A quick and simple method for the analysis of putative recombinants during plaque purification

CHRISTINA B. MCCARTHY; VICTOR ROMANOWSKI

Foz do Iguassu

Congreso; XXXVth Annual Meeting of the Society for Invertebrate Pathology, VI International Conference on Bacillus thuringiensis, VII International Colloquium on Invertebrate Pathology and Microbial Control; 2002

Society for Invertebrate Pathology (SIP)

Amplification of specific segments of DNA by pCR can be extremely useful, both for identifying putative recombinants and for confirming that selected recombinants have the appropriate genome structure. The judicious choice of primers can enable the researcher to differentiate the desired recombinant virus from single crossover recombinants and/or contaminating parental virus. However, in previous work, PCR amplification of viral DNA extracted directly from a plaque has not been considered efficient enough to permit the routine use of this approach. Instead, it has been stated that it is necessary to scale up the plaque pick by infection of cultured cells. In the present study, an easily reproducible method has been adapted for a rapid and highly sensitive analysis of DNA extracted directly from plaques, which greatly simplifies the inherently tedious task of screening for recombinants. The method consists in treating an aliquot of the resuspended plaque with a lysis buffer (10 mM Tris-Cl pH 7,6; l0 mM EDTA, 0.25% SDS), extracting the lysate with chloroform and precipitating the DNA with ethanol. The DNA pellet, redissolved in water, is suitable for PCR amplification. This method has been tested on wt and recombinant AgMNPV plaques formed on UFLAG-286 monolayers. The possibility of directly screening plaques by PCR represents a sensible reduction in time and cost involved in selecting recombinants, similar but more significant than that involved in the direct screening of recombinant E. coli colonies, since time lapses when working with insect cell cultures are inherently greater than those for bacteria.