A phage T4 in vitro packaging system for cloning long DNA molecules.
Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia coli would be useful in the analysis of complex genomes. We tested a bacteriophage T4 in vitro DNA packaging system that has the capacity to package about 170 kb of DNA into its capsid for cloning long DNA fragments. We first asked whether the T4 in vitro system can package foreign DNA such as concatemerized lambda imm434 DNA and phage P1-pBR322 hybrid DNA. The data suggest that the T4 system can package foreign DNA as efficiently as the mature phage T4 DNA. We then tested the system for its ability to clone foreign DNA fragments using the P1-pBR322 hybrid vectors constructed by Sternberg [Proc. Natl. Acad. Sci. USA 87 (1990) 103-107]. E. coli genomic DNA fragments were ligated with the P1 vectors containing two directly oriented loxP sites, and the ligated DNA was packaged by the T4 in vitro system. The packaged DNA was then transduced into E. coli expressing the phage P1 cyclization recombination protein recombinase to circularize the DNA by recombination between the loxP sites situated at the ends of the transduced DNA molecule. Clones with long DNA inserts were obtained by using this approach, and these were maintained as single-copy plasmids under the control of the P1 plasmid replicon. Clones with up to about 122-kb size inserts were recovered using this approach.
Rao
V B
VB
Department of Biology, Catholic University of America, Washington, DC 20064.
Thaker
V
V
Black
L W
LW
D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015698:Genomic Library; D008970:Molecular Weight; D010957:Plasmids; D015183:Restriction Mapping; D013604:T-Phages / Q000235:genetics*; D014161:Transduction, Genetic
D004279:DNA, Viral; D004247:DNA; D004262:DNA Restriction Enzymes