range and the signal-to-noise ratio of the assay can be optimized by the probe concentration, the quencher-to-fluorophore ratio, and the probe strand sequence. By proper design of the sequence, the probe discriminates single nucleotide mismatches in a single step without any separation step or measurement of melting profile.

Original languageEnglish (US)
Pages (from-to)1013-1019
Number of pages7
JournalAnalyst
Volume133
Issue number8
DOIs
StatePublished - 2008

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nucleic acid analysis
Molecular Probes
Nucleic acids
Signal-To-Noise Ratio
Nucleic Acids
Freezing
Nucleotides
Fluorescence
probe
Fluorophores
Assays
assay
Labels
Signal to noise ratio
signal-to-noise ratio
Melting
fluorescence
melting

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

A double-stranded molecular probe for homogeneous nucleic acid analysis. / Meserve, Daniel; Wang, Zhaohui; Zhang, Donna; Wong, Pak Kin.

In: Analyst, Vol. 133, No. 8, 2008, p. 1013-1019.

Research output: Contribution to journalArticle

Meserve, Daniel ; Wang, Zhaohui ; Zhang, Donna ; Wong, Pak Kin. / A double-stranded molecular probe for homogeneous nucleic acid analysis. In: Analyst. 2008 ; Vol. 133, No. 8. pp. 1013-1019.
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