Simplified cloning techniques utilizing kanamycin resistant plasmids --Roger E. Mitchell II, John Hunsperger, and Irwin Rubenstein We have developed methods and vectors which utilize differential antibiotic resistance to manipulate DNA sequences. The beta-lactamase (ampicillin resistance gene, Ampr) was removed from the pUC118 and pUC119 plasmids and replaced with the aminoglycoside 3'-phosphotransferase (kanamycin resistance, Kanr) gene. Site-directed mutagenesis was used to eliminate the HindIII site present within the Kanr gene, without changing its amino acid sequence. The resulting plasmids are termed pXC118 and pXC119, according to whether they were derived from pUC118 or pUC119. These pXC (eXChange) plasmids, pXC118 and pXC119, retain all of the standard features of their parent plasmids, including poly-cloning sequences containing several unique restriction sites, an M13 viral origin of replication allowing the synthesis of single-stranded DNA, a high-copy-number plasmid origin of replication which does not require chloramphenicol amplification to make double stranded DNA, and a unique EcoO109I restriction site in the vector backbone. As with the pUC vectors, the poly-cloning site is situated within the lacZ gene, with the orientation of the cloning site in the 118 form being opposite to that of the 119 form. Despite these similarities, however, the pXC plasmids are readily distinguishable from the pUC plasmids as a result of their different antibiotic resistance genes they encode and the different unique restriction sites which the resistance genes contain. In the case of the Kanr gene of the pXC plasmids, the most useful distinguishing site remaining after mutagenesis is that of XhoI.

These plasmids have been used to develop a method to subclone DNA by sequence exchange. In the standard subcloning procedure, the recipient plasmid is cleaved with one or two restriction enzymes and the DNA fragments dephosphorylated to prevent direct ligation of the backbone or re-insertion of any fragments which may have been released. The donor plasmid is digested with the same enzymes, and the DNA fragment which is to be transferred is purified on an agarose gel, to prevent its re-insertion into the donor backbone upon ligation. This purification step is time consuming and can be difficult if the DNA fragment is small or if the donor plasmid is only available in small quantities. The pXC plasmids overcome these problems. By using a pXC plasmid as a recipient vector, the digested, dephosphorated recipient pXC DNA can be mixed directly with unpurified, digested donor pUC plasmid DNA and ligated. After the resultant constructs are transformed into E. coli, clones are selected on media containing kanamycin. Any construct not containing the recipient (pXC) backbone will fail to grow. It is, of course, possible for the donor (pUC) backbone to be inserted into the recipient rather than the intended fragment, but such clones occur infrequently and can be easily detected by virtue of their resistance to ampicillin. All of our sequence exchanges using these new plasmids have been successful. Exchanged fragment sizes have ranged from 51 up to 3000 base pairs in size. Furthermore, some fragments have been exchanged that were only available in such small quantities that they were barely visible on an agarose gel. Even in cases where the donor construct was available in abundance and the fragment to be transferred was large, this approach saves hours, and sometimes a full day. Of course, the roles of the pXC and pUC plasmids as recipient and donor can be reversed, and this has allowed us to make sequential transfers, simply by alternating their use.


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