title: In situ detection of a PCR-synthesized human pancentromeric DNA hybridization probe by color pigment immunostaining: application for dicentric assay automation.
abstract: We report a low cost and efficient method for synthesizing a human pancentromeric DNA probe by the polymerase chain reaction (PRC) and an optimized protocol for in situ detection using color pigment immunostaining. The DNA template used in the PCR was a 2.4 kb insert containing human alphoid repeated sequences of pancentromeric DNA subcloned into pUC9 (Miller et al. 1988) and the primers hybridized to internal sequences of the 172 bp consensus tandem repeat associated with human centromeres. PCR was performed in the presence of biotin-11-dUTP, and the product was used for in situ hybridization to detect the pancentromeric region of human chromosomes in metaphase spreads. Detection of pancentromeric probe was achieved by immunoenzymatic color pigment painting to yield a permanent image detected at high resolution by bright field microscopy. The ability to synthesize the centromeric probe rapidly and to detect it with color pigment immunostaining will lead to enhanced identification and eventually to automation of various chromosome aberration assays.
authors.affiliation: Naval Research Laboratory, Washington, DC 20375, USA. kolanko@ccf.nrl.navy.mil
authors.forename: C J
authors.initials: CJ
authors.lastname: Kolanko
authors.forename: M D
authors.initials: MD
authors.lastname: Pyle
authors.forename: J
authors.initials: J
authors.lastname: Nath
authors.forename: P G
authors.initials: PG
authors.lastname: Prasanna
authors.forename: H
authors.initials: H
authors.lastname: Loats
authors.forename: W F
authors.initials: WF
authors.lastname: Blakely
mesh_terms: D001331:Automation / Q000191:economics / Q000379:methods*; D001399:Azure Stains; D002503:Centromere / Q000235:genetics*; D002869:Chromosome Aberrations / Q000235:genetics; D020223:Chromosome Painting / Q000191:economics / Q000379:methods; D002877:Chromosomes, Human / Q000235:genetics; D003116:Color; D015342:DNA Probes / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry / Q000191:economics / Q000379:methods*; D017403:In Situ Hybridization / Q000191:economics / Q000379:methods*; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D008297:Male; D008677:Metaphase / Q000235:genetics; D008853:Microscopy; D010860:Pigments, Biological; D016133:Polymerase Chain Reaction; D020080:Tandem Repeat Sequences / Q000235:genetics; D013698:Templates, Genetic
chemical_list: D001399:Azure Stains; D015342:DNA Probes; D010860:Pigments, Biological