Abstract
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. Two 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. A non-ligated control was included, and overhangs with 0%, 50%, and 100% GC content were compared. The 100% GC-content overhang displayed approximately 25% greater ligation efficiency than lower GC-content overhangs, suggesting that higher GC content may promote more efficient ligation.