Increasing Overhang GC-Content Increases Sticky-End Ligation Efficiency

The ligation efficiency of sticky-ends differs between overhangs generated by different restriction enzymes. Differences between the composition of these overhangs include length and GC-content, thus indicating that these properties may play a role in ligation efficiency. Although ligases are a common and widely used tool in molecular biology, a method to accurately quantify ligation efficiency has yet to be fully developed and standardized. In this study, ligation efficiency of synthetic oligonucleotides with four-nucleotide overhangs containing varying GC-content was quantitatively investigated using qPCR. 2 sets of synthetic, double-stranded oligonucleotides with complementary overhangs were created. The complementary oligonucleotides were mixed with ligase, and the efficiency of ligation was quantified by measuring an increase in fluorescence intensity due to SYBR Green intercalation between amplified polymerase chain reaction products, where ligated oligonucleotide served as template. Using a control oligonucleotide, semi-quantitative qPCR analysis of ligations involving oligonucleotides with 0%, 50%, and 100% GC-content in their overhangs was developed. The ligation of oligonucleotides with overhangs containing 100% GC-content resulted in ~25% more ligated product than oligonucleotides with overhangs containing 0% GC-content. These findings suggest that oligonucleotides possessing overhangs with higher GC-content are more efficiently ligated.