Design of a Molecular Beacon DNA Probe with Two Fluorophores

Zhang, Peng; Beck, Terry; Tan, Weihong

doi:10.1002/1521-3773(20010119)40:2<402::aid-anie402>3.0.co;2-i

Cited by 200 publications

(118 citation statements)

References 16 publications

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“…Such working feature enables real-time, direct detection of nucleic acids. Although the intrinsic simplicity and convenience makes direct detection attractive, the sensitivity of direct detection is limited, normally around nanomolar range (23,24). Detection of DNA/RNA of lower concentrations necessitates indirect detection, which involves target amplification.…”

Section: Discussionmentioning

confidence: 99%

“…For direct detection, molecular beacons can directly identify DNA/RNA in solution, on surfaces, and in live cells. Solution-based detection is the most convenient one, and the results showed nice reproducibility as well as good linear relationship between target concentration and fluorescence intensity (23,24). Surface-based detection has been practiced in order to develop new types of sensors and microarrays, for which the traditional washing step is eliminated (25,26).…”

Section: Introductionmentioning

confidence: 99%

“…While the 1:1 stoichiometry in hybridization provides an excellent linearity for target quantitation, it also sets a limitation for the sensitivity of detection, because the number of beacon molecules one target molecule can open cannot exceed one. Normally, molecular beacons are sensitive enough for direct quantitation of nucleic acids down to nanomolar range (23,24); to detect lower concentration, however, a target amplification technique, usually PCR, is needed.…”

Section: Introductionmentioning

confidence: 99%

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Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Chu

Lee

et al. 2008

Nucleic Acids Research

216

173

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Molecular beacons represent a new family of fluorescent probes for nucleic acids, and have found broad applications in recent years due to their unique advantages over traditional probes. Detection of nucleic acids using molecular beacons has been based on hybridization between target molecules and molecular beacons in a 1:1 stoichiometric ratio. The stoichiometric hybridization, however, puts an intrinsic limitation on detection sensitivity, because one target molecule converts only one beacon molecule to its fluorescent form. To increase the detection sensitivity, a conventional strategy has been target amplification through polymerase chain reaction. Instead of target amplification, here we introduce a scheme of signal amplification, nicking enzyme signal amplification, to increase the detection sensitivity of molecular beacons. The mechanism of the signal amplification lies in target-dependent cleavage of molecular beacons by a DNA nicking enzyme, through which one target DNA can open many beacon molecules, giving rise to amplification of fluorescent signal. Our results indicate that one target DNA leads to cleavage of hundreds of beacon molecules, increasing detection sensitivity by nearly three orders of magnitude. We designed two versions of signal amplification. The basic version, though simple, requires that nicking enzyme recognition sequence be present in the target DNA. The extended version allows detection of target of any sequence by incorporating rolling circle amplification. Moreover, the extended version provides one additional level of signal amplification, bringing the detection limit down to tens of femtomolar, nearly five orders of magnitude lower than that of conventional hybridization assay.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Chu

Lee

et al. 2008

Nucleic Acids Research

216

173

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“…There have been several approaches to the synthesis of oligonucleotides labeled by fluorophores with useful properties [6, 7, 15, 23(a)]. Notable approach has been reported on hairpinforming oligonucleotides attached to a fluorescent dye at one end and a fluorescence quencher at the other [33][34][35][36]. The fluorescence increase upon hybridization to a target DNA has facilitated real-time analysis of DNA sequences being amplified in polymerase chain reaction (PCR) and detection of mutant DNA.…”

Section: Bis-pyrene Modified Oligonucleotidesmentioning

confidence: 93%

Nucleic Acid Seqeuence Analysis Using Oligonucleotide Probes

Nakamura¹,

Kanaori²,

Yamana³

2005

Frontiers in Organic Chemistry

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We have designed and synthesized oligonucleotides possessing a pyrene or a bis-pyrene at the defined position using phosphoramidite chemistry. The pyrene probes exhibit strongly enhanced fluorescence upon binding to specific sequences of DNA/RNA. The attractive features of our probes are that the pyrene fluorescence is sensitive to local base sequences and structures of probe-nucleic acid complexes around the pyrene modification. The pyrene probes would thus be useful in fluorescence recognition of nucleic acid sequences and structures.

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“…Probes with a long stem can be too stable and might not hybridize effectively to the target. To circumvent this problem, another strategy which uses two Fs instead of one F and one Q has been developed (42) [177]. Another problem arises from the fact that stem arms should not interact with the flanking target sequences.…”

Section: Uni-probe With Two Different Fluorescent Fs (Or a F And A Q)mentioning

confidence: 99%

Development and Applications of Fluorescent Oligonucleotides

Asseline

2006

COC

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Fluorescent oligonucleotides (FONs) are used in a wide variety of areas such as molecular and mechanistics biological studies, molecular diagnostics, therapeutic development, biotechnology and nanotechnology. Ever since the post-genome era, there has been an ever-increasing demand for more rapid and accurate nucleic acid detection and quantification methods. Genetic information analyses require highly sensitive and specific detection of certain sequences, single nucleotide changes, specific structures and varied reactions in different formats in vitro, in living cells and, ultimately in animals and in human beings. Ideally, a unique event could be detected and quantified using the genomic information without amplification of the nucleic acids to be analyzed. Recent developments enabling detection at the single-molecule (SM) level have opened new perspectives for applications. This review reports on the development and applications of different families of FON probes. Specific insight is given to those leading to an important fluorescent signal change upon hybridization with their targets. Applications of FON probes include real time polymerase chain reaction (PCR) quantification, detection of single-nucleotide polymorphisms (SNP), fluorescence in situ hybridization (FISH) including detection of specific messenger RNAs in living cells, analysis of gene expression, analysis of nucleic acid structures and reactions, and in nanotechnology, the assessment of molecular machine motion and functioning.

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Design of a Molecular Beacon DNA Probe with Two Fluorophores

Cited by 200 publications

References 16 publications

Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Nucleic Acid Seqeuence Analysis Using Oligonucleotide Probes

Development and Applications of Fluorescent Oligonucleotides

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