In molecular biology workflows—whether cloning, NGS library preparation, or repair of fragmented DNA—ligation efficiency is fundamental. T4 DNA Ligase serves as the “molecular glue” that joins DNA fragments by catalyzing phosphodiester bond formation in the presence of ATP. However, two critical limitations of conventional T4 DNA ligase have long constrained both experimental efficiency and result quality.

Figure 1. Schematic illustration of the mechanism of T4 DNA ligase

Limitations of Traditional T4 DNA Ligase

Pain 1. Poor thermal stability

Conventional T4 DNA ligase is highly temperature-sensitive—its activity rapidly declines at room temperature and is quickly inactivated at elevated temperatures. This thermal instability restricts its use in high-temperature ligation protocols and introduces significant variability in standard workflows. For instance, during high-throughput pre-aliquoting of reaction mixes, enzyme activity may degrade while sitting at room temperature, leading to inconsistent ligation efficiency across samples. Similarly, in multi-step protocols where ligation is delayed, reduced enzymatic activity can result in failed reactions, undermining data reproducibility and reliability.

Pain 2. High adapter self-ligation

In traditional enzyme-based DNA library preparation, adapters frequently undergo self-ligation (adapter dimerization), generating substantial amounts of unwanted byproducts. This not only consumes valuable adapter reagents—reducing the effective capture efficiency of target DNA in low-input or rare samples—but also introduces a high proportion of non-informative reads in sequencing data. Such artifacts severely interfere with downstream bioinformatic analysis and critically impair the detection sensitivity and accuracy of low-abundance variants, particularly in precision applications like liquid biopsy or single-cell genomics.

Innovative Ligation Solution: Overcoming Enzyme Thermolability and Adapter Dimerization

To address these persistent challenges, Yeasen has developed a next-generation solution: Premium T4 DNA Ligase (Cat#14966ES)—engineered through a novel optimization process to overcome both limitations simultaneously.

• Outstanding Heat Stability: Maintains over 60% residual activity after 4 hours at 42°C.
• Ultra-Low Adapter Self-Ligation: Cleaner Libraries, Higher Sensitivity
• Excellent batch-to-batch consistency: Stable and uniform performance.

Adapter dimers are one of the most problematic artifacts in NGS library construction, particularly in low-input applications like: Liquid biopsy, Single-cell sequencing, cfDNA analysis, Low-cell-number tumor samples.

In the 0.5 ng gDNA library prep test, Yeasen’s Premium T4 DNA Ligase produced substantially lower adapter self-ligation compared with other suppliers.

Performance Validation

1. Exceptional Thermostability: Retains >60% Activity After 4 Hours at 42°C

To evaluate thermostability, three independent lots of Yeasen Premium T4 DNA Ligase (Lots 1, 2, and 3) were compared against T4 DNA ligases from Supplier A and Supplier B. All enzymes were subjected to heat treatment at 42°C and 45°C for 0, 2, and 4 hours, respectively.

Following heat exposure, each enzyme was used in a standardized whole-genome library preparation workflow using 1 µg sheared bovine genomic DNA and Yeasen DNA Library Prep Kit (Cat# 12927ES) to assess functional ligation efficiency under thermal stress.

Results:

Yeasen Premium T4 DNA Ligase demonstrated significantly superior thermostability compared to both Supplier A and Supplier B—retaining over 60% residual activity after 4 hours at 42°C. Even at 45°C, Yeasen’s enzyme consistently outperformed competitors, confirming its enhanced suitability for high-temperature or prolonged ligation applications.

Figure 2. Thermostability assessment at 42°C and 45°C

2. Ultra-Low Adapter Dimerization: Minimal Self-Ligation with 0.5 ng gDNA in Library Prep

To evaluate adapter dimer formation, three batches of Yeasen Premium T4 DNA Ligase were compared against T4 DNA ligases from Supplier A and Supplier B. The experiment involved constructing whole-genome DNA libraries using 0.5 ng sheared bovine genomic DNA and Yeasen DNA Library Prep Kit (Cat# 12927ES).

Results:

Yeasen’s product exhibited significantly lower adapter dimerization rates compared to Supplier A and Supplier B, effectively minimizing non-specific ligation interference. This ensures higher yields of usable sequencing data and enhances the reliability of downstream analyses.

Figure 3. Adapter dimerization rates with 0 and 0.5 ng gDNA inputs.

3. Minimal Host gDNA Residuals: Ensuring Reliable Experimental Outcomes

The presence of residual host genomic DNA (gDNA) can significantly impact experimental results. To assess this, three batches of Premium T4 DNA Ligase were tested for E.coli host gDNA contamination using Yeasen E.coli Host Cell DNA Residual Detection Kit (Cat# 41318).

Results:

All three batches showed extremely low levels of host gDNA residuals, far below the standards set by imported brands. This confirms the high purity and reliability of Yeasen’s enzyme, ensuring clean and trustworthy experimental outcomes.

Figure 4. Host gDNA residual detection results for Premium T4 DNA Ligase.

4. No Nuclease Contamination: Ensuring Purity and Integrity

T ensure the absence of contaminating nucleases such as exonucleases or nicking enzymes, 6000 U of Premium T4 DNA Ligase was incubated with nucleic acid substrates and analyzed via agarose gel electrophoresis

Results:

Aalysis confirmed that all three batches of Premium T4 DNA Ligase showed no detectable exonuclease or nicking enzyme activity, verifying the absence of potential nuclease contamination. This guarantees the integrity of your samples and the reliability of your results. 

Figure 5. Exonuclease and nicking enzyme contamination test results for Premium T4 DNA Ligase. 

Applications: From Molecular Cloning to High-Throughput Genomics

With its dual advantages of high heat stability and low self-ligation, Premium T4 DNA Ligase is ideal for:

• Routine molecular cloning
• High-throughput or automated library prep
• Low-input NGS applications
• cfDNA and liquid biopsy workflows
• Challenging ligation reactions requiring long incubation
• Protocols involving room-temperature operations
• Whole-genome library construction

Whether you're building high-complexity libraries or running time-sensitive automation, Premium T4 DNA Ligase provides the reliability and precision needed to produce clean, high-quality data.

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