NEB Releases a New Site-Directed Mutagenesis Kit for Both Standard and Novel Uses, Including Long Insertions and Deletions

New England Biolabs (NEB), a world leader in the discovery and production of reagents for the life science industry, launched a new reagent kit for site-directed mutagenesis (SDM). The Q5® Site-Directed Mutagenesis Kit (Q5 SDM Kit) is capable of introducing long insertions and deletions, thus allowing a broader range of applications for this widely-used laboratory technique.

The Q5 SDM Kit can make standard single-base modifications, as well as novel manipulations, such as deletions or additions of any length, including the addition of nuclear localization signals to cloned proteins.“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,” says John Pezza, Ph.D., an Applications and Product Development Scientist at NEB.

NEB adopted a non-overlapping 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 facilitates the successful modification of highly repetitive or GC-rich sequences.

NEB has also released an SDM primer design tool, NEBaseChanger™, which supports the Q5 SDM Kit. Developed by Sanjay Kumar, Ph.D., Senior Research Scientist at NEB, the new web tool 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,” says 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 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, 14.3 kb.