PCR-directed formation of viral hybrids in vitro.
When constructing viruses that have desired hybrid phenotypes, anticipated difficulties include the nonviability of many, possibly most, of the hybrid genomes that can be constructed by incorporation of DNA fragments. Therefore, many different hybrid genomes may have to be constructed in order to find one that is viable. To perform this combinatorial work in a single experiment, we have used bacteriophage T7-infected cell extracts to transfer DNA in vitro. In an extract, we have incubated T7 DNA, together with DNA obtained by polymerase chain reaction (PCR) amplification of the gene (gene 17) for the tail fiber of the T7-related bacteriophage, T3. After in vitro packaging of DNA in the extract, hybrid progeny bacteriophage were detected by probing with a T3-specific oligonucleotide; hybrids are found at a frequency of 0.1%. By determination of the nucleotide sequence of the entire gene 17 of 14 independently isolated hybrids, both right and left ends of the PCR fragment 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 different locations. The nonrandom location of the ends is explained by selection among different inserts for viability; that is, most of the hybrid genomes are nonviable. Some hybrids acquire from T3 the desirable phenotype of nonadherence to agarose gels during agarose gel electrophoresis.
Khan
S A
SA
Department of Biochemistry, The University of Texas Health Science Center, San Antonio 78284-7760, USA.
Watson
R H
RH
Hayes
S J
SJ
Serwer
P
P
D000595:Amino Acid Sequence; D017903:Bacteriophage T3 / Q000235:genetics* / Q000302:isolation & purification; D017123:Bacteriophage T7 / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003433:Crosses, Genetic; D017931:DNA Primers; D004279:DNA, Viral / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000821:virology; D005838:Genotype; D006824:Hybridization, Genetic; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping
D017931:DNA Primers; D004279:DNA, Viral