Fidelity of DNA ligation: a novel experimental approach based on the polymerisation of libraries of oligonucleotides.
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.
Housby
J N
JN
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. housby@bioch.ox.ac.uk
Southern
E M
EM
D017104:Bacteriophage M13 / Q000737:chemistry; D020029:Base Pairing; D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism*; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D016208:Databases, Factual; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism*; D013698:Templates, Genetic; D016963:Thermus thermophilus / Q000201:enzymology*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D011088:DNA Ligases