Isolation method for human metaphase chromosomes

Abstract

Given that DNA on which genomic information is written exists aschromosomes in a cell, handling chromosomes in vitro asexperimental materials can provide varieties of informationthroughout life sciences. Metaphase chromosomes are highlydelicate under in vitro conditions, moreover, it has beendifficult to prepare massive chromosomes as experimentalmaterials. These inconvenient points have prevented researchersto use chromosomes as the materials for in vitro experiments,although numerous microscopic observations have been so farperformed. There is a standard protocol to prepare mitoticmetaphase chromosomes, i.e., PA method1, 2, 5-7. However thechromosomes prepared by the method have been found to containlots of contaminated proteins4. Ordinary purification processes,i.e., the sucrose density gradient centrifugation4 often orusually result in tremendous decrease in chromosome yield. Thusthe purity and the quantity have never met in chromosome samplepreparation. Recently we have developed a new method based onseveral previously published protocols1-4. The method enablesobtainment of intact and highly purified chromosomes in largequantities. The protocol consists of two steps; isolation ofchromosomes from the synchronized mitotic human cells by theimproved PA procedure, and purification of the chromosomes byPercoll density gradient centrifugation (PDGC).