PCR in situ followed by microdissection allows whole chromosome painting probes to be made from single microdissected chromosomes.
Whole-chromosome painting probes (WCPs) and chromosome-arm painting probes (CAPs) are an integral part of the cytogenetic analysis of chromosome abnormalities. While these are routinely made by chromosome microdissection, multiple copies of the dissected region have been necessary to achieve a library sufficiently complex to provide adequate painting. Performing multiple dissections of chromosomes or chromosome regions is time consuming and occasionally impossible, such as when working with species whose banded karyotype is not well defined. We have developed a method whereby chromosome paints can be reliably generated by dissecting single chromosomes. The technique consists of performing degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) in situ on the chromosomes, prior to dissection. Enough amplification occurs to enable a single dissected chromosome to be used to create a painting probe sufficiently complex for use in fluorescence in situ hybridization (FISH). The amplification products remain localized on the chromosomes; this allows region-specific chromosome paints to be made. We detail this novel technique and show whole-chromosome, arm-specific, and contiguous region-specific probes for human and rat, each created from single dissected fragments of chromatin.
Christian
A T
AT
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, P.O. Box 808, L-452, Livermore, California 94551, USA.
Garcia
H E
HE
Tucker
J D
JD
D000818:Animals; D020223:Chromosome Painting / Q000379:methods*; D002877:Chromosomes, Human; D017931:DNA Primers; D004210:Dissection / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008677:Metaphase / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D012235:Rhodamines
D017931:DNA Primers; D012235:Rhodamines