Construction of band-specific painting probes from a single microdissected chromosome

초록

Chromosome microdissection has become a very powerful approach to generate chromosome band-specific libraries and painting probes for physical mapping or cytogenetic analysis. This study demonstrates a modified strategy for the generation of band-specific probes for human chromosomes by microdissection and polymerase chain reaction (PCR). The microdissected fragments were pretreated with Topoisomerase I (Topo I), which catalyzed the relaxation of supercoiled DNA, and two initial rounds of DNA synthesis with T 7 DNA polymerase were performed followed by conventional PCR to enable the reliable preparation of fluorescent in situ hybridization (FISH) probe from a single microdissected chromosome. With this method, it was possible to construct region-specific painting 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 analysis.

키워드