Journal Article

High sequence fidelity in a non-enzymatic DNA autoligation reaction

Yanzheng Xu and Eric T. Kool

in Nucleic Acids Research

Volume 27, issue 3, pages 875-881
Published in print February 1999 | ISSN: 0305-1048
Published online February 1999 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/27.3.875
High sequence fidelity in a non-enzymatic DNA autoligation reaction

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The success of oligonucleotide ligation assays in probing specific sequences of DNA arises in large part from high enzymatic selectivity against base mismatches at the ligation junction. We describe here a study of the effect of mismatches on a new nonenzymatic, reagent-free method for ligation of oligonucleotides. In this approach, two oligonucleotides bound at adjacent sites on a complementary strand undergo autoligation by displacement of a 5′-end iodide with a 3′-phosphorothioate group. The data show that this ligation proceeds somewhat more slowly than ligation by T4 ligase, but with substantial discrimination against single base mismatches both at either side of the junction and a few nucleotides away within one of the oligonucleotide binding sites. Selectivities of >100-fold against a single mismatch are observed in the latter case. Experiments at varied concentrations and temperatures are carried out both with the autoligation of two adjacent linear oligonucleotides and with intramolecular autoligation to yield circular ‘padlock’ DNAs. Application of optimized conditions to discrimination of an H-ras codon 12 point mutation is demonstrated with a single-stranded short DNA target.

Journal Article.  4491 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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