Identification and quantitation of human chromosomes by primed in situ synthesis.

Abstract

Identification of cases of chromosomal aneuptoidy represents a major area for prenatal diagnosis. In the past, analysis of chromosome complements has been the province of skilled cytogeneticists, with many years of training and experience. Recently the techniques of molecular biology have been applied to make such analyses available to laboratories without such experienced workers. The initial approach has been to use the method of fluorescent in situ hybridization (FISH). When the material to be analysed can be cultured to produce metaphase chromosomes, in situ hybridization probes derived from complete chromosomal DNA libraries have been extremely valuable (Bienz et aI. 1993; Pandya et al. 1994). A recent publication claims to be able to analyse the complete human karyotype (23 chromosome pairs) in a single step by labelling the individual chromosome probes with differing ratios of haptens, and using a sophisticated fluorescence microscope and computer image analysis system to distinguish the different labels (Speicher et al. 1996). In addition, these whole chromosome 'paints' are a robust and reliable method for analysing chromosome translocations (Delaroche et al. 1995), capable of resolving complex rearrangements beyond the skill of even the most experienced cytogeneticists. However, if the target material cannot be cultured, or when the time available does not permit such a necessarily lengthy procedure, the whole chromosome paint analysis is not appropriate. Using the paints on intact, undividing interphase nuclei produces smears of colour that cannot normally be resolved. In such cases, one must employ an alternative method of labelling and identifying the constituent chromosomes. The simplest and most robust approach is based on chromosome-specific differences in the tandem~repe ated DNA sequences located at or near the centromeres of all the human chromosomes - the alphoid and classical satellites. Labelled probes derived from the chromosome-specific sequences are annealed to fixed cells, and the number of spots in the nuclei counted to determine the number of the specific chromosome in that material. This method is susceptible to analysis by FISH, using fluorescent reporters to detect the label in the chromosome centromeres (or labelling the probes directly with fluorochromes) but there is an alternative approach, with a number of advantages over FISH, that forms the subject of this article. The chromosome-specific sequences used as probes for FISH are derived from those members of repeated sequence families that have diverged sufficiently to have distinct hybridization patterns, annealing only to a specific target chromosome. Although it has been possible to identify such sequences for a number of human chromosomes, the limitation has always been in finding a sufficiently long distinct sequence to function as a probe for in situ hybridization. In 1989 Koch et al. showed that it was possible to generate detectable fluorescent signals in situ by annealing an unlabelled oligonucleotide derived from the human alphoid satellite sequence to the denatured DNA of chromosome preparations on microscope slides, and extending this primer sequence enzymically in situ, with the incorporation of labelled nucleotide triphosphates in the newly synthesized DNA. The location of the labelled DNA can then be visualized with a fluorescent reporter. This is essentially the principle of oligonucleotide primed in situ synthesis (PRINS).