Application of laser scanning image cytometry for human chromosome microdissection and DNA analysis

Abstract

P. METEZEAU, G. TACHDJIAN*, H. KIEFER, L. DRUART, V. CACHEUX*, C. NESSMANN* and G. LIZARD Biochimie Cellulaire et Cytometrie Analytique et Preparative, SC 9 INSERM-Pasteur, 28 rue du DrRoux, 75724 Paris cedex 15, France * Unite de Cytogenetique, Service de Biologie du Developpement et de la Reproduction, Hopital Robert Debre, 48 Boulevard Serurier, 75019 Paris, France Centre Commun Cytometrie en Flux, HopitalE. Herriot, Place d'Arsonval, 69437Lyon cedex 03, France INTRODUCTION Recent progress in molecular biology have increased the use of chromosomal microdissection in human genetics (Kao and Yu, 1991; Seki et al., 1993). Indeed, generation of DNA probes from genome is of great interest in Genetics. This objective can be obtained by using microdissection and amplification of a desired chromosomal region (Ludecke et al., 1989; Meltzer et al., 1992). We describe here application of laser scanning image cytometry for human chromosome microdissection and DNA analysis. MATERIALS AND METHODS Chromosome preparation Phytohemagglutinin-stimulated human lymphocyte cultures were established from healthy donors. Metaphase spreads were prepared according to our standard protocoles. Laser microdissection The laser microdissection of the chromosomes was performed on chromosomes stained with Giemsa 0.5%. Conditions adopted for current microdissection were obtained with