Rolling circle amplification revolutionizes diagnosis and genomics of geminiviruses.
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.
Haible
Daniela
D
University of Stuttgart, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, 70550 Stuttgart, Germany.
Kober
Sigrid
S
Jeske
Holger
H
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
D004279:DNA, Viral; D004262:DNA Restriction Enzymes