PCR in situ followed by microdissection allows whole chromosome painting probes to be made from single microdissected chromosomes

Abstract

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.

Chromosomal rearrangements in�cattle and�pigs revealed by�chromosome microdissection and�chromosome painting

“…The quality of the spreading was especially poor, due to the particular fixation procedure carried out, which is necessary in order not to compromise the amplification of the microdissected material. A new strategy has been recently proposed [4] that allows the production of efficient paint probes from only one copy of microdissected material, using in situ DOP-PCR. This approach could be of help to overcome the problems discussed above, mainly due to the difficult detection of the target chromosomes on poor quality preparations.…”

Differential gene expression of mammalian SPO11/TOP6A homologs during meiosis1

“…The resulting digoxigeninlabeled painting probe was hybridized to metaphase chromosome spreads and developed according to standard protocols [19]. The metaphase chromosomes were then counterstained with 4P,6-diamidino-2-phenylindole (DAPI) and an antifade solution.…”

Use of chromosome microdissection in fish molecular cytogenetics

“…Since Sequenase is not thermo-stable, small quantities (0.3 U) must be added at each of the eight cycles during annealing. The use of Thermosequenase has also been reported (Christian et al, 1999). Since this enyme is thermostable, its use minimizes risk of contamination involved in adding additional enzyme each cycle.…”

“…However, protocol modifications have been made in order to reduce the number of chromosomes (one to five chromosomes are sufficient depending on the application), turning microdissection into a routine procedure whilst maintaining probe quality (Guan et al, 1994;Engelen et al, 1998;Christian et al, 1999;Weimer et al, 1999).…”