Synthetic Polynucleotides
Enzymic Synthesis of Ribonucleotide Terminated Oligodeoxynucleotides and Their Use as Primers for the Enzymic Synthesis of Polydeoxynucleotides
Note. The abbreviations and symbols presented in this paper follow recommendations of IUPAC‐IUB Commission on Biochemical Nomenclature, published in Eur. J. Biochem. 15 (1970) 203.
Abstract
- 1
The use of chemically synthesized hexathymidylate as primer for terminal deoxynucleotidyl transferase in the presence of a ribonucleoside triphosphate and Mg2+ results in a limited addition of ribonucleotides at the 3′‐end of the primer. The incorporation is absolutely dependent upon the presence of Mg2+ and primer, and the pH optimum is similar to that observed for deoxynucleotide incorporation.
- 2
The priming efficiency decreases when oligothymidylates of smaller chain length are used according to the order: hexa > penta > tetra > tri. The incorporation into the trinucleotide is very small.
- 3
Depending upon the primer: triphosphate ratio, the reaction mixture after incubation contains either a mono‐ or a diaddition product, or both and such products can be synthesized under controlled conditions. The upper limit of this terminal addition has been found to be up to two nucleotide units. A ribonucleotide to ribonucleotide linkage is formed in the case of the diaddition product.
- 4
The ribonucleotide terminated hexathymidylates are capable of accepting dAMP residues from dATP in the presence of terminal deoxynucleotidyl transferase to give rise to a polymeric product whereby the newly synthesized polymer remains covalently linked to the original hexathymidylate by a ribonucleotide linkage.
- 5
Due to the presence of this ribonucleotide linkage, the polymeric product can be cleaved from the primer by alkaline hydrolysis, resulting in a separation of the primer sequences from the product sequences. The use of ribonucleotide terminated oligodeoxynucleotides thus provides a valuable tool for enzymic synthesis of polydeoxynucleotides, whereby the material used as primer (necessary for an enzyme catalyzed reaction) can be removed from the product.
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