Abstract:
The manipulation of large (>10 kb) plasmid systems amplifies problems common to
traditional cloning strategies. Unique or rare restriction enzyme recognition sequences are
uncommon and very rarely located in opportunistic locations. Making site-specific deletions and
insertions in larger plasmids consequently leads to multiple step cloning strategies that are often
limited by time-consuming, low efficiency linker insertions or blunt-end cloning strategies.
Manipulation ofthe adenovirus genome and the genomes ofother viruses as bacterial plasmids are
systems that typify such situations.
Recombinational cloning techniques based on homologous recombination in
Saccharomyces cerevisiae that circumvent many ofthese common problems have been
developed. However, these techniques are rarely realistic options for such large plasmid systems
due to the above mentioned difficulties associated with the addition ofrequired yeast DNA
replication, partitioning and selectable marker sequences.
To determine ifrecombinational cloning techniques could be modified to simplify the
manipulation of such a large plasmid system, a recombinational cloning system for the creation of
human adenovirus EI-deletion rescue plasmids was developed.
Here we report for the first time that the 1,456 bp TRP1/ARS fragment ofYRp7 is alone
sufficient to foster successful recombinational cloning without additional partitioning sequences,
using only slight modifications of existing protocols. In addition, we describe conditions for
efficient recombinational cloning involving simultaneous deletion of large segments ofDNA (>4.2
kb) and insertion of donor fragment DNA using only a single non-unique restriction site.
The discovery that recombinational cloning can foster large deletions has been used to develop a novel recombiliational cloillng technique, selectable inarker 'kilockouf" recombinational
cloning, that uses deletion of a yeast selectable marker coupled with simultaneous negative and
positive selection to reduce background transformants to undetectable levels.
The modification of existing protocols as described in this report facilitates the use of
recombinational cloning strategies that are otherwise difficult or impractical for use with large
plasmid systems. Improvement of general recombinational cloning strategies and strategies
specific to the manipulation ofthe adenovirus genome are considered in light of data presented
herein.
