Robust Site-Directed Mutagenesis Kit Enables Standard and Novel Applications

A new kit that enables rapid site-directed mutagenesis (SDM) of plasmid DNA has been designed to include unique features that expand the range of applications, including a more effective method for introduction of long insertions and deletions. A new SDM primer design web tool that supports the kit has also been developed.

New England Biolabs (NEB; Ipswich, MA, USA) introduces the “Q5 Site-Directed Mutagenesis Kit,” capable of making standard single-base modifications as well as novel manipulations such as deletions or additions of almost any length, including for the addition of nuclear localization signals to cloned proteins. It also includes unique features for difficult-to-amplify GC-rich and repetitive sequences. "The Q5 SDM Kit makes applications possible for which you might not have thought of using SDM. For example, SDM can be a much quicker and less error-prone way to add tags, make large deletions, add or remove restriction sites, and many other sequence modifications," said John Pezza, PhD, scientist at NEB’s Applications and Product Development.

NEB adopted a nonoverlapping primer design to allow for longer modifications. The most popular SDM methods use overlapping primers that limit the length of insertions or deletions. NEB's approach enables exponential amplification and more efficient plasmid transformation, which also facilitates the successful modification of highly repetitive or GC-rich sequences. NEB has also released “NEBaseChanger,” an SDM primer design web tool that supports the Q5 SDM Kit. Developed by Sanjay Kumar, PhD, Senior Research Scientist at NEB, it includes a novel feature - the application of nearest neighbor thermodynamics for matched and mismatched bases to design mutational SDM primers and calculate custom annealing temperatures. "These adjustments increase the odds that the experiment will work. Most, if not all other design tools, are not able to take mismatches into account," said Dr. Pezza. NEBaseChanger also provides live primer design updates, so users can make iterative changes to their primers and watch how details like reading frame and Tm are affected.

The Q5 SDM Kit includes the robust Q5 Hot Start High-Fidelity Polymerase, which has been tested to amplify sequences with a fidelity 100-times greater than Taq and twice that of “Phusion” DNA Polymerase. The kit also includes high-efficiency NEB 5-alpha Competent E. coli. Robust results have been observed with plasmids up to at least 14 kb.

Related Links:
New England Biolabs
Q5 Site-Directed Mutagenesis Kit