ABSTRACT

Centrifugation ...........................................................................161 12.3.3 Isolation of PA Chromosomes by Glycerol Density Gradient Centrifugation ...........................................................................161 12.3.4 Isolation of CA Chromosomes ................................................. 162

12.4 Isolation of Specific Chromosomes ....................................................... 163 12.5 Mass Isolation of Plant Chromosomes .................................................. 165 12.6 Conclusion ............................................................................................. 165 Acknowledgment .............................................................................................. 166 References ......................................................................................................... 166

In higher eukaryotes, when cells enter mitosis the chromosomes start to condense and at metaphase they take on their most condensed structure. Their characteristic X-shaped structure is common to all higher eukaryotes despite the differences in DNA sequence among species, and the formation of this structure is essential to cell viability. From the early stages of chromosome research in the late 19th century until recently, chromosomes have been mainly studied from morphological and genetic points of view. There are two main reasons for this. One is the fascinating structure of chromosomes visible under the light microscope with or without

dye staining, thus rendering them well suitable for morphological study. The second reason is that DNA has been a primary research interest until the completion of sequencing of the human genome, and therefore preparation and handling techniques for chromosomes as large macromolecular complexes have been left behind the study of the genome. Difficulties in isolation of chromosomes in large quantities and their inherently fragile nature are also reasons for the current lack of information on chromosomal material. In the case of human chromosomes, each chromatid has dimensions of several micrometers in length and about 1 µm in width, which makes them suitable targets for on-chip devices produced using recent nanotechnology (see Chapter 4). Chromosomes of some plants such as barley and rye are larger in size than those of human chromosomes. Furthermore, the plant Crepis

capillaries, for example, has only three chromosomes. These plant chromosomes are therefore easily distinguished and handled under optical microscopy. Hence, as summarized in Figure 12.1, chromosomes from different sources can be chosen depending on the purpose of the experiment. In this chapter, the isolation, purification and handling of human and plant chromosomes as nanomaterials for differing purposes are described, based on our recent studies.