title: Rolling circle amplification revolutionizes diagnosis and genomics of geminiviruses.
abstract: Better, easier and cheaper than polymerase chain reaction (PCR) or antibody detection, rolling circle amplification (RCA) using the bacteriophage varphi 29 DNA polymerase allows for a reliable diagnosis of geminiviruses and presumably all viruses with small single-stranded circular DNA genomes. The results show the efficiency of this technique in characterizing viral DNA components of several geminiviruses from experimental and natural host plant sources. The advantages are: (a) that no expensive devices are necessary, (b) simple handling, (c) detection of all infecting circular DNA components without any knowledge of sequence information in a single step, and (d) low costs per reaction. In addition, RCA-amplified viral DNA can be characterized by restriction fragment length polymorphism analysis and directly sequenced up to 900 bases in a single run circumventing cloning and plasmid purification. This shortcut will considerably accelerate genomics of at least gemini-, nano- and circoviruses in the future.
authors.affiliation: University of Stuttgart, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, 70550 Stuttgart, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Haible
authors.forename: Sigrid
authors.initials: S
authors.lastname: Kober
authors.forename: Holger
authors.initials: H
authors.lastname: Jeske
mesh_terms: D004262:DNA Restriction Enzymes / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D017791:Geminiviridae / Q000235:genetics* / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques; D010935:Plant Diseases / Q000821:virology*; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000821:virology
chemical_list: D004279:DNA, Viral; D004262:DNA Restriction Enzymes