Product Survey: Cloning kits

Seamless Integration
by Harald Zähringer, Labtimes 02/2014

Figures: Gheorghe Christols

The recent boost in cloning techniques has give rise to a plethora of new cloning kits.

In the old days of molecular cloning, DNA fragment and vector were digested with restriction endonucleases (RE) to create sticky or blunt ends that were joined together with a T4 DNA ligase. This traditional restriction enzyme or ligase-dependent cloning approach is simple and still in use today. However, defined restriction sites are needed at the right places of the DNA fragment and multiple, time consuming steps are required. To circumvent these shortcomings, molecular biologists have developed various ligation-independent cloning (LIC) methods based on different PCR and recombineering techniques.

The granddaddy of all subsequent LIC methods, dubbed LIC-PCR, was presented by Charalampos Aslanidis and Pieter J. de Jong, then at the Lawrence Livermore National Laboratories, back in 1990, already. As the name implies, Jong and Aslanidis developed LIC-PCR to clone PCR products into a PCR-amplified plasmid vector. Instead of using restriction enzymes and T4 ligase to “cut and paste” the gene of interest into a plasmid, they added homologous overlapping ends to DNA fragment and vector via PCR, applying specially-designed primers.

But this alone won't do the trick. The smart part of LIC-PCR is the clever usage of the (3'-5') exonuclease activity of T4 DNA ­polymerase or exonuclease III, to trim the added tails of fragment and vector into complementary cohesive ends. The resulting single-stranded overhangs may easily pair under proper reaction conditions, to form a circularised vector containing the cloned gene of interest (GOI).

Countless variations

Over the years, various ligation-independent cloning approaches have evolved from the original LIC-PCR procedure and the term LIC cloning has been generally adopted for restriction enzyme-free cloning strategies. Several of them, such as the Gateway, Creator or Gibson assembly cloning systems are based on in vitro recombination. The Gateway system, for example, utilises the site-specific recombination system of phage I to shuttle DNA fragments from a holding or entry vector into different kinds of destination vectors. The cloning strategy of the Creator system is very similar but relies on the Cre-loxP recombinase for the rapid transfer of genes from donor to acceptor vector.

Gibson assembly falls into the category of seamless cloning approaches, which allow sequence-independent and scarless insertion of DNA fragments into vectors. The basic idea of Gibson assembly is as simple as it can get: apply a PCR to add complementary overlapping ends to both, your gene(s) of interest and your linearised vector and join them together at 50°C in a one-pot reaction starring T5 exonuclease, Phusion polymerase and Taq ligase. The exonuclease chews back the 5' ends of the fragments and enables the complementary strands to hybridise. The remaining gaps are filled in by the polymerase and the nicks in the single DNA strands are finally sealed by the ligase.

Convertible ligase

Sequence- and ligation-independent cloning (SLIC) and Golden Gate cloning are two other prominent examples of seamless cloning techniques. SLIC is very akin to the original LIC-PCR method with some slight modifications. DNA fragment and linearised vector with homologous ends (added as usual with specific PCR primers) are treated separately with T4 DNA polymerase in the absence of dNTP. Under these conditions, T4 polymerase acts as a 3' exonuclease and chews back the ends from 3' to 5'.

The reaction is stopped by adding dCTP, which transforms the enzyme back into a polymerase, however, the polymerase activity immediately stalls due to the lack of dNTPs. Subsequently, DNA and vector are mixed to anneal the created complementary single-stranded 5' overhangs and the gaps are finally repaired by E. coli cells after transformation with the construct.

Golden Gate assembly relies upon the use of type II endonucleases, such as BsaI. Two properties of type II endonucleases come in very handy for molecular cloning: firstly, the recognition sites are distal from the cut sites and secondly, they are not palindromic but directional. How can this be exploited for cloning? Pretty easily. Just add a BsaI recognition site with an overhang sequence of four arbitrary nucleotides to both ends of the linearised vector and the gene of interest (with the recognition sites pointing “inwards” in the direction of the overhang sequence), using specific PCR primers. Put both fragments into one pot, mixing them with BsaI and T4 DNA ligase. BsaI nibbles away the BsaI recognition sites at the ends of the linearised vector as well as the GOI and produces cohesive ends. The ends are joined together by the ligase, leaving an assembly product without BsaI sites but with a defined sequence.

Most of the aforementioned cloning techniques found entry into commercial cloning kits. However, new cloning approaches are coming out of laboratories almost on a weekly basis.

Take, for example, the brand new Golden Gate cloning variation presented by Carsten Grötzinger's group from the Molecular Cancer Research Center at the Charité clinic in Berlin, which they call pHeaven's Door cloning (Schefer et al., Mol. Biotechnol., DOI 10.1007/s12033-014-9736-2). According to the authors, pHeaven's Door (pHD) cloning allows very fast, cheap, easy and reliable sub-cloning of PCR products with zero background.
Knocking on pHeaven's Door

Similar to Golden Gate cloning, two BsaI sites are first added to the ends of the gene of interest via PCR. The difference lies in the pHD vector that harbours a ccdB suicide insert, flanked by two BsaI sites, as well as a kanamycin resistance marker. GOI and pHD vector are simultaneously incubated at room temperature with BsaI and T4 ligase, and the resulting construct is transferred into E.coli cells. Streaking out the cells on kanamycin plates gives rise to two possibilities: the cells are viable since the suicide insert has been replaced by the GOI – or they are not, as the ccdB insert is still there.

To further rule out any background from the original PCR, the template vector contains an AmpR marker, which doesn't support survival of the cells on kanamycin plates. Sounds pretty cool, however, heaven only knows whether pHeaven's Door cloning will deliver you from cloning hell and leads you directly into cloning heaven.

First published in Labtimes 02/2014. We give no guarantee and assume no liability for article and PDF-download.

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