An efficient method for the physical mapping of transgenes in barley using in situ hybridization
| Autor: | Silvia Travella, Wendy Harwood, Trude Schwarzacher, Haroldo Salvo-Garrido, John W. Snape |
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| Rok vydání: | 2001 |
| Předmět: |
Genetics
medicine.diagnostic_test Transgene Hordeum General Medicine Computational biology Genetically modified crops In situ hybridization Agrobacterium tumefaciens Biology Genes Plant Physical Chromosome Mapping biology.organism_classification Plasmid Labelling medicine Transgenes Nick translation DNA Probes Molecular Biology In Situ Hybridization Biotechnology Fluorescence in situ hybridization |
| Zdroj: | Genome. 44:104-110 |
| ISSN: | 1480-3321 0831-2796 |
| DOI: | 10.1139/gen-44-1-104 |
| Popis: | The genetic transformation of crops by particle bombardment and Agrobacterium tumefaciens systems have the potential to complement conventional plant breeding programmes. However, before deployment, transgenic plants need to be characterized in detail, and physical mapping is an integral part of this process. Therefore, it is important to have a highly efficient method for transgene detection by fluorescence in situ hybridization (FISH). This study describes a new approach, which provides efficient control of probe length and labelling, both of which play an important role in in situ hybridization of transgenes. The approach is based on reducing the size of the plasmid prior to labelling by nick translation, rather than using the whole or linearized plasmid, or varying the amounts of DNaseI in the nick translation mixture. This provided much more efficient labelling of the probe, which yielded optimal hybridization, minimal fluorescent background, and accurate physical location of the transgene.Key words: barley, transformation, FISH, transgene detection, probe design. |
| Databáze: | OpenAIRE |
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