Comment on Blachinsky et al. "Procedure for controlling number of repeats, orientation, and order during cloning of oligonucleotides" Biotechniques 36:933-936 (June 2004).

BIOFEEDBACK Blachinsky et al. (1) described a procedure they term controlled and ordered oligonucleotides ligations (COOL), which we agree will be an extremely useful procedure for scientists attempting to create tandemly reiterated sequences in plasmids. We are writing to point out that we described a technique very similar to this one in a series of articles starting with a paper published in 1993 (2). In that paper, we used the technique to reiterate copies of various transcription control sites derived from a Ty2 retrotransposon in the yeast Saccharomyces cerevisiae. Using the technique, we were able to create tandem reiterations of up to 48 copies of a 47 bp oligonucleotide, amounting to over 2200 bp or about one-fifth of the total plasmid size (2). Clearly, without a way to direct reiteration of the oli-gonucleotides, it would be difficult to achieve even modest reiteration. Our procedure differs from that described by Blachinsky et al. (1) in ways that we think improve the ability to reiterate an oligonucleotide. They describe a process by which they repeatedly insert single oligonucleotides into the same site in a plasmid, each new oligonucle-otide inserting adjacent to the last. This technique limits the increase of the number of copies during each step to a single oligonucleotide; that is, from 1, 2, 3, and so forth. In our technique, we use the fact that flanking the oligonucleotide are sites recognized by restriction enzymes that create compatible sticky ends, for example SalI and XhoI, which both create a 5′-extension of 5′-TCGA-3′. Digestion of such a plasmid alternatively with SalI or XhoI in combination with an enzyme that recognizes a single site elsewhere in the plasmid creates fragments that each end with a copy of the region between the SalI and XhoI sites. Ligation of the two fragments reconstitutes the plasmid while also reiterating the region between the restriction sites. Applying this procedure to a plasmid carrying a single oligonucleotide insert results in duplication of the oligonucle-otide. However, because even multiple tandemly reiterated oligonucleotides are flanked by the same two restriction sites means that multimers can be combined to create very large reiterations. By combining plasmids carrying appropriately reiterated copies, one can build a plasmid with essentially any number of copies. In our hands, the structure of the plasmids do not break down by recom-bination in either bacteria or yeast. The technique may be of more general interest than simply the …