Study On Alignment Efficiency Of Four Different Ligation Methods: A Comparative Study

Abstract

Background: DNA ligation is a pivotal process in molecular cloning, enabling the covalent joining of DNA fragments. The efficiency of ligation depends on both the enzyme and reaction conditions, which directly impact downstream applications such as cloning and synthetic biology.

Methods: A total of 240 ligation reactions were conducted using four ligation systems, T4 DNA ligase (standard conditions), T4 DNA ligase with polyethylene glycol (PEG) enhancement, E. coli DNA ligase, Taq DNA ligase under thermostable conditions. Both cohesive-end and blunt-end DNA substrates were utilized. Ligation efficiency was assessed via gel electrophoresis and quantified using real-time PCR. Statistical comparisons were performed using one-way ANOVA (p < 0.001).

Results:T4 DNA ligase with PEG enhancement showed the highest efficiency, achieving 87.3 ± 4.2% for cohesive ends and 72.8 ± 5.1% for blunt ends. Standard T4 DNA ligase followed with 76.4 ± 3.8% (cohesive) and 58.2 ± 4.6% (blunt). E. coli DNA ligase exhibited moderate efficiency at 68.9 ± 4.3% and 42.1 ± 3.9% respectively. Taq DNA ligase was least effective, with 45.2 ± 3.7% for cohesive ends and negligible activity for blunt-end ligation. All inter-group comparisons were statistically significant (p < 0.001).

Conclusion: The use of PEG with T4 DNA ligase significantly enhances ligation efficiency, especially for blunt-end ligation. These findings support the strategic selection of ligation methods based on end-type and experimental goals in molecular biology workflows.