Chapter 10 Analytical Techniques for Isolated Metaphase Chromosome Fractions

Publisher Summary This chapter discusses the analytical techniques for isolated metaphase chromosome fractions. Metaphase chromosomes represent reproducible precise subfractions of the total genome of an organism. The technology described in this chapter was developed to evaluate isolated chicken chromosomes in experiments designed to locate specific viral DNA sequences integrated into the chicken genome. In principle, the same approach can identify proteins peculiar to specific chromosomes. The chapter presents a discussion on the analysis of chromosome fractions. All chromosome isolation procedures require a hypotonic treatment step to disrupt any spindle remnants and to make the cell susceptible to shear disruption. Isolated chromosomes offer few advantages over isolated whole nuclei, unless some type of sorting of the individual kinds of chromosomes can be achieved. The techniques for fraction analysis are visual inspection and flow microfluorometry. Visual inspection of the various fractions with a phase-contrast microscope confirms the general impression that the chromosomes are distributed in the gradient according to size. In contrast to the qualitative data which visual examination provides, precise quantitative data are readily obtained by the particle analysis technique—flow microfluorometry (FMF)..