Procedure for controlling number of repeats, orientation, and order during cloning of oligonucleotides

Abstract

COOL Cloning Insertion of short DNA sequences into plasmids is a widely applied technique, but it can be complicated by lack of precision and the need to screen and separate clones in order to identify the insert of interest. Blachinsky et al. (p. 933) describe a straightforward procedure for introducing oligonucleotides into plasmids in the desired number, orientation, and order. The protocol entails the sequential insertion of sequences in separate ligation reactions, each of which restores the original restriction sites of the plasmid. The procedure requires only basic cloning skills, can be used with any combination of restriction enzymes, is applicable to inserts of any length (with some caveats), and, although demonstrated here in yeast, can be used in any system. The authors dub their method Controlled and Ordered Oligonucleotides Ligations (COOL).

Engineered Circular RNA Sponges Act as miRNA Inhibitors to Attenuate Pressure Overload-Induced Cardiac Hypertrophy

“…The miRNA sponge oligos were cloned between these fragments to generate circmiRs carrying 2 or 6 binding sites. For circmiRs carrying 8, 12, or 16 binding sites, additional miRNA sponge oligos were sequentially cloned 45 between the inverted intronic repeats. The linear sponge sequence comprised of 12 bulged miRNA binding sites, constructed by sequential cloning 45 of miRNA sponge oligos carrying 6 binding sites into the AAV9-cTnT-eGFP plasmid, without inverted intronic repeats.…”

A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale

“…For that, we constructed variants of the expression plasmid Twin_11B1 with up to 4 copies of the Adx cDNA by successively integrating additional copies including a 5′-ribosomal binding site at the end of the trancription unit according to Blachinsky et al [34]. The relative increase of Adx expression was estimated by Western Blot (Figure 5) and evaluation of the Adx signal with an imaging software.…”

“…In order to consecutively insert additional copies of the Adx cDNA into the Twin_11B1 plasmid behind the pre-existing copy, the following strategy, which takes advantage of restriction site compatibility of EcoRI and MfeI, was pursued referring to [34]. In a first step, Twin_11B1 was used as a template to amplify the Adx cDNA by PCR including its ribosomal binding site and to introduce MfeI and XhoI restriction sites at the 5′ and 3′ end, respectively.…”

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

“…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).…”

“…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 oligonucleotide inserting adjacent to the last.…”