Oestradiol, a new hapten for detecting nucleic acid sequences by FISH.

Oestradiol has been conjugated to allylamine-dUTP with an 11-atom spacer to allow enzymatic incorporation of the label into DNA sequences. In a comparative DNA and mRNA FISH study we have used DNA probes that were either labelled with digoxigenin, biotin or oestradiol. Results show that oestradiol-labelled probes can detect DNA and RNA sequences in FISH equally well as digoxigenin- and biotin-labelled probes. Further, no crossreactivity between the various hapten-specific antibodies and the three haptens were observed. Binding of the rabbit anti-oestradiol antibody to endogenous oestrogen in various tissues was not observed under the conditions tested. In view of the increasing demands for multi-colour DNA and mRNA FISH applications, oestradiol is a welcome addition to the collection of haptens employed in FISH.

The digoxigenin (DIG) system for non-radioactive labelling and detection of nucleic acids--an overview.

The use of non-radioactive nucleic acid probes has increased dramatically over the last years. The convenience of not having to deal with radioactive isotopes combined with the stability and economy of the non-radioactive systems has led to applications in various techniques. One of the most successful labelling and detection systems is based on the hapten digoxigenin. Here, the different methods for labelling nucleic acids with digoxigenin as well as the various possibilities for detection are described. Some typical applications illustrate the utility of the DIG system.

An infrared fluorescent dATP for labeling DNA.

Near-infrared fluorescence provides a nonradioactive method of detection with high sensitivity and low background. An infrared fluorophore has been attached covalently to the nucleotide deoxyadenosine triphosphate (dATP) to provide a reagent for enzymatic labeling of various types of DNA molecules and for facilitating their detection with an automated DNA sequencing and analysis system. DNA sequencing reaction products can be labeled internally by performing limited polymerization utilizing infrared-labeled dATP (IR-dATP) as the sole source of adenine deoxynucleotide prior to a dideoxy-specific termination reaction. PCR products can be labeled fluorescently by the addition of limited quantities of IR-dATP to the amplification reaction. This latter strategy has been utilized for detection of short tandem repeat polymorphisms (STRPs) which are useful for gene mapping, genetic diagnostics, forensic analysis, and paternity testing. Restriction fragments can be labeled also by fill-in reactions of appropriate 5' overhangs. Diminutive amounts of such fluorescently labeled DNA molecules can be visualized rapidly and conveniently using infrared detection technology.

Identification of Whole Fixed Bacterial Cells with Nonradioactive 23S rRNA-Targeted Polynucleotide Probes.

Polyribonucleotide probes (ca. 200 to 300 nucleotides in length) carrying multiple reporter molecules were produced by in vitro transcription with labeled UTP derivatives (fluorescein-12-UTP, 7-amino-4-methyl-coumarin-3-acetyl-6-UTP, tetramethylrhodamine-6-UTP, or digoxigenin-11-UTP). Despite their length, these molecules penetrated into whole fixed gram-negative cells and hybridized specifically to their target sites on the 23S rRNA. Fluorescence intensities were quantified for target and nontarget cells by the combination of a charge-coupled device videocamera and an image-processing system. Polyribonucleotide probes confer up to 26 times more fluorescence to target cells than oligonucleotide probes do. Probe sensitivity and specificity were strongly influenced by the stringency of hybridization. The use of differently labeled probes allowed the simultaneous detection of three populations. Identification of introduced test organisms in activated-sludge samples proved the applicability of this method for the in situ identification of microorganisms in complex microbial communities.

Internal fluorescence labeling with fluorescent deoxynucleotides in two-label peak-height encoded DNA sequencing by capillary electrophoresis.

Fluorescently labeled deoxynucleotides were used for internal labeling of DNA sequencing fragments generated in a two-color peak-height encoded protocol. Sequenase and a manganese-containing buffer were used to generate uniform peak heights. Tetramethyl rhodamine - dATP was used in a labeling step, followed by termination with ddATP and ddCTP in a 3:1 ratio. Fluorescein - dATP was used in a second reaction, followed by termination with ddGTP and ddTTP in a 3:1 ratio. The fragments were pooled and separated by capillary gel electrophoresis. The results were compared with peak-height encoded sequencing based on fluorescently labeled primers. The dye-labeled primers produced higher resolution separations for shorter fragments. However, dye-labeled primer fragments suffered from an earlier onset of biased reptation and produced shorter sequencing reads. Fragments from 50 to at least 500 bases in length could be sequenced with the internal labels.

Nonradioactive HLA class II typing using polymerase chain reaction and digoxigenin-11-2'-3'-dideoxy-uridinetriphosphate-labeled oligonucleotide probes.

We describe a new, simple, rapid, and sensitive nonradioactive technique for the analysis of genetic variations. Genomic DNA was amplified using polymerase chain reaction and amplified DNA was hybridized, with digoxigenin (DIG)-labeled sequence-specific oligonucleotides. High specificity and sensitivity was achieved when labeling the sequence-specific oligonucleotide at the 3' end with only one DIG using digoxigenin-11-2',3'-dideoxy-uridine-5'-triphosphate and DNA deoxynucleotidylexotransferase. The hybridized probes were detected using antidigoxigenin alkaline phosphatase, fab fragments, and X-phosphate/NBT for visualization. This method was applied to the analysis of HLA-DR4-DRB1 alleles in polymerase chain reaction-amplified genomic DNA and resulted in highly specific and sensitive hybridization signals discriminating even in cases of a one-base-pair mismatch. This technique is particularly suited for HLA oligotyping because it allows the use of tetramethylammonium chloride for the simplification of hybridization and washing conditions.

Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system).

A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Following hybridization of membrane-bound target-DNA with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CIAP]. This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deep-blue coloured, water-insoluble precipitate directly adhering to the membrane. The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot- and Southern-blots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations.

Non-radioactive labeling and detection of nucleic acids. II. Optimization of the digoxigenin system.

The random-primed DNA labeling technique was modified for the incorporation of digoxigenin into DNA as basic component of the digoxigenin-based non-radioactive DNA labeling and detection system. Digoxigenin molecules act as reporter groups for highly sensitive DNA detection by a digoxigenin-specific antibody:alkaline phosphatase-conjugate-catalysed color reaction. The parameters affecting the individual reaction steps of the digoxigenin labeling, hybridization and detection reactions were optimized to maximal sensitivity and specificity.

Non-radioactive labeling and detection of nucleic acids. IV. Synthesis and properties of digoxigenin-modified 2'-deoxyuridine-5'-triphosphates and a photoactivatable analog of digoxigenin (photodigoxigenin).

The chemical syntheses of novel digoxigenin-derivatized compounds are described which are modified substrates for enzymatically or photochemically non-radioactive digoxigenin labeling of nucleic acids. Various activated digoxigenin-haptens are coupled to 5-aminoallyl-substituted 2'-deoxyuridine-5'-triphosphate. This results in digoxigenin-modified nucleoside triphosphates of variable spacer lengths (Dig-[4]-dUTP/Dig-[11]-dUTP/Dig-[16]-dUTP) which can be used as substrates for enzymatic labeling of DNA with digoxigenin-haptens by Klenow enzyme-catalysed random-primed synthesis. In addition the synthesis of N-[4-azidobenzoyl]-N'-[(3-O-digoxigeninyl)methylcarbonyl)]-1 ,8-diamino- 3,6-dioxaoctane (photodigoxigenin), a photoactivatable analog of digoxigenin, is described which can be applied for photolabeling of DNA and RNA with digoxigenin-haptens leaving the nucleic acid molecules intact.

Studies on the role of tetrazole in the activation of phosphoramidites.

The mechanism of the tetrazole-activated coupling step in the synthesis of oligonucleotides via phosphoramidites is studied with the help of model reactions: Treatment of diethoxydiisopropylaminophosphane with two equivalents of tetrazole resulted in a diethoxy-tetrazolophosphane, whose (31P)-NMR shift of 126 ppm is identical with the signal observed during internucleotide bond formation. A series of different related diethoxy-phosphorous-acid derivatives were also synthesized; their (31P)-NMR signals between 123.9 and 130.8 ppm are additional evidence for the intermediacy of a tetrazolide species. Further NMR investigations with more basic azoles showed that tetrazole is also active as a proton donor.

X-ray study of the lithium complex of NAD.

The Li+-NAD+ complex exists as a 'dimer' of two molecules arranged head-to-tail with Li+ coordinated tetrahedrally to adenine N(7) and three pyrophosphate oxygens. Adenine is stacked intermolecularly on nicotinamide. The conformation of NAD+ is 'extended' and similar to that found in holoenzyme complexes. This is in contrast to the 'folded' structure proposed from spectroscopic studies.