Abstract

The DNA duplex (designated [A]) corresponding to the nucleotides 1 to 20 of the major yeast alanine transfer RNA (Fig. 1) has been synthesized. The first step involved the T4 ligase-catalyzed joining of d-(5′- 32P)-C-C-G-G-A-A-T-C (segment 4, Fig. 1) to the dodecanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T (segment 1, Fig. 1), in the presence of the complementary decanucleotide d-(5′-OH)-C-C-G-G-A-C-T-C-G-T (segment 3, Fig. 1). The resulting icosanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T-C-C-G-G-A-A-T-C, was isolated free from the decanucleotide (segment 3). The synthesis of [A] was then completed by the ligase-catalyzed joining of 5′- 32P or 33P-labeled hexanucleotide d-(5′-P)-C-C-A-C-C-A (segment 2) to the 5′- 32P or 33P-labeled decanucleotide, d-(5′-P)-C-C-G-G-A-C-T-C-G-T (segment 3), in the presence of the above icosanucleotide. During the above work, the intermediate in the T4 ligase-catalyzed joining was identified as the pyrophosphate formed by the adenylation of the 5′-phosphate group of the oligodeoxynucleotide. Furthermore, the T4 ligase was found to bring about the joining reaction when the hydrogen-bonded duplexes contained mismatched (non-Watson-Crick) base pairs.

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