Nucleic Acids Research, Vol 26, Issue 18 4259-4266, Copyright © 1998 by Oxford University Press
JN Housby and EM Southern
Complete libraries of oligonucleotides were used as substrates for Thermus
thermophilus DNA ligase, on a M13mp18 ssDNA template. A 17mer primer was
used to start a polymerisation process. Ladders of ligation products were
analysed by gel electrophoresis. Octa-, nona- and decanucleotide libraries
were compared. Nonanucleotides were optimum for polymerisation and up to 15
monomers were ligated. The fidelity of incorporation was studied by
sequencing 28 clones (2268 bases) of nonanucleotide polymers, 12 monomers
in length. Of the ligated monomers, 79% were the correct complementary
sequence. In a total of 57 (2.5%) mispaired bases, there was a strong bias
to G.T, G.A, G.G and A.G mismatches. Of the mismatches, 86% were found to
be purines on the incoming oligonucleotide, of which 71% were G. There is
evidence for clustering of mismatches within specific 9mers and at specific
positions within these 9mers. The most frequent mismatches were at the
5'-terminus of the oligonucleotide, followed by the central position. We
suggest that sequence selection was imposed by the ligase and not just by
base pairing interactions. The ligase directs polymerisation in the 3' to
5' direction which we propose is linked to its role in lagging strand DNA
replication.
ARTICLES
Fidelity of DNA ligation: a novel experimental approach based on the polymerisation of libraries of oligonucleotides
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. housby@bioch.ox.ac.uk
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