Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes.
Leaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers' fields in 23 districts in Uganda in May/June 2005 by leaf-pressing infected samples onto FTA Classic Cards. Viral DNA was successfully extracted from cards stored at room temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage phi29 DNA polymerase using the TempliPhi system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced. Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned and that no errors were introduced by the phi29 polymerase. This is the first successful large-scale application of FTA card technology to the field, and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as diversity analysis and cloning of potentially new virus genomes.
Owor
Betty E
BE
Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch 7701, South Africa.
Shepherd
Dionne N
DN
Taylor
Nigel J
NJ
Edema
Richard
R
Monjane
Adérito L
AL
Thomson
Jennifer A
JA
Martin
Darren P
DP
Varsani
Arvind
A
D017103:Bacillus Phages / Q000201:enzymology / Q000235:genetics*; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D005374:Filtration / Q000295:instrumentation; D005822:Genetic Vectors; D016679:Genome, Viral; D026762:Maize streak virus / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D010209:Paper; D010802:Phylogeny; D018515:Plant Leaves / Q000821:virology; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D013997:Time Factors; D003313:Zea mays / Q000821:virology*
D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase