Microdissection of Chromosomes and Reverse FISH

Abstract

Microdissection of chromosomes or chromosomal regions was first published by Scalenghe et al. in 1981. They dissected fragments from the polytene X chromosome of Drosophila melanogaster. The DNA was extracted and ligated to a lambda vector. Already in this early work, it was demonstrated that the resulting clones can be hybridized back to the original section of the X chromosome, where the dissection was carried out. However, application of this method to mammalian chromosomes was hampered due to the fact that normal metaphase chromosomes contain only two double helices of DNA and hence a large number of fragments (more than 100) had to be collected in order to obtain sufficient DNA for successful cloning in a lambda vector. A significant improvement of the original technique was achieved by the application of G-banding prior to the dissection procedure (Senger et al. 1990) and by establishing an efficient micro-cloning system (Lüdecke et al. 1989). However, the real breakthrough for the microdissection technique was the invention of the sequence-independent DNA amplification. Different protocols have been shown to be efficient in amplifying a DNA pool with no specific priming sites (Bohlander et al. 1992, Telenius et al. 1992). However, it turns out that the polymerase chain reaction using a degenerate oligonucleotide primer (DOP-PCR) (Telenius et al. 1992) is the preferred amplification technique which is applied in most experiments.