Ligation of Hexamers on Hexamer Templates to Produce Primers for Cycle Sequencing or the Polymerase Chain Reaction
✍ Scribed by J.J. Dunn; L.L. Butlerloffredo; F.W. Studier
- Publisher
- Elsevier Science
- Year
- 1995
- Tongue
- English
- Weight
- 954 KB
- Volume
- 228
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
A method is described for the ordered ligation of hexanucleotides (hexamers) in solution to produce unique longer oligonucleotides. To form an 18-mer, for example, six different hexamers are selected that can base pair unambiguously to form a double-stranded complex of indefinite length. In the most efficient arrangement, each hexamer forms three complementary base pairs with two other hexamers, generating complementary chains of contiguous hexamers with strand breaks staggered by three bases. Two adjacent hexamers in one chain contain (5^{\prime}) phosphate groups and the others are unphosphorylated. Both (\mathrm{T} 4) and (\mathrm{T} 7) DNA ligase can ligate the phosphorylated hexamers to their neighbors in such a complex at hexamer concentrations in the 50-100 mM range, producing an 18-mer and leaving three unphosphorylated hexamers. Twenty-nine of 34 complexes that satisfied the requirements for unambiguous ligation generated the desired 18-mers, which could be used directly for cycle sequencing or, after removal of the unreacted hexamers, for polymerase chain reactions (PCR). Comparable ligation reactions also produced 12 -, 24 -, and 30 -mers. With a library of all 4096 possible hexamers, unambiguous ligation has the potential to produce more than (82 %) of all possible 18 -mers and could readily supply the oligonucleotides needed for DNA sequencing by primer walking, for PCR, or for gene synthesis. 1995 Academic Press, Inc.