Abstract
We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.
Highlights
One of the most common tasks in molecular cloning is the insertion of a defined DNA fragment into a target plasmid
sequence and ligation-independent cloning (SLIC) requires linearizing the target plasmid by enzymatic cleavage or PCR, and the addition of RecA, for highest efficiency
In analogy to how RF cloning relates to QuikChange, Exponential Megapriming PCR (EMP) cloning relates to inverse PCR [39] (Fig. 2). iPCR can be used for the same manipulations QuikChange was developed for, but it uses non-overlapping primers for exponential target amplification, resulting in drastically higher product amounts and more positive clones. iPCR was originally problematic due to the relatively high error-rate of native, thermostable DNA polymerases, such as Taq
Summary
One of the most common tasks in molecular cloning is the insertion of a defined DNA fragment into a target plasmid This is achieved by restriction enzyme mediated sticky end [1,2,3,4] or blunt end [5,6] ligation, greatly facilitated by the advent of PCR [7,8,9]. Instead recombination is facilitated by in vitro generation of single strand overhangs For this purpose ligation-independent cloning (LIC) [18] uses the 39–59 exonuclease activity of T4 DNA polymerase. SLIC requires linearizing the target plasmid by enzymatic cleavage or PCR, and the addition of RecA, for highest efficiency Another recent in vitro recombination technique was presented by Gibson and coworkers [20]. A major disadvantage of RF cloning is low product yield due to linear PCR amplification, which becomes prohibitive for larger inserts
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