Rapid production of full-length, infectious geminivirus clones by abutting primer PCR (AbP-PCR).
The application of the polymerase chain reaction (PCR) method of DNA amplification for the isolation of full-length, infectious clones of geminiviruses is described. Non-overlapping, abutting 20-mer oligonucleotide primers were used to produce a linear product from the circular geminivirus genomic template. Clones of African cassava mosaic virus (ACMV) DNA A, obtained by this method, were infectious following mechanical inoculation (in the presence of ACMV DNA B) onto Nicotiana benthamiana. Normal ACMV symptoms resulted and typical geminate viral particles were detected by electron microscopy. The use of PCR for the detection and production of full-length, infectious geminivirus clones is discussed.
Briddon
R W
RW
Department of Virus Research, John Innes Institute, John Innes Centre for Plant Science Research, Norwich, UK.
Prescott
A G
AG
Lunness
P
P
Chamberlin
L C
LC
Markham
P G
PG
D000595:Amino Acid Sequence; D016477:Artifacts; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004270:DNA, Circular / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D014644:Genetic Variation; D007630:Kenya; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D009549:Nigeria; D010935:Plant Diseases / Q000382:microbiology; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017385:Sequence Homology; D019914:Taq Polymerase; D013698:Templates, Genetic; D014026:Nicotiana / Q000382:microbiology; D014162:Transfection
D004270:DNA, Circular; D004279:DNA, Viral; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase