Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

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Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying ... continued below

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Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F. & Weier, Heinz-Ulli G. December 4, 2008.

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Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

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  • Report No.: LBNL-3116E
  • Grant Number: DE-AC02-05CH11231
  • DOI: 10.2172/982916 | External Link
  • Office of Scientific & Technical Information Report Number: 982916
  • Archival Resource Key: ark:/67531/metadc1013868

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  • December 4, 2008

Added to The UNT Digital Library

  • Oct. 14, 2017, 8:36 a.m.

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  • Oct. 18, 2017, 12:54 p.m.

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Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F. & Weier, Heinz-Ulli G. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA, report, December 4, 2008; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc1013868/: accessed September 19, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.