Linked Life Data

9222398

Source:http://linkedlifedata.com/resource/pubmed/id/9222398

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1997-10-21
pubmed:abstractText
It has been shown for several DNA probes that the recently introduced Fast-FISH (fluorescence in situ hybridization) technique is well suited for quantitative microscopy. For highly repetitive DNA probes the hybridization (renaturation) time and the number of subsequent washing steps were reduced considerably by omitting denaturing chemical agents (e.g., formamide). The appropriate hybridization temperature and time allow a clear discrimination between major and minor binding sites by quantitative fluorescence microscopy. The well-defined physical conditions for hybridization permit automatization of the procedure, e.g., by programmable thermal cycler. Here, we present optimized conditions for a commercially available X-specific alpha-satellite probe. Highly fluorescent major binding sites were obtained for 74 degrees C hybridization temperature and 60 min hybridization time. They were clearly discriminated from some low fluorescent minor binding sites on metaphase chromosomes as well as in interphase cell nuclei. On average, a total of 3.43 +/- 1.59 binding sites were measured in metaphase spreads, and 2.69 +/- 1.00 in interphase nuclei. Microwave activation for denaturation and hybridization was tested to accelerate the procedure. The slides with the target material and the hybridization buffer were placed in a standard microwave oven. After denaturation for 20 sec at 900 W, hybridization was performed for 4 min. at 90 W. The suitability of a microwave oven for Fast-FISH was confirmed by the application to a chromosome 1-specific alpha-satellite probe. In this case, denaturation was performed at 630 W for 60 sec and hybridization at 90 W for 5 min. In all cases, the results were analyzed quantitatively and compared to the results obtained by Fast-FISH. The major binding sites were clearly discriminated by their brightness.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0100-879X
pubmed:author
pubmed:issnType
Print
pubmed:volume
30
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
15-23
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1997
pubmed:articleTitle
Optimized Fast-FISH with alpha-satellite probes: acceleration by microwave activation.
pubmed:affiliation
Institute of Applied Physics, University of Heidelberg, F.R. Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't