Čeština
EnglishFlow sorting–assisted optical mapping
Šimková H., Tulpová Z., Cápal P.
Plant cytogenetics and cytogenomics: Methods and Protocols 2672: 465-483, 2023
Keywords: Optical mapping, flow cytometry and sorting, HMW DNA preparation, ultralong high-molecular-weight DNA, Bionano genome map, nuclei, chromosomes
Abstract: Optical mapping—a technique that visualizes short sequence motives along DNA molecules of hundred kilobases to megabase in size—has found an important place in genome research. It is widely used to facilitate genome sequence assemblies and analyses of genome structural variations. Application of the technique is conditional on availability of highly pure ultra-long high-molecular-weight DNA (uHMW DNA), which is challenging to achieve in plants due to the presence of the cell wall, chloroplasts, and secondary metabolites, just as a high content of polysaccharides and DNA nucleases in some species. These obstacles can be overcome by employment of flow cytometry, enabling a fast and highly efficient purifica-tion of cell nuclei or metaphase chromosomes, which are afterward embedded in agarose plugs and used to isolate the uHMW DNA in situ. Here, we provide a detailed protocol for the flow sorting–assisted uHMW DNA preparation that has been successfully used to construct whole-genome as well as chromosomal optical maps for 20 plant species from several plant families.
DOI: 10.1007/978-1-0716-3226-0_28
Fulltext: contact IEB authors
IEB authors: Petr Cápal, Hana Šimková, Zuzana Tulpová
Plant cytogenetics and cytogenomics: Methods and Protocols 2672: 465-483, 2023
Keywords: Optical mapping, flow cytometry and sorting, HMW DNA preparation, ultralong high-molecular-weight DNA, Bionano genome map, nuclei, chromosomes
Abstract: Optical mapping—a technique that visualizes short sequence motives along DNA molecules of hundred kilobases to megabase in size—has found an important place in genome research. It is widely used to facilitate genome sequence assemblies and analyses of genome structural variations. Application of the technique is conditional on availability of highly pure ultra-long high-molecular-weight DNA (uHMW DNA), which is challenging to achieve in plants due to the presence of the cell wall, chloroplasts, and secondary metabolites, just as a high content of polysaccharides and DNA nucleases in some species. These obstacles can be overcome by employment of flow cytometry, enabling a fast and highly efficient purifica-tion of cell nuclei or metaphase chromosomes, which are afterward embedded in agarose plugs and used to isolate the uHMW DNA in situ. Here, we provide a detailed protocol for the flow sorting–assisted uHMW DNA preparation that has been successfully used to construct whole-genome as well as chromosomal optical maps for 20 plant species from several plant families.
DOI: 10.1007/978-1-0716-3226-0_28
Fulltext: contact IEB authors
IEB authors: Petr Cápal, Hana Šimková, Zuzana Tulpová