Nucleotide Composition Immediately Flanking the DNA Overhangs Generated by Restriction Endonucleases Was Not Shown to Contribute to the Ligation Efficiency of T4 DNA Ligase

Abstract

The apparent efficiency of T4 DNA ligase has been observed to vary for fragments cut by different restriction enzymes. In this study, restriction enzymes that leave the same overhang were used to investigate whether nucleotide content immediately flanking the overhangs generated by the restriction endonuclease digestion affect the ligation efficiency of T4 DNA ligase on pBR322 and pACYC177. Two sets of enzymes were used to digest the plasmids, one set resulted in G,C nucleotides immediately flanking the staggered ends, while the other set had A,T nucleotides. Our results suggest that on average T4 ligase showed higher calculated number for ligation efficiencies of fragments cut by G,C-enzymes than A,Tenzymes. However, the small sample size limited our ability to obtain statistically significant results.