Use of a linear multicopy vector based on the mini-replicon of temperate coliphage N15 for cloning DNA with abnormal secondary structures

Abstract

A new cloning vector pN15L is described. It is a linear 13.8 kb plasmid based on the coliphage N15 minireplicon. The vector capacity exceeds 50 kb and the copy number is 250 per Escherichia coli chromosome. We show that some artificial and natural palindromes and~5% of human DNA BglII fragments can be cloned effectively in linear vector pN15L, whereas they either sharply reduce the copy number of circular vector pUC19 or cannot be cloned at all. We conclude that pN15L may be usefully employed to clone large imperfect palindromes and some abnormal sequences of human DNA.

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells

“…The first generation of linear cloning vector, termed pN15L (Table , no. 10), was constructed by ligating the partially digested N15 genome with an antibiotic selective selection marker . pN15L shared the same end structures and replicon as the linear N15 genome.…”

“…In contrast, insertion of human palindromic sequences as short as 460 bp into the typical circular vector pUC19 had caused unstable propagation (mutations include deletion, rearrangement, or unclonable at all) and drastic reduction of copy number compared to the nonpalindromic insert. , These undesirable molecular events were also detected in vectors with relatively large transgene capacity, such as cosmids (capacity of 35–45 kb) ,, and yeast artificial chromosomes (YACs) (capacity of up to 2.3 Mb). , The difference in stability and copy number between circular and N15-based linear vectors could be explained by the vector topology. Insertion of unstable palindromic or other repetitive DNA sequences into circular plasmids could contribute to the release of superhelical tension and strand slippage during the replication of lagging strand, thereby inducing the formation of cruciform or other secondary structures during the replication event .…”

“…Several advantages of the TelN-tos module have led to its adaptation for the development of linear cloning vectors. Primarily, it was reported that unstable repetitive DNA are more stably propagated in N15-based linear vectors (pN15L 34 and pJAZZ 35 plasmids, Table 1, nos. 10−11) than the conventional cloning vectors (e.g., pUC-based plasmids 38 ).…”

“…10), was constructed by ligating the partially digested N15 genome with an antibiotic selective selection marker. 34 as the linear N15 genome. Interestingly, pN15L was found to have a remarkable stability and cloning fidelity in propagating artificial (11 kb) and natural (12.5 kb) palindromic sequences as well as a large, nonpalindromic sequence (50 kb) without hampering its copy number (250 copies/E.…”

“…In contrast, insertion of human palindromic sequences as short as 460 bp into the typical circular vector pUC19 had caused unstable propagation (mutations include deletion, rearrangement, or unclonable at all) and drastic reduction of copy number compared to the nonpalindromic insert. 34,35 These undesirable molecular events were also detected in vectors with relatively large transgene capacity, such as cosmids (capacity of 35−45 kb) 26,34,35 and yeast artificial chromosomes (YACs) (capacity of up to 2.3 Mb). 39,40 The difference in stability and copy number between circular and N15-based linear vectors could be explained by the vector topology.…”