Use of DOP-PCR for amplification and labeling of BAC DNA for FISH

Abstract

Fluorescence in situ hybridization (FISH) is a powerful molecular cytogenetic method that permits rapid detection of specific chromosomal rearrangements. It is based on the hybridization of fluorescent labeled probes to metaphase chromosomes or interphase nuclei. The DNA probes commonly are generated from cloned sources such as bacterial artificial chromosomes (BACs). The major disadvantage of this approach is that it requires laborious and time-consuming work. We used a degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) for both amplification and labeling of very small amounts of purified BAC DNA for FISH. The DOP-PCR reaction was processed in two steps: pre-amplification followed by simultaneous amplification and labeling of BAC DNA. The DOP-PCR probes obtained provided good hybridization signals and low background. Thus, DOP-PCR can be used to produce unlimited quantities of FISH probes with decreased cost and labor.