A simple method for cloning the complete begomovirus genome using the bacteriophage phi29 DNA polymerase.
The bacteriophage phiDNA polymerase amplifies circular DNA in a rolling circle amplification mechanism. This characteristic was applied to amplify and clone the complete circular DNA genome of a begomovirus. Total DNA extracted from infected tissue was used as the template of an amplification reaction using the commercial kit TempliPhi (Amersham Biosciences). The amplified DNA could be used for direct sequencing and was cloned after digestion with a single cutting restriction endonuclease. The use of this enzyme simplified the cloning steps and increased the cloning efficiency of the complete genome of a circular plant DNA virus.
Inoue-Nagata
Alice K
AK
Embrapa Hortaliças, Virology, Km. 9, BR060, C. Postal 218, 70359-970 Brasília, DF, Brazil. alicenag@cnph.embrapa.br
Albuquerque
Leonardo C
LC
Rocha
Wesley B
WB
Nagata
Tatsuya
T
D017103:Bacillus Phages / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D017791:Geminiviridae / Q000235:genetics*; D016679:Genome, Viral; D016133:Polymerase Chain Reaction / Q000379:methods
D017931:DNA Primers; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase