Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
It’s easier than ever these days to clone and sequence DNA. Thanks to CRISPR/Cas and related technologies, it’s even straightforward to rewrite genomic sequences in living cells and organisms. But as powerful as it is, CRISPR, et al., cannot induce genetic rewrites in a test tube—genome editing requires cellular machinery to repair the DNA breaks the methods produce. Instead, researchers interested in mutating cloned genes on plasmids must revert to a tried-and-true method, site-directed mutagenesis.
First described in the 1970s—and earning its inventor a share of the Nobel Prize in Chemistry in 1993—site-directed mutagenesis uses short oligonucleotides to introduce single base changes, as well as insertions and deletions, to DNA plasmids. Researchers can use the method to swap amino acids in expressed proteins, test clinically relevant mutations and tweak promoters.
But there’s more than one way to change a base, and molecular-tools vendors have commercialized multiple strategies. Here, we review some of the more popular approaches to site-directed mutagenesis.
All about that base
Most site-directed mutagenesis protocols work in one of two basic ways. A short oligonucleotide, usually containing the desired mutation in its center, is annealed to single-stranded template DNA and extended by DNA polymerase. Alternatively, two outward-facing primers, designed either to introduce a point mutation or to add or remove nucleotides, are used to replicate and amplify the intervening plasmid DNA. The products of the reaction are then introduced into cells, at which point mutants must be identified.
How easy it is to identify those mutants depends on the mutagenesis strategy. Mutagenesis protocols originally had no mechanism to eliminate the wild-type (i.e., unmodified) plasmid, but later strategies have evolved. One involves growing the template DNA in a mutant E. coli strain that introduces the RNA base uracil into the DNA. Upon mutagenesis, extension and transformation into another bacterial strain, the uracil-containing wild-type strand is destroyed, leaving only the mutant sequence.
Newer alternatives exploit DNA methylation, including New England Biolabs’ (NEB’s) Q5® Site-Directed Mutagenesis Kit. Plasmid DNA propagated in E. coli typically is methylated, but DNA polymerized in vitro is not, so “you can treat with [the methylation-sensitive restriction enzyme] DpnI to get rid of the wild-type plasmid,” says John Pezza, NEB applications and product development scientist. That fact, coupled with the company’s high-fidelity Q5 DNA polymerase and a primer configuration that enables efficient amplification of mutant sequences, yields a mutational efficiency of 85% to 95%, Pezza says. “I generally recommend customers select three [clones] for sequencing, and they’ll have their product.”
Agilent Technologies’ QuikChange Site-Directed Mutagenesis Kit also exploits DpnI to remove methylated plasmid sequences, while Thermo Fisher Scientific’s GeneArt® Site-Directed Mutagenesis System relies on the transformed cells to do likewise.
Clontech Laboratories’ Transformer™ Site-Directed Mutagenesis Kit selects against wild-type sequences by exploiting a unique restriction site in the plasmid. In this case, mutagenesis is achieved using two oligos, one to create the desired mutation and the other to inactivate that unique cut site, thereby protecting mutant plasmids from enzyme degradation in a later step.
Phusion™ Site-Directed Mutagenesis Kit from Thermo Fisher Scientific, uses no mutation selection step at all. According to the product literature, “[b]ecause minute amounts of template DNA are exponentially amplified in this method, the fraction of non-mutated template is minimal. Thus there is no need to destroy it in a separate step.”
Design differences
One attribute that distinguishes mutagenesis strategies is the design/placement of the primers. Traditional mutagenesis used a single primer annealed to a single-stranded template. But the Q5 kit, for instance, uses two primers arranged “back to back” on the plasmid, thereby enabling exponential amplification of the mutant plasmid DNA.
Phusion also uses the back-to-back configuration, whereas QuikChange uses two directly complementary oligonucleotides, each containing the mutation; annealing and extension of these primers yields a “nicked” plasmid that is repaired following transformation, says Ben Borgo, global product manager at Agilent. (QuikChange is a family of kits, Borgo notes; these include QuikChange Lightning, with a one-hour protocol and the ability to introduce three mutations in one step, and QuikChange HT, for generating mutant libraries.)
Clontech’s In-Fusion® HD Cloning Plus Kit uses PCR primers containing both a point mutation and 15 bases of overlapping sequence. Amplification produces a linearized molecule with 15 identical bases on either end. That molecule is then circularized using the company’s In-Fusion enzyme, a “proprietary enzyme mix with 3’ [to] 5’ exonuclease activity,” says Karen Martin, senior product manager at Clontech. In-Fusion chews back the 15-base overhangs, she explains, creating a recombination template that can be sealed by the transformed bacteria, similar in concept to Thermo Fisher Scientific’s GeneArt Seamless Cloning and Assembly kits.
Generally speaking, says Adam Clore, manager of synthetic biology design, support and biosecurity at Integrated DNA Technologies (IDT), mutagenesis primers can be purchased with no special modifications. The only exception, he says, is phosphorylation. “Anything that needs to be ligated needs to have a 5’ phosphate.” Thermo Fisher Scientific’s Phusion kit, for instance, requires phosphorylated primers, as the kit includes DNA ligase, but not a kinase.
Another key difference between kits is the polymerase, which determines the maximum size and complexity of the template that can be used and the fidelity of base incorporation. NEB’s mutagenesis kit features the company’s Q5 DNA polymerase, which Pezza calls “the highest fidelity polymerase available.” NEB has applied the method to plasmids up to 14 kb, but customers have had success with templates as long as 20 kb, he says. Agilent’s QuikChange XL kit (PfuTurbo DNA polymerase) also supports plasmids up to 14 kb, and the Phusion kit (Phusion Hot Start II DNA polymerase) can handle up to 10 kb. Clontech’s In-Fusion cloning kit uses the company’s ClonAmp HiFi DNA polymerase, which copies 200 bases per second with a fidelity of about one error per 45,200 bases on “challenging GC-rich” templates, Martin says.
Applications
Researchers can use site-directed mutagenesis to drive a range of in vitro applications, but according to Pezza, the “number-one use” is modifying the amino acids in an expression vector.
Another application is inserting relatively short sequences (up to about 100 bases), such as a protein tag or, increasingly common, guide RNA sequences for CRISPR/Cas applications . “That’s a great novel use, and one that will be used more and more,” Pezza says. In the Q5 and Phusion kits, this addition is accomplished by appending the appropriate sequence as a “tail” at the 5’ ends of the mutagenesis primers. (Deletions are achieved simply by separating the two primers rather than having them positioned directly next to each other.)
Q5- and Phusion-kit users can insert even more DNA by using longer oligos, such as IDT's Ultramer oligonucleotides, which can run as long as 200 bases. According to Clore, Ultramer oligonucleotides are synthesized using a more efficient process than traditional oligos, which typically max out at about 100 bases.
Of course, as DNA synthesis gets ever more efficient and inexpensive, researchers could simply opt to synthesize their mutant genes from scratch rather than rewriting existing sequences. IDT, for instance, can produce so-called gBlocks (constructed using Ultramer oligonucleotides) that are as long as 2 kb. A 200-base Ultramer, Clore says, costs $150, and a 500-base gBlock costs just $89.
“If you are doing basic site-directed mutagenesis, it’s much easier to just use oligos,” he says. “But if you’re [inserting] more than a few hundred bases of sequence, it’s generally more cost-effective to use gBlocks.”
Often, says Martin, the choice between mutagenesis and de novo synthesis comes down to budget. Gene synthesis “is still relatively expensive for a number of labs,” she says. That could change as prices fall, she adds, but in the short term, “I don’t see these very specific site-directed mutagenesis methods going away.”
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