Molecular Beacons: Probes that Fluoresce upon Hybridization

Tyagi, Sanjay; Kramer, Fred Russell

doi:10.1038/nbt0396-303

Cited by 3,937 publications

(2,925 citation statements)

References 21 publications

Supporting

Mentioning

2,855

Contrasting

Unclassified

Order By: Relevance

“…This rate is typical for molecular beacon detection in aqueous buffers. 19 The hybridization kinetics become progressively faster as the ethanol content is increased to 56% (Figure 1A), where the signal saturates in less than 16 sec (the fastest time that can be achieved by manual pipetting and the fluorescence plate 7 reader). In the case of 75% ethanol (pink curve), no fluorescence increase was observed and the background fluorescence was lower compared to other samples.…”

Section: The Effect Of Varying Ethanol Percentagementioning

confidence: 99%

See 1 more Smart Citation

Fast Molecular Beacon Hybridization in Organic Solvents with Improved Target Specificity

Dave

Liu

2010

J. Phys. Chem. B

View full text Add to dashboard Cite

DNA hybridization is of tremendous importance in biology, bionanotechnology, and biophysics.Molecular beacons are engineered DNA hairpins with a fluorophore and a quencher labeled on each of the two ends. A target DNA can open the hairpin to give an increased fluorescence signal. To date, the majority of molecular beacon detections have been performed only in aqueous buffers. We describe herein DNA detection in nine different organic solvents, methanol, ethanol, isopropanol, acetonitrile, formamide, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethylene glycol, and glycerol, varying each up to 75% (v/v). In comparison to detection in water, the detection in organic solvents showed several important features. First, the molecular beacon hybridizes to its target DNA in the presence of all the nine solvents up to a certain percentage. Second, the rate of this hybridization was significantly faster in most organic solvents compared to water. For example, in 56% ethanol the beacon showed a 70-fold rate enhancement. Third, the ability of the molecular beacon to discriminate single base mismatch is still maintained. Lastly, the DNA melting temperature in the organic solvents showed a solvent concentrationdependent decrease. This study suggests that molecular beacons can be used for applications where organic solvents must be involved or organic solvents can be intentionally added to improve the molecular beacon performance.

show abstract

Section: The Effect Of Varying Ethanol Percentagementioning

confidence: 99%

“…15,19,28 The beacon in the closed hairpin form has low fluorescence. In the presence of target DNA, the hairpin is opened resulting in an increase in the fluorescence.…”

Section: Introductionmentioning

confidence: 99%

Fast Molecular Beacon Hybridization in Organic Solvents with Improved Target Specificity

Dave

Liu

2010

J. Phys. Chem. B

View full text Add to dashboard Cite

show abstract

“…The separation step can be eliminated by a probe whose signal changes upon hybridization (1,2) or which is degraded during the PCR amplification reaction (3,4). One such probe is the molecular beacon (5,6), which is an oligonucleotide having a fluorophore and a quencher attached to the 5Ј and 3Ј ends, respectively, designed to form a stem-andloop structure. Upon hybridization the oligonucleotide is straightened, which separates the quencher from the fluorophore, resulting in an increase in fluorescence.…”

mentioning

confidence: 99%

Light-Up Probes: Thiazole Orange-Conjugated Peptide Nucleic Acid for Detection of Target Nucleic Acid in Homogeneous Solution

Svanvik

Westman

Wang³

et al. 2000

Analytical Biochemistry

237

147

View full text Add to dashboard Cite

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25°C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.

show abstract

“…Molecular beacons are hybridization probes that can bind to target amplification products during PCR and generate a fluorescent signal (Tyagi and Kramer, 1996). In the molecular beacon-based real-time PCR, the amplification cycle at which fluorescence becomes detectable is termed the 'cycle threshold' (C T ).…”

Section: Real-time Pcr Analysis Of Host Gene Expression Using Lcm-promentioning

confidence: 99%

“…In order to evaluate in vivo gene expression in the granuloma of a host infected with the biohazardous M. tuberculosis , we developed a system, based on LCM (Emmert-Buck et al ., 1996;Bonner et al ., 1997) and the molecular beacon-based, real-time PCR (Tyagi and Kramer, 1996) technology, which allows proficient and accurate analysis of gene expression in granulomas obtained from formalin-fixed tissues. The rationale for the LCM approach is based on the assumption that the use of RNAs extracted from whole organs of an M. tuberculosis -infected host to examine gene expression in the granuloma may yield confounding information secondary to the inclusion of non-granulomatous tissues.…”

Section: Introductionmentioning

confidence: 99%

Gene expression in the tuberculous granuloma: analysis by laser capture microdissection and real-time PCR

et al. 2003

Self Cite

View full text Add to dashboard Cite

SummaryWe have assessed the kinetics of host gene expression in granulomas of mice infected with virulent Mycobacterium tuberculosis , using an approach that incorporates the laser capture microdissection (LCM) and real-time PCR technology in conjunction with a newly derived mathematical equation. The results have provided evidence indicating that conventional use of whole infected lungs to study granulomaspecific gene expression can yield data that may not genuinely reflect intralesional events. Significantly, the expression of nine host genes known to regulate the inflammatory response to M. tuberculosis , as determined by real-time PCR analysis of microdissected granuloma-derived cDNAs, was downregulated (up to 27-fold) at around the time when the rapid growth phase of the bacilli in the lungs of infected mice ends. This downregulation was masked when whole infected lungs were used for the studies. The data suggest that the host immune system can adjust and respond to, or can be modulated by specific physiological states of the tubercle bacillus in vivo . The LCM/real-time PCR-based system described in this study can be applied to safely and accurately evaluate gene expression in any lesions that can be microscopically visualized, including those contained in biohazardous tissues.

show abstract

Molecular Beacons: Probes that Fluoresce upon Hybridization

Cited by 3,937 publications

References 21 publications

Fast Molecular Beacon Hybridization in Organic Solvents with Improved Target Specificity

Fast Molecular Beacon Hybridization in Organic Solvents with Improved Target Specificity

Light-Up Probes: Thiazole Orange-Conjugated Peptide Nucleic Acid for Detection of Target Nucleic Acid in Homogeneous Solution

Gene expression in the tuberculous granuloma: analysis by laser capture microdissection and real-time PCR

Contact Info

Product

Resources

About