Development of a Real-Time Polymerase Chain Reaction Method to Measure Ligation Efficiency

09/05/2015

Jessica Qiu, Priscilla Chen, Serena Lin

Department of Microbiology and Immunology, University of British Columbia

Volume 19
Fall 2014 / Winter 2015

An efficient method to measure ligation efficiency would be a useful tool in experiments that use the ligase enzyme. In this study, real-time polymerase chain reaction (qPCR) was used to determine whether the ligation of synthetic oligonucleotides by T4 DNA ligase had occurred. qPCR is a technique used to simultaneously amplify and quantify DNA, and is widely used in many research settings for gene expression analysis. As an alternative to gel electrophoresis, qPCR was used to confirm ligation since it not only requires less time to produce results, but also is a more sensitive assay capable of detecting a few femtogram quantities of DNA. A second objective was to determine the effect of cohesive ends’ GC (guanine and cytosine) content on the ligation efficiency of T4 DNA ligase. The results of the initial experimental setup resulted in no amplification, which may be due to complementary 5’ and 3’ ends of ligated oligonucleotides forming concatemers. Modified oligonucleotide sequences resulted in DNA amplification during qPCR, indicating that ligation had occurred. qPCR can be used as a method to analyze ligation by T4 DNA ligase, and can be utilized in further experiments to determine ligation efficiencies of cohesive ends with different GC content. However, caution must be taken when designing oligonucleotides to avoid unfavourable formation of DNA structures that may hinder the amplification of DNA during qPCR.