Use of the polymerase chain reaction for screening and evaluation of recombinant baculovirus clones.
We report the application of the PCR for screening and high-resolution characterization of recombinant baculovirus clones. Starting with less than 10 nanograms of viral DNA, it is possible to 1) demonstrate that the DNA sequence to be expressed has not been deleted or rearranged during the co-transfection or homologous recombination events, 2) test for the presence of wild-type virus in the isolate and 3) generate amplified DNA that can be used for nucleotide sequence analysis or high-resolution restriction analysis. The method is based upon PCR of genomic viral DNA prepared from primary amplified stocks of extracellular virus using a small-scale procedure. The approach has special relevance for definitive characterization of recombinant virus used to express point mutant proteins and for characterization of recombinant virus generated through use of mixed oligonucleotides or random mutagenesis.
Webb
A C
AC
Wellesley College.
Bradley
M K
MK
Phelan
S A
SA
Wu
J Q
JQ
Gehrke
L
L
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis; D008969:Molecular Sequence Data; D009036:Moths; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic; D014162:Transfection
D004274:DNA, Recombinant; D004279:DNA, Viral