PCR-mediated whole genome amplification of phytoplasmas
| Autor: | Michel Ruiz Rosquete, Djaouida Rekab, Meritxell Garcia-Chapa, Giuseppe Firrao, Assumpció Batlle |
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| Rok vydání: | 2004 |
| Předmět: |
DNA
Bacterial Microbiology (medical) Phytoplasma Polymerase Chain Reaction Microbiology law.invention Nucleic acid thermodynamics law Centrifugation Density Gradient Cloning Molecular Molecular Biology Polymerase chain reaction Plant Diseases Whole Genome Amplification Genetics Base Composition Base Sequence biology Multiple displacement amplification Nucleic Acid Hybridization Vinca Amplicon biology.organism_classification AT Rich Sequence Molecular biology Restriction enzyme Recombinant DNA Genome Bacterial |
| Zdroj: | Journal of Microbiological Methods. 56:231-242 |
| ISSN: | 0167-7012 |
| DOI: | 10.1016/j.mimet.2003.10.010 |
| Popis: | A method was developed for genome analysis of phytoplasmas, bacterial plant pathogens that cannot be cultivated in vitro in cell-free media. The procedure includes a CsCl-bisbenzimide gradient buoyant centrifugation followed by polymerase chain reaction (PCR)-mediated whole genome amplification. The latter step involves digestion of the DNA by a restriction enzyme with an A/T-rich recognition sequence. Due to the different A/T content in the DNA of the pathogen and its plant host, the fragments originating from phytoplasma are shorter and are preferentially amplified in the PCR reaction. Products obtained were cloned and screened by dot-blot hybridization. Results showed that about 90% of recombinant clones appeared to harbor phytoplasma specific DNA inserts. Sequencing of randomly selected clones was carried out and comparison with the NCBI database confirmed the bacterial origin for the sequences, which have been assigned a putative function. The origin of the recombinant clones was further confirmed by the generation of specific amplicons from the phytoplasma-infected plant and not from the healthy control, using PCR primers devised from the sequences of the recombinant clones. This method could be used for genome-wide comparisons between phytoplasmas. |
| Databáze: | OpenAIRE |
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