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Abstract
T4 DNA ligase has been shown to perform ligation reactions more efficiently with HindIIIdigested λ DNA fragments than NdeI-digested λ fragment. The reasons for this observation are currently under investigation. One hypothesis is that ligation efficiency might be dependent on sequences adjacent to the restriction endonuclease nick site. To test this hypothesis, an experiment was designed to compare ligation efficiency between a control DNA fragment and a substrate with an insertion mutation of 3 nucleotides in length at the position five bases away from the Restriction endonuclease nick site. In order to generate these substrates, it was necessary to amplify a control and a mutagenized DNA fragments by polymerase chain reaction (PCR) and clone these inserts into pUC19 to produce plasmids pJW3 and pJW4. Several attempts had been made through a 1-step cloning procedure, but no clones were obtained. This cloning failure is potentially due to low restriction enzyme activity at the end of PCR-amplified DNA fragments. As a result, an alternative cloning strategy was employed to clone the inserts into intermediate products via single 3’-thymidine overhangs. This new strategy yielded 3 clones with the control insert and 2 clones containing the mutagenized insert, which were named pJW1 and pJW2 respectively. The successful generation of pJW1 and pJW2 will provide more convenient means to construct the pJW3 and the pJW4 plasmids. These plasmids will eventually allow the isolation of substrates needed for the ligation efficiency test.