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Paint‐assisted microdissection‐FISH: Rapid and simple mapping of translocation breakpoints in the embryonal rhabdomyosarcoma cell line RD
Author(s) -
Roberts Ian,
Foster Nicola,
Nacheva Elisabeth,
Coleman Nicholas
Publication year - 2004
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20025
Subject(s) - microdissection , fluorescence in situ hybridization , biology , chromosomal translocation , breakpoint , karyotype , chromosome , cytogenetics , microbiology and biotechnology , molecular cytogenetics , genetics , comparative genomic hybridization , computational biology , gene
Background Spectral karyotyping and multiple fluorophore fluorescence in situ hybridisation (M‐FISH) facilitate identification of inter‐chromosomal rearrangements, but are of low cytogenetic resolution in mapping translocation breakpoints. Reverse chromosome painting yields increased cytogenetic information but isolation of aberrant chromosomes is technically difficult. We have developed the technique of paint‐assisted microdissection FISH (PAM‐FISH), which enables microdissection of aberrant chromosomes to be carried out easily and rapidly using relatively simple apparatus. Methods A selected chromosome paint is hybridised to abnormal metaphases to label a chromosome of interest, which is then microdissected, amplified, labelled by polymerase chain reaction (PCR), and reverse painted onto extended normal metaphases. Results PAM‐FISH was used to reassess structural chromosomal abnormalities identified by molecular cytogenetics in the rhabdomyosarcoma cell line RD. PAM‐FISH improved the analysis of virtually all structural abnormalities, identifying six novel translocations and indicating that seven previously described rearrangements were in fact not present in RD. Accuracy of the breakpoint mapping obtained was confirmed by bacterial artificial chromosome‐FISH. Conclusions PAM‐FISH is ideally suited to analysis of tumour metaphases as it is not affected by poor chromosome morphology. Reagents generated by PAM‐FISH are also suitable for other investigations, such as mapping using sequence tagged‐site PCR or genomic microarrays. PAM‐FISH is technically straightforward and could readily be adopted in a routine cytogenetics laboratory for accurate high‐throughput analysis of chromosome breakpoints. © 2004 Wiley‐Liss, Inc.