Amplification and cloning of complete enterovirus genomes by long distance PCR.
A method for amplification and cloning of complete enterovirus cDNA genomes is described. Viral RNA was reverse transcribed using an optimized protocol and a reverse transcriptase with reduced RNase H activity. Amplicons corresponding to complete genomes of 14 prototype strains of group B coxsackieviruses and echoviruses were amplified using oligonucleotide primers derived from the Coxsackievirus B3 genomic sequence of the 5' and 3' ends and a mixture of thermostable DNA polymerases. Coxsackievirus B2 amplicon was then cloned and the terminal sequences of the insert were determined. Lipofection of individual clones resulted in productive. Coxsackievirus B2 infection. The method described makes it possible to obtain large amounts of complete enterovirus cDNAs and simplifies the construction of infectious full-length cDNA clones. Successful amplification of all enterovirus prototype strains tested emphasizes the general use of the method described, which provides a rapid and efficient alternative to traditional cloning strategies.
Lindberg
A M
AM
Department of Natural Sciences, University of Kahmar, Sweden. Michael.Lindberg@ng.hik.se
Polacek
C
C
Johansson
S
S
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000302:isolation & purification; D004770:Enterovirus / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D029822:Enterovirus B, Human / Q000235:genetics / Q000302:isolation & purification; D016679:Genome, Viral; D007668:Kidney / Q000166:cytology / Q000821:virology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014162:Transfection
D018076:DNA, Complementary