A Practical Comparison of Ligation-Independent Cloning Techniques

Abstract

The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.

A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis

“…The pTK-SM N49-GFP-V5 construct was generated previously (8). All SM N100 truncation, domain replacement, and domain deletion constructs were generated from the pTK-SM N100-GFP-V5 plasmid using polymerase incomplete primer extension cloning (42). Nucleotide sequences coding for chicken SM N100 (NCBI accession no.…”

“…NP_009868.3) were codon-optimized for mammalian expression obtained in a pUC19 backbone (GenScript). These coding sequences were cloned into the human pTK-SM N100-GFP-V5 plasmid using the polymerase incomplete primer extension method (42), replacing the coding sequence of human SM N100. The identity of all cloned constructs were confirmed by Sanger sequencing.…”

Universal CG cloning of polymerase chain reaction products

“…1). Tandem AhdI sites [5] separated by an 8-bp random linker sequence were introduced into pcDNA4 Myc His B (Life Technologies) using primers 5 0 -GACTGTCGGTCGCGAGTATGACCGACAGTCCA GCACAGTGGCGGCCGCTCGAGT-3 0 and 5 0 -GACTGTCGGTCATACTCGC GACCGACAGTCGATATCTGCAGAATTCCACCAC-3 0 or 5 0 -GACTGCC GGTCGCGAGTATGACCGGCAGTCCAGCACAGTGGCGGCCGCTCGAGT-3 0 and 5 0 -GACTGCCGGTCATACTCGCGACCGGCAGTCGATATCTGCAGA-ATTCCACCAC-3 0 (recognition sequences are underlined, and the resulting overhanging base is in boldface and italicized) with polymerase incomplete primer extension site-directed mutagenesis [6,7] with Phusion High Fidelity Polymerase (all enzymes from New England Biolabs) using a lower primer concentration (0.2 lM instead of 0.5 lM) because primer dimer formation due to the presence of long complementary 5 0 overhangs led to no amplification. AhdI was selected because it cuts any sequence between its two recognition sequences and can be used for prolonged digestion without star activity.…”

“…Vectors (3 lg) were digested with 10 U of AhdI (for single-nucleotide overhangs) or EcoRV HF (for blunt ends) for 16 h in CutSmart Buffer at 37°C, incubated for a further 1 h with 10 U of Antarctic Phosphatase, and purified using a HiYield Gel/PCR DNA Mini Kit (Real Biotech). A 1.4-kb kanamycin resistance cassette from pUC18/Kan was prepared by PCR using phosphorylated primers pUC18-Kan F and pUC18-Kan R and Phusion High Fidelity Polymerase [7]. Purification and treatment of blunt product with dGTP is highly recommended for CG cloning because Taq strongly prefers to add a 3 0 A [3].…”

“…However, none of these methods achieves directional cloning. Alternatively, ligation-independent cloning methods are simpler and more robust than cloning with restriction enzymes and ligation and achieve very high efficiencies with limited manipulations for a wide range of insert sizes [7], but they require the addition of long 5 0 overhangs to PCR primers, increasing the cost of cloning projects. However, for higher throughput cloning of short sequences for library construction, which do not tolerate the presence of concatemers, CG cloning is most suitable.…”