Introduction to NEBuilder HiFi DNA Assembly
11
March

By Adem Lewis / in , , , , /


NEBuilder® HiFi DNA Assembly, developed by
New England Biolabs, is an effective method for the high-fidelity assembly of multiple
DNA fragments. Advantages over Gibson Assembly Master Mix include higher efficiency, higher
accuracy, the ability to assemble both 5´- and 3´-restriction enzyme mismatches, the
ability to bridge two double-stranded DNA fragments with single-stranded DNA and no
licensing fees from NEB. This is accomplished in a single-tube isothermal reaction with
NEBuilder HiFi DNA Assembly Master Mix. The method utilizes adjacent DNA fragments with
complementary ends, which can be added, for example, by PCR. Then, the overlapping fragments
are added to the NEBuilder HiFi DNA Assembly Master Mix and incubated for 15 minutes to
one hour at 50°C. During the one-hour incubation, the Master Mix’s three enzyme activities
set to work on the fragments. First, a 5´ to 3´ exonuclease activity creates single
stranded 3´ overhangs. These complementary sequences then anneal, creating the double-stranded
DNA of interest. A high-fidelity DNA polymerase then extends the 3´ ends, filling in the
gaps, and a DNA ligase seals the remaining nicks. The end result is a fully-sealed, double-stranded
DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology
applications, including direct transformation. In addition to joining two double-stranded
DNA fragments together with an overlap, NEBuilder HiFi DNA Assembly Master Mix can also join
two non-overlapping double-stranded DNA together with an overlapping single-stranded oligonucleotide.
NEBuilder HiFi DNA Assembly has been successfully used to reliably join up to six DNA fragments
into a single molecule, as well as create a library (gRNA or linker insertion). This
remarkably straightforward and powerful technique makes quick work of large multi-fragment assemblies,
but it is also useful for more routine applications, such as cloning. The NEBuilder HiFi DNA Assembly
Cloning Kit, which includes both NEBuilder HiFi DNA Assembly Master Mix and NEB 5-alpha
competent cells, has been optimized for efficient assembly and cloning of multiple fragments
into any vector in just over an hour.


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