Joining of short DNA oligonucleotides with base pair mismatches by T4 DNA ligase

Oligonucleotide-directed mutagenesis is a widely used method for studying e nzymes and improving their properties. The number of mutants that can be ob tained with this method is limited by the number of synthetic 25-30mer olig onucleotides containing the mutation mismatch, becoming impracticably large with increasing size of a mutant Library. To make this approach more pract ical, shorter mismatching oligonucleotides (7-12mer) might be employed. How ever, the introduction of these oligonucleotides in dsDNA poses the problem of sealing a DNA nick containing 5'-terminal base pair mismatches. In the present work we studied the ability of T4 DNA ligase to catalyze this react ion. It was found that T4 DNA ligase effectively joins short oligonucleotid es, yielding dsDNA containing up to five adjacent mismatches. The end-joini ng rate of mismatching oligonucleotides is limited by the formation of the phosphodiester bond, decreasing with an increase in the number of mismatchi ng base pahs at the 5'-end of the oligonucleotide substrate, However, in th e case of a 3 bp mismatch, the rate is higher than that obtained with a 2 b p mismatch. Increasing the matching length with the number of mismatching b ase pairs fixed, or moving the mismatching motif downstream with respect to the joining site increases the rate of ligation, The ligation rate increas es with the molar ratio [oligonucleotide:dsDNA]; however, at high excess of the oligonucleotide, inhibition of joining was observed. In conclusion, 9m er oligonucleotides containing a 3 bp mismatch are found optimal substrates to introduce mutations in dsDNA, opening perspectives for the application of T4 DNA Ligase in mutagenesis protocols.