PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae
| Autor: | Minetaka Sugiyama, Shigehito Ikushima, Toshimasa Nakazawa, Yoshinobu Kaneko, Satoshi Harashima |
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| Jazyk: | angličtina |
| Rok vydání: | 2005 |
| Předmět: | |
| Zdroj: | BioTechniques, Vol 38, Iss 6, Pp 909-914 (2005) |
| Druh dokumentu: | article |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/05386RR01 |
| Popis: | Chromosome engineering is playing an increasingly important role in the functional analysis of genomes. A simple and efficient technology for manipulating large chromosomal segments is key to advancing these analyses. Here we describe a simple but innovative method to split chromosomes in Saccharomyces cerevisiae, which we call PCR-mediated chromosome splitting (PCS). The PCS method combines a streamlined procedure (two-step PCR and one transformation per splitting event) with the Cre/loxP system for marker rescue. Using this novel method, chromosomes I (230 kb) and XV (1091 kb) of a haploid cell were split collectively into 10 minichromosomes ranging in size from 29-631 kb with high efficiency (routinely 80%) that were occasionally lost during mitotic growth in various combinations. These observations indicate that the PCS method provides an efficient tool to engineer the yeast genome and may offer a possible approach to identify minimal genome constitutions as a function of culture conditions through further splitting, followed by combinatorial loss of minichromosomes. |
| Databáze: | Directory of Open Access Journals |
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