Comparison of Fluorescence Energy Transfer Primers with Different Donor–Acceptor Dye Combinations

Hung, Su-Chun; Mathies, Richard A.; Glazer, Alexander N.

doi:10.1006/abio.1997.2451

Cited by 44 publications

(20 citation statements)

References 19 publications

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“…Because it is essential to have labels with as little spectral overlap as possible, ET primers, with a cyanine donor dye and ROX or R110 as the acceptor dyes, were chosen. Both ET primer sets absorb at the common laser excitation wavelength of 488 nm, while ET-ROX emits at 605 nm and ET-R110 at 530 nm (Hung et al 1996(Hung et al , 1998Ju et al 1997). Figure 2 presents the results of a two-color LOH analysis.…”

Section: Two-color Str Analysesmentioning

confidence: 99%

“…Four sets of two-color, energy-transfer fluorescent PCR primers were synthesized according to the methodology of Hung et al (1996Hung et al ( , 1998 and Ju et al (1995). The labeled primers were all conjugated with a common fluorescent donor cyanine dye (CYA), 3-(-carboxypentyl)-3Ј-ethyl-5-5Ј-dimethyloxacarbocyanine, at the 5Ј end of the primer, and the acceptor dyes were conjugated onto a modified T nucleotide within the sequence.…”

Section: Pcr Primers and Amplification Of Str Locimentioning

confidence: 99%

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Loss of Heterozygosity Assay for Molecular Detection of Cancer Using Energy-transfer Primers and Capillary Array Electrophoresis

Medintz¹,

Lee

Wong³

et al. 2000

Genome Res.

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Microsatellite DNA loci are useful markers for the detection of loss of heterozygosity (LOH) and microsatellite instability (MI) associated with primary cancers. To carry out large-scale studies of LOH and MI in cancer progression, high-throughput instrumentation and assays with high accuracy and sensitivity need to be validated. DNA was extracted from 26 renal tumor and paired lymphocyte samples and amplified with two-color energy-transfer (ET) fluorescent primers specific for loci associated with cancer-induced chromosomal changes. PCR amplicons were separated on the MegaBACE-1000 96 capillary array electrophoresis (CAE) instrument and analyzed with MegaBACE Genetic Profiler v.1.0 software. Ninety-six separations were achieved in parallel in 75 minutes. Loss of heterozygosity was easily detected in tumor samples as was the gain/loss of microsatellite core repeats. Allelic ratios were determined with a precision of ± 10% or better. Prior analysis of these samples with slab gel electrophoresis and radioisotope labeling had not detected these changes with as much sensitivity or precision. This study establishes the validity of this assay and the MegaBACE instrument for large-scale, high-throughput studies of the molecular genetic changes associated with cancer.

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Section: Two-color Str Analysesmentioning

confidence: 99%

Section: Pcr Primers and Amplification Of Str Locimentioning

confidence: 99%

Loss of Heterozygosity Assay for Molecular Detection of Cancer Using Energy-transfer Primers and Capillary Array Electrophoresis

Medintz¹,

Lee

Wong³

et al. 2000

Genome Res.

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“…The TaqMan DNA probe used to detect specific DNA sequences, for example, employs the energy transfer pair of fluorescein and rhodamine [10]. The growing awareness that matrix metalloproteinases play a significance role in various cancers has spurred the development of new laboratory assays based on fluorescence energy transfer pairs for specific matrix metalloproteinases.…”

Section: Periodontal Disease Assaysmentioning

confidence: 99%

<title>Intraoral fiber-optic-based diagnostic for periodontal disease</title>

et al. 2000

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“…[5] Several studies on the application of FRET to DNA duplexes have reported an increase in the "apparent" Stokes shift. [6][7][8][9][10][11][12] For example, pyrene and perylene, which are known as a FRET pair, [13] were introduced into an ODN to increase the apparent Stokes shift. The emission band of pyrene appeared at 370-430 nm, whereas the absorption band of perylene appeared at 390-470 nm.…”

Section: Introductionmentioning

confidence: 99%

An Efficient Fluorescence Resonance Energy Transfer (FRET) between Pyrene and Perylene Assembled in a DNA Duplex and Its Potential for Discriminating Single‐Base Changes

Kashida

Takatsu

Sekiguchi

et al. 2010

Chemistry A European J

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To increase the apparent Stokes' shift of perylene, pyrene (donor) and perylene (acceptor) were assembled in a DNA duplex to achieve the efficient fluorescence resonance energy transfer (FRET) from pyrene to perylene. Multiple donors were introduced in the vicinity of acceptors through D-threoninol and natural base pairs were inserted between the dyes. Accordingly, donors and acceptors could be accumulated inside the DNA without forming an undesired excimer/exciplex. When two pyrene moieties were located in proximity to one perylene with one base pair inserted between them, efficient FRET occurred within the duplex. Thus, strong emission at 460 nm was observed from perylene when excited at 345 nm at which pyrene has its absorption. The apparent Stokes' shift became as large as 115 nm with a high apparent FRET efficiency (Phi>1). However, the introduction of more than two pyrenes did not enhance the fluorescence intensity of perylene, due to the short Förster radius (R(0)) of the donor pyrene. Next, this FRET system was used to enlarge the Stokes' shift of the DNA probe, which can discriminate a one-base deletion mutant from wild type with a model system by incorporation of multiple donors into DNA. Two perylene moieties were tethered to the DNA on both sides of the intervening base, and two pyrenes were further inserted in the vicinity of the perylenes as an antenna. Hybridization of this FRET probe with a fully matched DNA allowed monomer emission of perylene when the pyrenes were excited. In contrast, excimer emission was generated by hybridization with a one-base deletion mutant. Thus, the apparent Stokes' shift was enhanced without loss of efficiency in the detection of the deletion mutant.

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Comparison of Fluorescence Energy Transfer Primers with Different Donor–Acceptor Dye Combinations

Cited by 44 publications

References 19 publications

Loss of Heterozygosity Assay for Molecular Detection of Cancer Using Energy-transfer Primers and Capillary Array Electrophoresis

Loss of Heterozygosity Assay for Molecular Detection of Cancer Using Energy-transfer Primers and Capillary Array Electrophoresis

<title>Intraoral fiber-optic-based diagnostic for periodontal disease</title>

An Efficient Fluorescence Resonance Energy Transfer (FRET) between Pyrene and Perylene Assembled in a DNA Duplex and Its Potential for Discriminating Single‐Base Changes

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