13 - Flow Cytometric Analysis and Sorting of Chromosomes

Abstract

Chromosome analysis by banding techniques is the standard way by which the cytogeneticist identifies the 24 human chromosomal types and looks for abnormalities. These methods are very useful and indispensable, although they are labour-intensive and qualitative. A method of chromosome analysis has been developed using the same flow cytometric approach developed originally for cell analysis. This new method proves to be a helpful adjunct to banding analysis. Flow sorting allows the purification of chromosomes of a single type for use in molecular genetics. For analysis by flow cytometry, metaphase chromosomes are isolated from mitotic cells and then stained while in suspension with one, or more usually two, DNA-specific fluorescent dyes. The chromosomes are then processed one at a time through a flow cytometer or sorter where their dye content is measured at high speed and with high precision. These measurements are the basis of chromosome classification by flow systems. Dual staining is used as bivariate analysis affords better discrimination among chromosomal types; two dyes commonly used are Hoechst 33258, which binds preferentially to AT-rich DNA, and chromomycin A3, which binds preferentially to GC-rich DNA.