CONSTRUCTION OF BAND-SPECIFIC PAINTING PROBES FROM A SINGLE MICRODISSECTED CHROMOSOME

Chromosome microdissection has become a very powerful approach to gene rate chromosome band-specific libraries and painting probes for physic al mapping or cytogenetic analysis. This study demonstrates a modified strategy for the generation of band-specific probes for human chromos omes by microdissection and polymerase chain reaction (PCR). The micro dissected fragments were pretreated with Topoisomerase I (Topo I), whi ch catalyzed the relaxation of supercoiled DNA, and two initial rounds of DNA synthesis with T 7 DNA polymerase were performed followed by c onventional PCR to enable the reliable preparation of fluorescent in s itu hybridization (FISH) probe from a single microdissected chromosome . With this method, it was possible to construct region-specific paint ing probes for 1q32, 1q42, 7q22, 19q13.1-q13.2, and for the breakpoint of marker chromosome der(2) in renal cell carcinoma cell line (Caki-1 ). The probe has been used successfully to determine the derivation of chromosome segments unidentifiable by standard chromosome banding ana lysis.