PCR-DIRECTED FORMATION OF VIRAL HYBRIDS IN-VITRO

When constructing viruses that have desired hybrid phenotypes, anticip ated difficulties include the nonviability of many, possibly most, of the hybrid genomes that can be constructed by incorporation of DNA fra gments. Therefore, many different hybrid genomes may have to be constr ucted in order to find one that is viable. To perform this combinatori al work in a single experiment, we have used bacteriophage T7-infected cell extracts to transfer DNA in vitro. In an extract, we have incuba ted T7 DNA, together with DNA obtained by polymerase chain reaction (P CR) amplification of the gene (gene 17) for the tail fiber of the T7-r elated bacteriophage, T3. After in vitro Packaging of DNA in the extra ct, hybrid progeny bacteriophage were detected by probing with a T3-sp ecific oligonucleotide; hybrids are found at a frequency of 0.1%. By d etermination of the nucleotide sequence of the entire gene 17 of 14 in dependently isolated hybrids, both right and left ends of the PCR frag ment are found to be truncated in all hybrids. For all 14 hybrids, the right end is in the same location; the left end is found at 3 differe nt locations. The nonrandom location of the ends is explained by selec tion among different inserts for viability; that is, most of the hybri d genomes are nonviable. Some hybrids acquire from T3 the desirable ph enotype of nonadherence to agarose gels during agarose gel electrophor esis. (C) 1997 Academic Press