Near-Infrared Fluorescence Instrumentation for DNA Analysis

Middendorf, Lyle R.; Amen, Jim; Bruce, Robert; Draney, Dan; DeGraff, Darryl; Gewecke, John; Grone, Dan; Humphrey, Pat G.; Little, Garrick; Lugade, A. G.; Narayanan, Narasimhachari; Oommen, Abe; Osterman, Harry L.; Peterson, Rex A.; Rada, John; Raghavachari, Ramesh; Roemer, Steve

doi:10.1007/978-94-011-5102-3_2

Cited by 19 publications

(16 citation statements)

References 104 publications

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“…Long wavelength excitation and emission are valuable because the background autofluorescence from biological samples and optical components typically decreases at longer incident wavelengths. Red and NIR probes are now routinely used in protein labeling (2,3), chromatography (4,5), measurements in blood (6,7), noninvasive medical testing (8 -10), and DNA analysis (11)(12)(13)). An advantage of the presently available red-NIR fluorophores is their high extinction coefficient, which in turn allows high sensitivity detection.…”

confidence: 99%

On the Possibility of Long-Wavelength Long-Lifetime High-Quantum-Yield Luminophores

Lakowicz

Piszczek

Kang

2001

Analytical Biochemistry

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We describe an approach to creating a new class of luminophores which display both long wavelength emissions exceeding 600 nm and long lifetimes. These luminophores are based on resonance energy transfer (RET) from a long lifetime donor to a short lifetime but long wavelength acceptor. We demonstrated the possibility of obtaining these desirable spectral properties using donors and acceptors noncovalently bound to DNA. The donor was a ruthenium (Ru) metal-ligand complex in which one of the diimine ligands intercalated into double-helix DNA. The acceptors were either nile blue, TOTO-3, or TO-PRO-3. Upon binding of the acceptor to donor-labeled DNA, we found that the acceptor quantum yield was remarkably enhanced so that the wavelength-integrated intensities of the donor and acceptor bound to DNA were many-fold greater than the intensity of the donor and acceptor alone when separately bound to DNA. The origin of this effect is efficient energy transfer from the donor. Under these conditions the effective overall quantum yield approaches that of the acceptor. Importantly, the increased quantum yield can be obtained while maintaining usefully long apparent acceptor lifetimes of 30 to 80 ns. The effect of an increased quantum yield from a low quantum yield donor may find use in assays to detect macromolecular binding interactions. These results suggest the synthesis of covalently linked donor-acceptor pairs with the desirable spectral properties of long wavelength emission, high quantum yield, and moderately long lifetimes for gated detection.

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confidence: 99%

On the Possibility of Long-Wavelength Long-Lifetime High-Quantum-Yield Luminophores

Lakowicz

Piszczek

Kang

2001

Analytical Biochemistry

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“…The procedure of dual dye labeling coupled with automated fluorescent detection described in this study provided an excellent system to retrieve the sequence information. The IRD provide satisfactory signal levels for DNA sequence analysis because of their high molar absorbtivity and satisfactory quantum efficiency (44). Meanwhile, the improved sensitivity for near infrared detection and a constant distance in band separation provided by the Li-cor automated DNA sequencer allow accurate determination of any band gain, band loss or band shift.…”

Section: Characterization Of Frameshift Mutations Using Fluorescent Dmentioning

confidence: 99%

Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR

Chen¹

2000

Nucleic Acids Research

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Mutational spectrum analysis has become an informative genetic tool to understand those protein functions involved in mutation avoidance pathways since specific types of mutations are often associated with particular protein defects involved in DNA replication and repair. In this study, we describe a novel, fluorescence-based procedure for direct determination of deletions and insertions with 100% accuracy. We performed two complementary directed termination PCR with near infrared dye-labeled primers, followed by visualization of termination fragments using an automated Li-cor DNA sequencer. This method is used for rapid analysis of mutational spectra generated in nuclease-defective strains of Saccharomyces cerevisiae to elucidate the role of RNase H(35) in RNA primer removal during DNA replication and in mutation avoidance. Strains deficient in RNH35 displayed a distinct spontaneous mutation spectrum of deletions characterized by a unique 4 bp deletion in a lys2-Bgl allele. This was in sharp contrast to strains deficient in rad27 that displayed duplication mutations. Further analysis of mutations in a rnh35/rad27 double mutant revealed a mixed spectrum. These results indicate that RNase H(35) may participate in a redundant pathway in Okazaki fragment processing and that mutational spectra caused by protein deficiencies may be more intermediate-specific than pathway-specific.

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“…This type of label has previously been shown in work with other immunoassay formats to provide good limits of detection (i.e., pM to nM range) and low background signals for biological samples [16,17,35]. The NIR fluorescent labeled antibodies that were prepared in this study had a final antibody concentration of 0.75-0.92 mg/mL (5.0-6.1 μM), and an average concentration of 0.86 (± 0.07) mg/mL ( n = 4 batches).…”

Section: Resultsmentioning

confidence: 99%

“…This system also included a custom-built NIR fluorescence detector supplied by LI-COR, as described previously [16,17,35]. A Rheodyne Advantage PF six-port switching valve (Cotati, CA, USA) was used to alternate passage of the analyte and buffer solutions through the columns during the frontal analysis studies.…”

Section: Methodsmentioning

confidence: 99%

Development of microcolumn-based one-site immunometric assays for protein biomarkers

Pfaunmiller

Anguizola

Milanuk

et al. 2014

Journal of Chromatography A

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One-site immunometric assays that utilize affinity microcolumns were developed and evaluated for the analysis of protein biomarkers. This approach used labeled antibodies that were monitored through on-line fluorescence or near-infrared (NIR) fluorescence detection. Human serum albumin (HSA) was utilized as a model target protein for this approach. In these assays, a fixed amount of labeled anti-HSA antibodies was mixed with samples or standards containing HSA, followed by the injection of this mixture onto an HSA microcolumn to remove excess antibodies and detect the non-retained labeled antibodies that were bound to HSA from the sample. The affinity microcolumns were 2.1 mm i.d. × 5 mm and contained 8-9 nmol of immobilized HSA. These microcolumns were used from 0.10-1.0 mL/min and gave results within 35 s to 2.8 min of sample injection. Limits of detection down to 0.10-0.28 ng/mL (1.5-4.2 pM) or 25-30 pg/mL (0.38-0.45 pM) were achieved when using fluorescein or a NIR fluorescent dye as the label, with an assay precision of ± 0.1-4.2%. Several parameters were examined during the optimization of these assays, and general guidelines and procedures were developed for the extension of this approach for use with other types of affinity microcolumns and protein biomarkers.

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Near-Infrared Fluorescence Instrumentation for DNA Analysis

Cited by 19 publications

References 104 publications

On the Possibility of Long-Wavelength Long-Lifetime High-Quantum-Yield Luminophores

On the Possibility of Long-Wavelength Long-Lifetime High-Quantum-Yield Luminophores

Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR

Development of microcolumn-based one-site immunometric assays for protein biomarkers

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