Cloning in the 21st century

It sometimes feels really nice to be old-school. But face it, a first-grader in a new school can out-do you. I'm talking about cloning today.

There are numerous mysterious 'non-traditional' cloning kits out there. One type that constantly gains popularity in recent years is the one that relies on the 3'->5' exonuclease activity of a DNA polymerase (most likely T4 DNA polymerase) to chew out long sticky ends on both the insert and the vector, which then hybridize to form a circular DNA (although not joined by covalent bonds) ready to be transformed. Therefore this procedure is usually called 'ligation-independent cloning'.

A popular example of this MAY be the In-Fusion kit now being sold by Clontech. I said 'MAY' because I don't really know whether the kit is based on this mechanism. I actually came across descriptions like this in literature:

"The In-Fusion mechanism is ligation-independent and while proprietary, likely uses the unique properties of the 3′–5′ exonuclease activity of poxvirus DNA polymerase." [Biotechniques. 2007 ;43(3):354-9, PMID: 17907578]

Now let's get less proprietary.

As far as I know, there are two types of ligation-independent cloning that are widely used. One is called 'LIC-PCR', which stands for 'Ligation-independent cloning of PCR products'. The original paper can be found here: [PMID: 2235490]. The limitation of this method is that at the junctions between the vector and the insert there has to be a long stretch of sequence lacking a particular base -- this is how you generate a well-defined sticky end.

Later it turned out you don't need well-defined sticky ends to do ligation-independent cloning. By optimizing the concentration of T4 DNA polymerase and duration of treatment, one can make roughly 15- to 20-nt overhangs, thus eliminating the sequence-dependence. This method is detailed in this paper: [PMID: 17293868]. The authors called this method 'sequence and ligation-independent cloning (SLIC)'. Unfortunately the method section of this paper is poorly written, and may contains several mistakes. They then clarified their method in Protocol Exchange on nature.com. [Here].

Both methods have gained pretty good reputation. Hopefully they work for you. Happy cloning!