BLUNT-END AND SINGLE-STRAND LIGATIONS BY ESCHERICHIA-COLI LIGASE - INFLUENCE ON AN INVITRO AMPLIFICATION SCHEME

A ligase-based, in vitro DNA amplication system (LAR) has been described by Wu and Wallace [Genomics 4 (1989) 560-569]. This strategy is based on the ability of a DNA ligase to join the 5′ phosphate of one DNA molecule to the 3′ hydroxyl of a second during a nick-closing reaction. Escherichia coli DNA ligase has been used in place of the T4 DNA ligase in our study in order to limit template-independent ligation activities, which lower the sensitivity of this amplication procedure. The results of this studyes indicate that E. coli ligase also joins blunt-ended DNA molecules and some single-stranded oligodeoxyribonucleotides, in the absence of a complementary template, with an efficiency which is sensitive to both the concentrations of DNA substrate and enzyme. © 1990.