Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Splicing, dicing, chopping and hopping genetic information has been the symphonic chant for DNA cloning since its introduction in 1972. DNA cloning continues to be an essential step in workflows, from characterizing gene expression to synthetic biology. And although classical restriction endonuclease (RE) cloning continues to be a familiar tune, it is joined by an increasing number of newer and progressive cloning techniques promising greater speed, accuracy and flexibility for many molecular biological applications.
Here we look into some of the tools that might best suit the cloning your lab is trying to do.
The classics
Researchers clone DNA for a host of reasons, from swapping out genetic regulatory regions or adding protein tags, for example, to inserting modular sequences or disrupting genes with a code, making the cell susceptible to antibiotics. Traditionally, they start by linearizing a vector at a specific location (the multiple cloning site, or MCS) using appropriately chosen REs and an insert containing the sequence of interest. The latter is constructed so that its ends are complementary to those generated by the RE digestion. The vector and insert are ligated together, the newly formed constructs transformed into bacteria, the bugs plated and colonies screened to identify positive recombinant clones.
“A lot of people are still doing the classic RE-and-ligase method,” says Tom Evans, scientific director in the DNA enzyme division of New England Biolabs (NEB). The basic mechanisms are the same, but there have been many improvements in the reagents used to carry out the processes: nearly instant ligases, faster REs, high-fidelity REs with reduced star activity and a single buffer enabling full activity with more than 200 REs. “In the old days, you’d have to go through and figure out which buffers the enzymes were compatible with. You don’t have to do that anymore—you can even use NEB’s CutSmart buffer for nearly all double digests,” he explains.
The RE-and-ligation approach is relatively inexpensive and perhaps still the most straightforward technique for things like moving varying-sized fragments of DNA between plasmids. Yet for many applications it requires more steps—an appropriate sequence needs to be found or created, for example, and the insert may need to be treated with phosphatase—and it takes longer than more recent innovations.
The new wave
Many techniques and reagents have recently come online that use a cocktail of enzymes to perform single-tube cloning, and then some. One example is Clontech Laboratories’ In-Fusion® system. As with classical cloning, you start with a linear vector and a linear insert with ends homologous to those of the vector. But in this case any site will do, and the insert typically is created by PCR using “primers designed in such a way that they contain the sequence of what you’re amplifying plus 15 base pairs of either end of the linearized vector,” says Suvarna Gandlur, senior product marketing manager at Clontech.
These proprietary master mixes tend to contain an exonuclease that chews back single strands of the insert to reveal its homology, allowing the insert’s ends to pair up with the ends of the vector in a directional manner. Some mixes contain a ligase, and others rely on endogenous bacterial enzymes to perform that function.
Perhaps the revolutionary advantage of such systems—which include Gibson Assembly® (named for its inventor Daniel Gibson, of the J. Craig Venter Institute and Synthetic Genomics (SGI), with versions marketed by NEB and SGI-DNA), GeneArt® Seamless Cloning and Assembly from Thermo Fisher Scientific and NEB’s NEBuilder® HiFi DNA Assembly—is that multiple different fragments can be assembled simultaneously in the same tube.
The procedure is also very quick—the website for System Biosciences’ (SBI’s) Cold Fusion Cloning Kit notes that the “entire process takes 20 minutes.” But the time savings are not just on reaction times, especially when researchers are assembling complex vectors. “With some old-school methods, you’ll spend probably a couple of months to figure out a four-fragment or four-gene expression, subcloning into a single vector,” notes SBI senior scientist Fangting Wu. “Here you just assemble four PCR products, and in three days you have your final construct, correct and accurate, in the desired orientation.”
Nearly all homologous recombination-based cloning kits claim very high efficiencies. When trying to stack these against each other, Gandlur points out that the percentage of colonies containing the correct clone is more important than the total number of colonies on the plate.
Wu notes that all the fidelity comes from the PCR reaction and recommends that customers use a “high-fidelity PCR DNA polymerase to amplify their products.”
Progressive rocks
“There are actually an amazingly large number of systems for assembling DNA,” notes Evans.
Among these is Life Technologies' lambda phage-based Gateway® Cloning system from Thermo Fisher Scientific, which allows for the facile shuttling of insert DNA into and out of Gateway vectors containing specific recognition sequences. This in turn enables easy sharing within and among research communities. MultiSite Gateway® kits, also from Thermo Fisher Scientific, are available to assemble multiple fragments into a single vector.
Similarly, Golden Gate Assembly from NEB, uses site-specific recognition to put together vectors. It’s popular with the TALEN and CRISPR communities, for example, which maintain plasmids with particular sub-domains, says Evans. “That’s a good instance of where Golden Gate really shines—you can take your plasmids out of the freezer, throw them together, put in the Golden Gate master mix, and it assembles them. You don’t have to go through PCR.”
Both of these methods require the use of RE digests to generate constructs. With the Gateway system, and like classical cloning, careful planning or lucky engineering in the RE digest will avoid leaving behing a "scar." Slightly different, Golden Gate assembly utilizes a type IIS restriction enzyme that cuts DNA outside of the recognition sequence. The fragments generated from the digest will have complimentary overhangs that can join in a “seamless” manner (like Gibson assembly) to form the appropriate construct. While based on the foundations of a classical method, these systems provide a powerful and effective cloning solution for scientist.
Ending on a high note
“All of these techniques have some learning curve—even classical RE cloning,” points out Evans. He recommends making use of online tools to help design the constructs and primers, for example, and especially using the considerable institutional knowledge that has developed in the literature and research communities. Follow these tips and you will be jamming to the tune of fist-pumping positive clones!