Energy Transfer Primers with 5- or 6-Carboxyrhodamine-6G as Acceptor Chromophores

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

doi:10.1006/abio.1996.0270

Cited by 32 publications

(22 citation statements)

References 3 publications

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“…133,134 Subsequently, improvements have been directed at enhancing the £uorescence intensity and matching the electrophoretic mobility of sequence extension products (e.g. replace FAM with 5-and 6-carboxyrhodamine, and replace JOE with R6G), 135 and minimiz ing spectral overlap (e.g. bi£uor-type dyes; 5CFB-dR110, 6CFB -dR6G, 5CFB-dTMR, 5CFB-dROX).…”

Section: Multi-colour Labellingmentioning

confidence: 99%

Stains, labels and detection strategies for nucleic acids assays

Kricka

2002

Ann Clin Biochem

134

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Selected developments and trends in stains, labels and strategies for detecting and measuring nucleic acids (DNA, RNA) and related molecules [e.g. oligo(deoxy)-nucleotides, nucleic acid fragments and polymerase chain reaction products] are surveyed based on the literature in the nal decade of the 20th century (1991--2000). During this period, important families of cyanine dyes were developed for sensitive detection of double-stranded DNA, single-stranded DNA, and oligo(deoxy)nucleotides in gels and in solution, and families of energy transfer primers were produced for DNA sequencing applications. The continuing quest for improved labels for hybridization assays has produced a series of candidate labels including genes encoding enzymes, microparticles (e.g. quantum dots, nanocrystals, phosphors), and new examples of the uorophore (e.g. cyanine dyes) and enzyme class of labels (e.g. re y luciferase mutants). Label detection technologies for use in northern and southern blotting assays have focused on luminescent methods, particularly enhanced chemiluminescence for peroxidase labels and adamantyl 1,2-dioxetanes for alkaline phosphatase labels. Sets of labels have been selected to meet the demands of multicolour assays (e.g. four-colour sequencing and single nucleotide primer extension assays). Non-separation assay formats have emerged based on uorescence polarization, uorescence energy transfer (TaqMan TM , molecular beacons) and channelling principles. Microanalytical devices (microchips), highthroughput simultaneous test arrays (microarrays, gene chips), capillary electrophoretic analysis and dipstick devices have presented new challenges and requirements for nucleic acid detection, and uorescent methods currently dominate in many of these applications.

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Section: Multi-colour Labellingmentioning

confidence: 99%

Stains, labels and detection strategies for nucleic acids assays

Kricka

2002

Ann Clin Biochem

134

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“…These results demonstrate that the sensitivity and precision of this assay on the MegaBACE instrument is not lost when differentially amplified samples are co-injected. The use of different ET dye combinations on the primers leads to slight electrophoretic migration differences of no more than 1 to 1.5 bp (Hung et al 1996). Also note the presence of stutter peaks that occur from polymerase slippage during amplification of these di-nucleotide repeat STR loci (Wang et al 1997).…”

Section: Two-color Str Analysesmentioning

confidence: 99%

“…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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“…We next designed a two-color assay in which PCR amplicons from normal DNA are labeled with one fluorescent primer, PCR amplicons from urine or tumor DNA are labeled with a second distinctive fluorescent primer, and both samples are coinjected into the same capillary for separation. To overcome the spectroscopic limitations of the available commercial dyes, we used energy-transfer (ET) dye-labeled PCR primers [23-251 that exploit cyanine dyes as the donor [26]. With these improved E r primers, we can excite the labeled normal and urine or tumor DNA at a common laser wavelength while …”

Section: Introductionmentioning

confidence: 99%

Microsatellite‐based cancer detection using capillary array electrophoresis and energy‐transfer fluorescent primers

et al. 1997

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The development of sensitive, rapid, and accurate methods and apparatus for high-throughput short tandem repeat (STR) analysis will be critical for the use of microsatellite alteration in cancer screening. Here we show that STR-based bladder cancer diagnosis can be performed using capillary array electrophoresis and two-color labeling with energy-transfer (ET) fluorescent primers. Rapid (< or = 35 min) separations are achieved on capillary arrays using replaceable separation matrices and the allelic ratios are quantitatively determined with a precision of +/- 10%. With this precision, a variation of 20% was considered diagnostically significant. These methods provide a significant improvement in the speed, ease, and precision of STR analyses compared to slab gel electrophoresis.

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Energy Transfer Primers with 5- or 6-Carboxyrhodamine-6G as Acceptor Chromophores

Cited by 32 publications

References 3 publications

Stains, labels and detection strategies for nucleic acids assays

Stains, labels and detection strategies for nucleic acids assays

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

Microsatellite‐based cancer detection using capillary array electrophoresis and energy‐transfer fluorescent primers

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