An efficient directional cloning system to construct cDNA libraries containing full-length inserts at high frequency.
We have developed a high efficiency cDNA cloning system which can direct the orientation of inserts in lambda-plasmid composite vectors with large cloning capacities. Cleavage of the vector DNA by SfiI creates two different nonsymmetrical 3' extensions at the ends of the vector arms. Using a linker-primer and an adaptor, cDNA is prepared so it has two different sticky ends which can be ligated to those of the vector arms. When the cDNA fragments and the vector arms are mixed, both the molecules can assemble without self-circularization due to base-pairing specificity. Ligation of the cDNA-vector mixture produces the concatemers from which phage clones carrying a single cDNA insert in the desired orientation can be formed very efficiently by in vitro packaging. This system provides: (1) high cloning efficiency [10(7)-10(8) clones/micrograms poly(A)+ RNA], (2) low background (more than 90% of the clones contain inserts), (3) directional insertion of cDNA fragments into the vectors, (4) presence of a single insert in each clone, (5) accommodation of long inserts (up to 10 kb), (6) a mechanism for rescue of the plasmid part from the lambda genome, and (7) a straightforward protocol for library preparation. Screenings of cDNA libraries constructed by this method demonstrated that cDNAs of up to 6.4 kb, containing complete coding sequences, could be isolated at high efficiency. Thus, this cloning system should be useful for the isolation of cDNAs of relatively long transcripts, present even at low abundance, in cells.
Miki
T
T
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD 20892.
Matsui
T
T
Heidaran
M A
MA
Aaronson
S A
SA
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000096:biosynthesis; D004274:DNA, Recombinant; D015698:Genomic Library; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D004274:DNA, Recombinant; D004247:DNA