Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing
| Autor: | Joanna Feehan, Shauna Somerville, Katherine E. Scheibel, Beat Keller, William Underwood, Salim Bourras |
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| Přispěvatelé: | University of Zurich, Somerville, Shauna C |
| Rok vydání: | 2017 |
| Předmět: |
Spores
0301 basic medicine genomic DNA General Chemical Engineering 580 Plants (Botany) Genome chemistry.chemical_compound 10126 Department of Plant and Microbial Biology 2400 General Immunology and Microbiology Psychology DNA Fungal Genetics Ascomycota biology General Neuroscience fungus Fungal genetics 2800 General Neuroscience sequencing Spores Fungal Issue 121 Fungal Cognitive Sciences Genome Fungal high molecular weight DNA Sequence Analysis Powdery mildew Biotechnology Sequence analysis DNA sequencing General Biochemistry Genetics and Molecular Biology 03 medical and health sciences 1300 General Biochemistry Genetics and Molecular Biology 1500 General Chemical Engineering Plant Diseases General Immunology and Microbiology Human Genome obligate pathogen Sequence Analysis DNA DNA biology.organism_classification Molecular Weight 030104 developmental biology chemistry powdery mildew Biochemistry and Cell Biology |
| Zdroj: | Journal of visualized experiments : JoVE, vol 2017, iss 121 |
| ISSN: | 1940-087X |
| DOI: | 10.3791/55463-v |
| Popis: | The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 µg DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., > 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen. |
| Databáze: | OpenAIRE |
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