Reverse chromosome painting: a method for the rapid analysis of aberrant chromosomes in clinical cytogenetics.

Abstract

We describe a method, termed reverse chromosome painting, which allows the rapid analysis of the content and breakpoints of aberrant chromosomes. The method involves the sorting of small numbers of the aberrant chromosome from short term blood culture preparations or cell lines by using bivariate flow karyotype analysis. The sorted chromosomes are amplified and biotin labelled enzymatically using a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), the product annealed to metaphase spreads from normal subjects, and hybridisation detected using fluorescence in situ hybridisation (FISH). We show the usefulness of this method for routine clinical cytogenetics by the analysis of cases involving an insertion, a deletion, a translocation, and two cases of a chromosome with additional material of unknown origin. The method has particular application for the rapid resolution of the origin of de novo unbalanced chromosome duplications.

Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe

“…With the advent of techniques for the general amplification of DNA by PCR using universal primers (e.g. degenerated oligonucleotide primed (DOP)-PCR, see Telenius et al 1992), it can be expected that even small tissue samples will be sufficient as source material.…”

“…Reverse chromosome painting has also been successfully used to identify marker chromosomes in disease cases. After flow sorting of a marker chromosome, its DNA was amplified and used as a probe for CISS hybridization to normal human chromosomes (Telenius et al 1992). This allows the rapid identification of the origin(s) of the marker chromosome.…”

Cytometry and genetics

“…Subsequently, methods based on polymerase chain reaction of chromosome amplification were developed (29), which allowed chromosome paints to be generated rapidly from only a small number of sorted or microdissected chromosomes (30). This in turn led to the development of reverse chromosome painting, where the paint used was constructed from a patient's rearranged chromosomes and hybridized back onto normal methaphase spreads (31). The pattern of hybridization on the normal chromosomes identifies not only the composition of the aberrant chromosome but also the position of any chromosome breakpoints.…”

Paint‐assisted microdissection‐FISH: Rapid and simple mapping of translocation breakpoints in the embryonal rhabdomyosarcoma cell line RD

“…Reverse chromosome painting overcomes this limitation in resolution by removing the need to produce highquality counterstained spreads of abnormal metaphases (3). Instead, an abnormal chromosome of interest is recovered by flow sorting or chromosome microdissection, then amplified and labelled by degenerate oligonucleotide primed-PCR (DOP-PCR) (4).…”