New England Biolabs® Scientists Push the Limits of Golden Gate Assembly

New publication maps ligase fidelity profiles with single molecule real-time sequencing to enable 20+ fragment assemblies with high efficiency and accuracy

IPSWICH, MA, USA (June 11, 2018) — Some of today’s most exciting molecular biology engineering — including the creation of customized CRISPR/Cas9 and TALEN constructs for genome editing — are led by synthetic biologists. These scientists are using methods such as Golden Gate Assembly. This molecular cloning technique uses a combination of Type IIS restriction enzymes and DNA ligase to assemble multiple DNA fragments simultaneously in a rapid “one-pot” reaction.

However, there are practical limitations to Golden Gate Assembly, which is highly dependent upon the efficient ligation of flanking four base pair overhangs with T4 DNA Ligase, where a difference in even one base may create unwanted ligation products. As a result, current best practices for the number of DNA fragments that can be joined in a single reaction have been typically limited to ten fragments or less.

A new ligase fidelity profile, developed and tested by researchers at New England Biolabs (NEB®), reported recently in bioRxiv, used single molecule real-time sequencing to identify and quantify the proportions of mismatched-prone junction pairs during ligation. The assay accurately predicted junction fidelity for all 256 possible 4-base overhangs, allowing users to choose optimal Golden Gate ligation junctions when designing assemblies.

“Without a proper understanding of ligase fidelity and bias, mismatched overhangs may be ligated — leading to the inclusion of mismatches, deletions or insertions into the final DNA vector assemblies,” said Gregory Lohman, Staff Scientist at NEB and author on the study. “The application of our ligation fidelity data effectively informed our choice of junctions during Golden Gate Assembly, enabling a highly flexible design and successful assembly of more than 20 fragments in a single reaction. Therefore, we’re making our ligase fidelity data publicly available to help scientists fine-tune their Golden Gate protocol.”

Additionally, NEB has introduced two new restriction enzymes — BsaI-HF®v2 and Esp3I; BsaI-HFv2 has been re-engineered and optimized for Golden Gate Assembly and Esp3I is an isoschizomer of BsmBI.

“NEB has an extensive history innovating in molecular biology enzymes and related technologies,” said Rebecca Kucera, Applications and Product Development Scientist at NEB. “In addition to the ligase fidelity profile data, the availability of BsaI-HFv2, a key type IIS restriction enzyme, has resulted in improved performance for Golden Gate Assembly, with highly efficient reactions yielding 99% accuracy for 12-fragment assemblies and greater than 90% accuracy for 24-fragment assemblies.”

The ligase fidelity research is being conducted as part of a broader effort to explore the fidelity of other commercially available enzymes, including transcriptases, polymerases, and exo/endonucleases, further enhancing standard molecular biology techniques.

“NEB’s research and development efforts will continue to focus on providing industry-leading reaction components for Golden Gate assembly, with the long-term vision of enabling efficient assembly of >50 fragments in a single reaction,” said Lohman.

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About New England Biolabs

Established in the mid 1970's, New England Biolabs, Inc. (NEB) is the industry leader in the discovery and production of enzymes for molecular biology applications and now offers the largest selection of recombinant and native enzymes for genomic research. NEB continues to expand its product offerings into areas related to PCR, gene expression, sample preparation for next generation sequencing, synthetic biology, glycobiology, epigenetics and RNA analysis. Additionally, NEB is focused on strengthening alliances that enable new technologies to reach key market sectors, including molecular diagnostics development. New England Biolabs is a privately held company, headquartered in Ipswich, MA, and has extensive worldwide distribution through a network of exclusive distributors, agents and seven subsidiaries located in Canada, China, France, Germany, Japan, Singapore and the UK. For more information about New England Biolabs visit

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Contact information: 

Deana D. Martin, Ph.D. 
Associate Director, Marketing Communications 
New England Biolabs 
240 County Road Ipswich, MA 01938 
Tel: 978-380-7464 



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