Yeasen and Molefuture Develops Thermotolerant, High-Fidelity T4 DNA Ligase to Combat False Mutations in NGS

Yeasen and Molefuture Introduce Advanced T4 DNA Ligase to Overcome Molecular Biology Challenges

Yeasen and Molefuture have jointly developed a cutting-edge T4 DNA ligase using their ZymeEditor platform, addressing key issues in molecular biology and DNA library construction for next-generation sequencing (NGS). Traditional T4 DNA ligase faces challenges such as poor stability, high adapter dimer formation, and high DNA fragment self-ligation. This novel enzyme aims to overcome these limitations.

T4 DNA ligase, an ATP-dependent enzyme, features a compact-helical DNA-binding domain (DBD), a nucleotidyl-transferase (NTase) domain, and an OB-fold domain. By analyzing protein structure, researchers identified key residues within 5 to 20ƅ of the DNA substrate. Using advanced computational tools, in silico screening, and structure-function analysis, they designed mutants through consensus sequence alignment, incorporating structural-functional insights.

In addition to rational design, the team employed the MTPS method for directed evolution, screening over 10,000 mutants for stability, activity, and residual activity. Promising candidates underwent rigorous purification and testing. Detailed characterization followed, evaluating activity, stability, adapter self-ligation, DNA fragment self-ligation, and DNA yield.

The researchers combined DNA rational design and directed mutation on G1 mutants to develop exceptional variants suited for diverse applications. This comprehensive approach has resulted in a highly efficient and reliable T4 DNA ligase, poised to enhance molecular biology research and DNA library construction in NGS.


Figure 1. Illustrates the evolution of Versatility T4 DNA Ligase. (A) Rational designĀ  of T4 DNA ligase. (B) Screening of thermotolerant T4 DNA ligase mutants at 45ā„ƒ, leading to the identification of mutants with significantly enhanced specific activity. (C) Screening of T4 DNA ligase mutants aimed at reducing adapter dimer formation during DNA library preparation.Ā  (D) The evaluation of library yields retention post heat treatment at 42ā„ƒ for 1 hour using DNA library preparation method.
Table 1. Yeasen Versatile T4 DNA ligase is thermostable. The enzyme was treated at 30ā„ƒ for 5 days and analyzed by DNA Lib Prep method.
Figure 2. Yeasen Versatile T4 DNA ligase has higher fidelityĀ compared to the wild type (WT) T4 DNA ligase indicated by less adapter dimer formation due to self mis-ligation (A) and (B) analyzed by DNA Lib Prep method.
Figure 3.Ā Yeasen Versatile T4 DNA ligase shows the lowest adapter dimer formation compared to the commercial T4 DNA ligases including thermotolerant ligases analyzed by DNA Lib Prep method.
This breakthrough demonstrates Yeasen and Molefuture's commitment to innovation and excellence in enzyme engineering.

Ordering information

Name Cat# Notes
Enzyme Hieffā„¢ Versatile T4 DNA Ligase (600 U/ĀµL) 12996ES Thermotolerant and High Fidelity for Tumor gene detection
DNA Hieff NGSĀ® DNA Library Prep Kit 13577ES Tumor/ Mechanic method
Hieff NGSĀ® OnePot Pro DNA Library Prep Kit V2 12194ES Tumor/ Enzymetic method
Hieff NGSĀ® OnePotĀ  II DNA Library Prep Kit for Illumina 13490ES Pathgen/ Enzymetic/ regular time (140min)
Hieff NGSĀ® OnePot Flash DNA Library Prep Kit 12316ES Pathgen/ Enzymetic/ UltrafastĀ  (100min)
Hieff NGSĀ® DNA&RNA Library Co-Prep Kit V2 12305ES Pathgen/ Enzymetic/ DNA & RNA Co-Prep
RNA Hieff NGSĀ® Ultima Dual-mode mRNA Library Prep KitĀ  12308ES Without oligo dT magnetic beads, 11 tubes
Hieff NGSĀ® Ultima Dual-mode mRNA Library Prep KitĀ Ā  12309ES oligo dT magnetic beads plus, 14 tubes
Hieff NGSĀ® Ultima Dual-mode RNA Library Prep KitĀ  12310ES Premixed version, 5 tubes
Hieff NGS Ā® EvoMax RNA Library Prep Kitļ¼ˆPremixed versionļ¼‰(actinomycin DĀ Free) 12340ES Premixed version,Ā (Actinomycin DĀ Free)
Hieff NGSĀ® MaxUp rRNA Depletion Kit (Plant)Ā  12254ES Plant
Hieff NGSĀ® MaxUp Human rRNA Depletion Kit (rRNA & ITS/ETS) 12257ES Human


Please leave a message to inquire about the performance comparison data between this enzyme and other top-tier thermostable and conventional T4 DNA ligases.

About Molefuture Biotechnology

Molefuture Biotechnology is a subsidiary of YeasenĀ Biotechnology (Shanghai) Co., Ltd., specializing in providing customized enzyme modification solutions driven by enzyme evolution technology. Leveraging the resources of YeasenĀ Biotechnology, Molefuture operates on six major technological platforms: the ZymeEditor innovative enzyme evolution platform, a multi-host high-efficiency expression platform, fermentation process development platform, purification process development platform, ultra-clean enzyme production platform, and enzyme analysis and quality control platform. With these six technological platforms, Molefuture can offer a complete set of customized solutions including enzyme-directed evolution, new enzyme development, enzyme process development, and GMP-level scale-up production to meet the application demands of enzymes in areas such as in vitro diagnostics, biomedicine, synthetic biology, medical aesthetics, pharmaceutical intermediates, et. al.