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J Virol Methods. 2004 Mar 15;116(2):209-11.

A simple method for cloning the complete begomovirus genome using the bacteriophage phi29 DNA polymerase.

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Embrapa Hortaliças, Virology, Km. 9, BR060, C. Postal 218, 70359-970 Brasília, DF, Brazil.


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.

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