Labeling of DNA probes with a photoactivatable hapten

Keller, George H.; Huang, Dao-Pei; Manak, Mark M.

doi:10.1016/0003-2697(89)90072-9

Cited by 51 publications

(22 citation statements)

References 9 publications

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“…Closely located polymorphisms substantially reduce the discrimination of the ASO probes in genotyping assays. (Keller and Manak 1993;Landegren et al 1998). …”

Section: Discussionmentioning

confidence: 99%

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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“…Closely located polymorphisms substantially reduce the discrimination of the ASO probes in genotyping assays. (Keller and Manak 1993;Landegren et al 1998). …”

Section: Discussionmentioning

confidence: 99%

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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“…Previous studies revealed that oligonucleotide probes consisting of 15-30 nucleotides produced good and specific hybridization patterns [47]. Thus, the whole length of an oligonucleotide probe synthesized (i.e., multiplying the number of nucleotides in a repeat unit and the number of tandem repeats) should be within this range (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30), suggesting that the number of tandem repeats in a VNTR locus is of no importance to design an oligonucleotide probe.…”

Section: Oligonucleotide Fingerprintingmentioning

confidence: 99%

Which genetic marker for which conservation genetics issue?

et al. 2004

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Conservation genetics focuses on the effects of contemporary genetic structuring on long-term survival of a species. It helps wildlife managers protect biodiversity by identifying a series of conservation units, which include species, evolutionarily significant units (ESUs), management units (MUs), action units (AUs), and family nets (FNs). Although mitochondrial DNA (mtDNA) evolves 5-10 times faster than single-copy nuclear DNA (scnDNA), it records few traces of contemporary events. Thus, mtDNA can be used to resolve taxonomic uncertainties and ESUs. Variable number of tandem repeats (VNTRs) evolve 100-1000 times faster than scnDNA and provide a powerful tool for analyzing recent and contemporary events. VNTR analysis techniques include polymerase chain reaction (PCR)-based microsatellite assays and oligonucleotide probing. Size homoplasy problems in PCR-based microsatellite assays can strongly affect the inference of recent population history. The high homozygosity in endangered species is reflected in a relatively low number and level of variability in microsatellite loci. This combined with "allelic dropout" and "misprinting" errors contributes to the generation of highly biased genetic data following analyses of natural populations. Thus, in conservation genetics, microsatellites are of limited use for identifying ESUs, MUs, and AUs. In contrast to PCR-based microsatellite analysis, oligonucleotide probing avoids errors resulting from PCR amplification. It is particularly suitable for inferring recent population history and contemporary gene flow between fragmented subpopulations. Oligonucleotide fingerprinting generates individual-specific DNA banding patterns and thus provides a highly precise tool for monitoring demography of natural populations. Hence, DNA fingerprinting is powerful for distinguishing ESUs, MUs, AUs, and FNs. The use of oligonucleotide fingerprinting and fecal DNA is opening new areas for conservation genetics.

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“…The indicated amounts of this mixture were loaded onto 1% (w/v) agarose gels, electrophoresed and transferred onto nitrocellulose or nylon membranes. 45 ' 461 or photo-DNP [47] ) or nucleic acid-intercalating substances (e.g. psoralen' 481 ).…”

Section: Specificity Of Dna Detection In Heterologous Dot-blots With mentioning

confidence: 99%

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)

Keßler

Höltke²,

Seibl³

et al. 1990

Biological Chemistry Hoppe-Seyler

122

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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-[ll]-dUTP). Following hybridization of membrane-bound target-DN A 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):CLAP].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 deepblue 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 Southernblots 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. intestine alkaline phosphatase; Dig, digoxigenin; Dig-[ll]-dUTP, digoxigenin-(9-succinyl-e-aminocaproyl[5-(3-aminoallyl)-2'-deoxyuridine-5'-triphosphate], Na4 (see Fig. 2); (Dig), digoxigenin-specific sheep polyclonal antibody (Fab-fragments); EDTA, ethylenediamine tetraacetic acid, disodium salt; ELISA, enzyme-linked immuno-sorbent assay; ß-Gal, ß-galactosidase from E. coli; Klenow enzyme, large fragment of E. coli DNA polymerase I; NBT, nitroblue tetrazolium salt (2,2'-di-p-nitrophenyl-5,5'-diphenyI-3,3'-dimethoxy-4,4'-diphenylene]ditetrazolium chloride); POD, peroxidase from horse-radish; SDS, sodium dodecyl sulphate (lauryl sulphate, sodium salt); SSC, standard saline citrate (0.15M NaCl, 0.015M Na citrate, pH 7.0);Tris, tris[hydroxymethyl]aminomethane. Zusammenfassung: Ein neues hochsensitives, nichtradioaktives DNA Markierungs-und Detektionssystem, das auf dem ELISA-Prinzip basiert, wurde entwickelt. DNA wird mit dem Cardenolid-Hapten Digoxigenin über enzymatischen Einbau von Digoxigenin-markierten Desoxyuridintriphosphaten mit Hilfe von Klenow-Enzym modifiziert. Digoxigenin ist mit dUTP über einen 11 Atome langen linearen Spacer verbunden (Dig-[ll]-dUTP). Im Anschluß an die Hybridisierung zwischen Membran-gebundener nachzuweisender DNA und einer Digoxigenin-markierten Sonde werden die Hybride über eine ELISA-Reaktion mit Hilfe von solchen Digoxigenin-spezifischen Antikörpern nachgewiesen, an die das Markierungsenzym Alkalische Phosphatase kovalent gebunden ist [

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Labeling of DNA probes with a photoactivatable hapten

Cited by 51 publications

References 9 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

Which genetic marker for which conservation genetics issue?

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)

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