Optimization of Spectroscopic and Electrophoretic Properties of Energy Transfer Primers

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

doi:10.1006/abio.1997.2287

Cited by 54 publications

(39 citation statements)

References 17 publications

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“…ET primers were synthesized and labeled as in Ju et al (1996) and Hung et al (1998). The donor dye utilized was 3-(-carboxypentyl)-3Ј-ethyl-5-5Ј-dimethyloxacarbocyanine, CYA, which was covalently attached to a modified amino linker at the 5Ј end of each ET primer (Ju et al 1996a;Hung et al 1998).…”

Section: Primer Synthesismentioning

confidence: 99%

“…The donor dye utilized was 3-(-carboxypentyl)-3Ј-ethyl-5-5Ј-dimethyloxacarbocyanine, CYA, which was covalently attached to a modified amino linker at the 5Ј end of each ET primer (Ju et al 1996a;Hung et al 1998). Acceptor dyes included 6-carboxyrhodamine-110, R110, carboxytetramethylrhodamine, TAM, and 6-carboxy-Xrhodamine, ROX (Molecular Probes).…”

Section: Primer Synthesismentioning

confidence: 99%

“…Acceptor dyes included 6-carboxyrhodamine-110, R110, carboxytetramethylrhodamine, TAM, and 6-carboxy-Xrhodamine, ROX (Molecular Probes). Acceptor dyes were attached to a modified T-nucleotide located within the sequence of each ET primer (Hung et al 1998). For specific acceptor dye positions within each ET primer sequence, see Table 2.…”

Section: Primer Synthesismentioning

confidence: 99%

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High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

Medintz¹,

Wong²,

Berti³

et al. 2001

Genome Res.

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An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population

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Section: Primer Synthesismentioning

confidence: 99%

Section: Primer Synthesismentioning

confidence: 99%

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High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

Medintz¹,

Wong²,

Berti³

et al. 2001

Genome Res.

Self Cite

View full text Add to dashboard Cite

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“…A 6% solution of acrylamide in 10 mL of water was degassed under helium for half an hour followed by addition of 5 mL of 10% TEMED (Bio-Rad, Hercules, CA) and 10 mL of 10% APS (ammonium persulfate; Bio-Rad) at 0 o C. The polymerization was allowed to proceed for 24 h at 0 o C. The polymerized gel was diluted to a final concentration of 3% in 7 M urea and 50 mM Tris, 50 mM Ntris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) and 2 mM EDTA buffer. DNA sequencing samples were synthesized using M13mp18 single-stranded templates, cyanine donor energy transfer (ET) primers [29] and Thermo Sequenase (Amersham Life Science, Cleveland, OH) following the Sanger dideoxy method [30]. Four DNA sequencing reactions were set up, one for each dye/ ddNTP pair.…”

Section: Capillaries Sieving Matrix and Samplesmentioning

confidence: 99%

Ultra-high throughput rotary capillary array electrophoresis scanner for fluorescent DNA sequencing and analysis

et al. 1999

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“…1A). DNA tagged with ET labels produces 2-14-fold higher fluorescent intensities for more readily and distinctively detected products [2][3][4]. Following their introduction and use for enhanced Figure 1.…”

Section: Introductionmentioning

confidence: 98%

Fluorescence energy transfer‐labeled primers for high‐performance forensic DNA profiling

et al. 2008

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A fluorescence energy transfer (ET) dye-labeled STR typing system (ET 16-plex) is developed for the markers used in the commercial STR typing kit PowerPlex 16, and its performance assessed using a 96-lane microfabricated capillary array electrophoresis (muCAE) system. The ET 16-plex amplicons displayed 1.6-9-fold higher fluorescence intensities compared to those produced using the single-dye (SD)-labeled multiplex kits. The ET multiplex delivered full STR profiles from 62.5 pg of DNA; half the input required for the SD kits while maintaining a similar heterozygote allele balance. This increased sensitivity should improve typing of poor-quality DNA samples by making minor or imbalanced alleles more readily detectable at the low copy number (LCN) threshold. The ET 16-plex also generated complete profiles with only 28 PCR cycles; this capability should improve LCN typing by reducing the amplification time and drop-in allele incidence. To confirm the practical advantages of ET-labeled primers, six previously problematic casework samples were tested and only the ET 16-plex kit was able to capture additional allele data. The successful development and demonstration of ET primers for higher sensitivity STR typing offers a simple solution to improving current commercial multiplex typing capability. The superior spectral properties and universal compatibility with any primer sequence provided by ET cassettes will make future multiplex construction more facile and straightforward. The pairing of ET cassette technology with the muCAE system illustrates not only an enhanced STR typing platform, but a significant step toward a higher-efficiency forensic laboratory enabled by better chemistry and microfluidics.

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Optimization of Spectroscopic and Electrophoretic Properties of Energy Transfer Primers

Cited by 54 publications

References 17 publications

High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

Ultra-high throughput rotary capillary array electrophoresis scanner for fluorescent DNA sequencing and analysis

Fluorescence energy transfer‐labeled primers for high‐performance forensic DNA profiling

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