SUBTRACTED, UNIQUE-SEQUENCE, IN-SITU HYBRIDIZATION - EXPERIMENTAL ANDDIAGNOSTIC APPLICATIONS

Nonrandom chromosomal aberrations, particularly in cancer, identify pa thogenic biological pathways and, in some cases, have clinical relevan ce as diagnostic or prognostic markers. Fluorescence and colorimetric in situ hybridization methods facilitate identification of numerical a nd structural chromosome abnormalities. We report the development of r obust, unique-sequence in situ hybridization probes that have several novel features: 1) they are constructed from multimegabase contigs of yeast artificial chromosome (YAC) clones; 2) they are in the form of a dapter-ligated, short-fragment, DNA Libraries that may be amplified by polymerase chain reaction; and 3) they have had repetitive sequences (eg, Alu and LINE elements) quantitatively removed by subtractive hybr idization. These subtracted probes are labeled conveniently, and the f luorescence or colorimetric detection signals are extremely bright. Mo reover, they constitute a stable resource that may be amplified throug h at least four rounds of polymerase chain reaction without diminishin g signal intensity, We demonstrate applications of subtracted probes f or the MYC and EWS oncogene regions, including 1) characterization of a novel EWS-region translocation in Ewing's sarcoma, 2) identification of chromosomal translocations in paraffin sections, and 3) identifica tion of chromosomal translocations by conventional bright-field micros copy.