The synthesis of oligodeoxyribonucleotides using RNA ligase.
T4 RNA ligase catalyzes the addition of a single deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of oligodeoxyribonucleotides (Hinton et al. (1978) Biochemistry 17, 5091). We have determined improved conditions for this reaction which give yields equal to or greater than 85% when any of five common deoxyribonucleoside bisphosphate (pdAp, pdCp, pdGp, pdTp, or pdUp) are added to dA(PDA)4. A low ATP concentration, which is constantly maintained by a regeneration system composed of phosphocreatine, creatine kinase, and myokinase, contributes to the attainment of high yields. The addition of RNase A and spermine also enhances the rates and yields of the reactions. These conditions facilitate the use of RNA ligase as a reagent for the stepwise synthesis of DNA of defined sequence.
D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008722:Methods; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis*; D009841:Oligonucleotides / Q000096:biosynthesis*; D011118:Polynucleotide Ligases / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000378:metabolism; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology
D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D004247:DNA; D011118:Polynucleotide Ligases; D011130:RNA Ligase (ATP)