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J. Biochem, 2001, Vol. 129, No. 1 61-68
© 2001 Japanese Biochemical Society

other

Joining of Short DNA Oligonucleotides with Base Pair Mismatches by T4 DNA Ligase1

Alexei Cherepanov, Eseng{diaeresis}l Yildirim and Simon de Vries2

Kluyver Department of Biotechnology, Delft University of Technology Julianalaan 67, 2628 BC Delft, The Netherlands

2To whom correspondence should be addressed. Phone: +31-15-2785139, Fax: +31-15-2782355, E-mail: S.deVries{at}tnw.tudelft.nl

Oligonucleotide-directed mutagenesis is a widely used method for studying enzymes and improving their properties. The number of mutants that can be obtained with this method is limited by the number of synthetic 25–30mer oligonucleotides containing the mutation mismatch, becoming impracticably large with increasing size of a mutant library. To make this approach more practical, shorter mismatching oligonucleotides (7– 12mer) might be employed. However, 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 reaction. It was found that T4 DNA ligase effectively joins short oligonucleotides, yielding dsDNA containing up to five adjacent mismatches. The end-joining rate of mismatching oligonucleotides is limited by the formation of the phosphodiester bond, decreasing with an increase in the number of mismatching base pairs at the 5'-end of the oligonucleotide substrate. However, in the case of a 3 by mismatch, the rate is higher than that obtained with a 2 by mismatch. Increasing the matching length with the number of mismatching base pairs fixed, or moving the mismatching motif downstream with respect to the joining site increases the rate of ligation. The ligation rate increases with the molar ratio [oligonucleotide: dsDNA]; however, at high excess of the oligonucleotide, inhibition of joining was observed. In conclusion, 9mer oligonucleotides containing a 3 by mismatch are found optimal substrates to introduce mutations in dsDNA, opening perspectives for the application of T4 DNA ligase in mutagenesis protocols.

1This work was supported by the Association of Biotechnology Centers in The Netherlands (ABON) (Project 1.2.8) and in part by the Netherlands Research Council for Chemical Sciences (CW), with financial aid from the Netherlands Technology Foundation (STW) (grant 349-3565).


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