Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids.

As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9.

Multiplex detection of RNA expression in Drosophila embryos.

We present a fluorescence-based, multiplex in situ hybridization method that permits the simultaneous detection of five differently labeled antisense RNA probes and up to seven differ-ent transcripts in a single Drosophila embryo. We also show that it should be possible to increase the number of detected transcripts substantially with nascent transcript multiplex fluorescent in situ hybridization. These multiplex methods fill a current technological gap between high-resolution in situ hybridization with one or two fluorescently labeled probes and low-resolution but genome-wide microarray RNA profiling and should be of great utility in establishing gene networks.

Rapid analysis of mitochondrial DNA depletion by fluorescence in situ hybridization and immunocytochemistry: potential strategies for HIV therapeutic monitoring.

Nucleoside reverse transcriptase inhibitors (NRTIs) have been a mainstay in the treatment of human immunodeficiency virus since the introduction of azidothymidine (AZT) in 1987. However, none of the current therapies can completely eradicate the virus, necessitating long-term use of anti-retroviral drugs to prevent viral re-growth. One of the side effects associated with long-term use of NRTIs is mitochondrial toxicity stemming from inhibition of the mitochondrial DNA (mtDNA) polymerase gamma, which leads to mtDNA depletion and consequently to mitochondrial dysfunction. Here we report the use of fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC) to monitor mtDNA depletion in cultured fibroblasts treated with the NRTI 2',3'-dideoxycytidine (ddC). These techniques are amenable to both microscopy and flow cytometry, allowing analysis of populations of cells on a single-cell basis. We show that, as mtDNA depletion progresses, a mosaic population develops, with some cells being depleted of and others retaining mtDNA. These techniques could be useful as potential therapeutic monitors to indicate when NRTI therapy should be interrupted to prevent mitochondrial toxicity and could aid in the development of less toxic NRTIs by providing an assay suitable for pharmacodynamic evaluation of candidate molecules.

Possible sources of dye-related signal correlation bias in two-color DNA microarray assays.

DNA microarray analyses commonly use two spectrally distinct fluorescent labels to simultaneously compare different mRNA pools. Signal correlation bias currently limits accepted resolution to twofold changes in gene expression. This bias was investigated by (i) examining fluorescence and absorption spectra and changes in relative fluorescence of DNAs labeled with the Cy3, Cy5, Alexa Fluor 555, and Alexa Fluor 647 dyes and by (ii) using homotypic hybridization assays to compare the Cy dye pair with the Alexa Fluor dye pair. Cy3 or Cy5 dye-labeled DNA exhibited reduced fluorescence and absorption anomalies that were eliminated by nuclease treatment, consistent with fluorescence quenching that arises from dye-dye or dye-DNA-dye interactions. Alexa Fluor 555 and Alexa Fluor 647 dye-labeled DNA exhibited little or no such anomalies. In microarray hybridization, the Alexa Fluor dye pair provided higher signal correlation coefficients (R2) than did the Cy dye pair; at the 95% prediction level, a 1.3-fold change in gene expression was significant using the Alexa Fluor dye pair. Lowered signal correlation of the Cy dye pair was associated with high variance in Cy5 dye signals. These results indicate that fluorescence quenching may be a source of signal bias associated with the Cy dye pair.

Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling.

Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized this latter two-step approach for maximal hybridization signal brightness using DNA probes labeled to varying degrees with different fluorescent dyes. Reverse transcriptase and DNA polymerase 1 efficiently incorporated aa-dUTP into DNA, and adjusting the aa-dUTP:dTTP ratio controlled the degree of substitution. With cDNA probes hybridized to dot blots, probes having approximately eight dyes per 100 bases gave the best sensitivity, irrespective of the dye label. alpha-Satellite probes generated by nick translation and hybridized to human chromosome spreads also showed that probes having approximately eight dyes per 100 bases provided the brightest overall signals. These data demonstrate that this labeling method generates highly sensitive DNA probes that are difficult to obtain by conventional direct incorporation approaches. The technique is inherently consistent and versatile by virtue of the efficient incorporation of primary amines and the reliable chemical labeling reaction.