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rdf:type |
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lifeskim:mentions |
umls-concept:C0012264,
umls-concept:C0012893,
umls-concept:C0028630,
umls-concept:C0185125,
umls-concept:C0205250,
umls-concept:C0205369,
umls-concept:C0220781,
umls-concept:C0314672,
umls-concept:C0332324,
umls-concept:C1704973,
umls-concept:C1706214,
umls-concept:C1883254
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pubmed:issue |
2
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pubmed:dateCreated |
1992-9-30
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pubmed:abstractText |
In this paper the in vitro synthesis and application of non-radioactively labelled strand specific DNA probes is described. The probe is labelled by incorporation of nucleotides with the hapten digoxigenin into single-stranded DNA during a 'run-off' reaction catalyzed by Thermus aquaticus (Taq) DNA-polymerase. The 'run-off' reaction requires a linearized plasmid template and one primer binding site at a defined distance from the restriction site. Single-stranded DNA can be synthesized during repeated cycles of denaturation, annealing, and extension. The conditions for the incorporation of digoxigenin-11-dUTP (dig-11-dUTP) during polymerization were optimized to generate strand specific DNA hybridization probes up to a length of 5000 nt. The strand specificity is demonstrated by a dot-blot, with in vitro-transcribed target RNA of c-sis. The sensitivity of the probe was tested in a Northern blot, and found to be identical to a probe radiolabelled by nick-translation (specific activity 6.5 x 10(8) cpm micrograms-1). The resolution of the signals and speed of development was even superior compared to the radiolabelled probe.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Actins,
http://linkedlifedata.com/resource/pubmed/chemical/DNA, Single-Stranded,
http://linkedlifedata.com/resource/pubmed/chemical/DNA Probes,
http://linkedlifedata.com/resource/pubmed/chemical/Deoxyuracil Nucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Digoxigenin,
http://linkedlifedata.com/resource/pubmed/chemical/Indicators and Reagents,
http://linkedlifedata.com/resource/pubmed/chemical/Platelet-Derived Growth Factor,
http://linkedlifedata.com/resource/pubmed/chemical/Polymers,
http://linkedlifedata.com/resource/pubmed/chemical/Proto-Oncogene Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Proto-Oncogene Proteins c-sis,
http://linkedlifedata.com/resource/pubmed/chemical/RNA,
http://linkedlifedata.com/resource/pubmed/chemical/digoxigenin-11-deoxyuridine...
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pubmed:status |
MEDLINE
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pubmed:month |
Apr
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pubmed:issn |
0890-8508
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
6
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
107-14
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pubmed:dateRevised |
2006-11-15
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pubmed:meshHeading |
pubmed-meshheading:1381047-Actins,
pubmed-meshheading:1381047-Base Sequence,
pubmed-meshheading:1381047-Blotting, Northern,
pubmed-meshheading:1381047-Cells, Cultured,
pubmed-meshheading:1381047-DNA, Single-Stranded,
pubmed-meshheading:1381047-DNA Probes,
pubmed-meshheading:1381047-Deoxyuracil Nucleotides,
pubmed-meshheading:1381047-Digoxigenin,
pubmed-meshheading:1381047-Electrophoresis, Agar Gel,
pubmed-meshheading:1381047-Enterovirus B, Human,
pubmed-meshheading:1381047-Genetic Vectors,
pubmed-meshheading:1381047-Humans,
pubmed-meshheading:1381047-Immunoblotting,
pubmed-meshheading:1381047-Indicators and Reagents,
pubmed-meshheading:1381047-Molecular Sequence Data,
pubmed-meshheading:1381047-Platelet-Derived Growth Factor,
pubmed-meshheading:1381047-Polymers,
pubmed-meshheading:1381047-Proto-Oncogene Proteins,
pubmed-meshheading:1381047-Proto-Oncogene Proteins c-sis,
pubmed-meshheading:1381047-RNA,
pubmed-meshheading:1381047-Sensitivity and Specificity
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pubmed:year |
1992
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pubmed:articleTitle |
'Run-off' polymerization with digoxigenin labelled nucleotides creates highly sensitive and strand specific DNA hybridization probes: synthesis and application.
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pubmed:affiliation |
Max-Planck-Institut für Biochemie, Department of Virus Research, Martinsried, Germany.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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