Nucleic Acids Research, Vol 26, Issue 21 4901-4909, Copyright © 1998 by Oxford University Press
Q Tao and HB Zhang
Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome
(PAC) systems were previously developed for cloning of very large
eukaryotic DNA fragments in bacteria. We report the feasibility of cloning
very large fragments of eukaryotic DNA in bacteria using conventional
plasmid-based vectors. One conventional plasmid vector (pGEM11), one
conventional binary plasmid vector (pSLJ1711) and one conventional binary
cosmid vector (pCLD04541) were investigated using the widely used BAC
(pBeloBAC11 and pECBAC1) and BIBAC (BIBAC2) vectors as controls. The
plasmid vector pGEM11 yielded clones ranging in insert sizes from 40 to 100
kb, whereas the two binary vectors pCLD04541 and pSLJ1711 yielded clones
ranging in insert sizes from 40 to 310 kb. Analysis of the pCLD04541 and
pSLJ1711 clones indicated that they had insert sizes and stabilities
similar to the BACs and BIBACs. Our findings indicate that conventional
plasmid-based vectors are capable of cloning and stably maintaining DNA
fragments as large as BACs and PACs in bacteria. These results suggest that
many existing plasmid- based vectors, including plant and animal
transformation and expression binary vectors, could be directly used for
cloning of very large eukaryotic DNA fragments. The pCLD04541 and pSLJ1711
clones were shown to be present at at least 4-5 copies/cell. The high
stability of these clones indicates that stability of clones does not seem
contingent on single-copy status. The insert sizes and the copy numbers of
the pCLD04541 and pSLJ1711 clones indicate that Escherichia coli can stably
maintain at least 1200 kb of foreign DNA per cell. These results provide a
new conceptual and theoretical basis for development of improved and new
vectors for large DNA fragment cloning and transformation. According to
this discovery, we have established a system for large DNA fragment cloning
in bacteria using the two binary vectors, with which several very
large-insert DNA libraries have been developed.
ARTICLES
Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors
Department of Soil and Crop Sciences and Crop Biotechnology Center, Texas A&M University, College Station, TX 77843-2123, USA.
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