The DNA duplexes representing nucleotide sequences 61-89 and 57-94 have been synthesized, isolated pure, and fully characterized. Synthesis of the duplex with the nucleotide sequence 61-89 involved the DNA ligase-catalyzed joining of chemically synthesized deoxyoligonucleotide segments 14 to 18 shown in Fig. 1A, while for the longer duplex (sequences 57-94) seven deoxyribooligonucleotides (segments 13 to 19, Fig. 1B) were used in one-step enzymatic joining. The joining of the short tetranucleotide (segment 16) to the segment 17 required the presence of the adjacent segment 14, even if the latter did not contain a 5'-phosphate group, to allow its joining to segment 16. However, in the synthesis of both of the DNA duplexes, the yields were comparatively low (30 to 40%) and could not be significantly increased although a variety of conditions was tried. The main cause in both cases evidently was the sluggish joining of segment 14 to 16 and of segment 16 to segment 17. Although the original plan for the total synthesis of this part of the gene for the tRNA precursor involved the DNA duplex consisting of segments 14 to 18, this duplex could not be quantitatively phosphorylated at the two 5'-OH ends for subsequent joining to the adjoining parts of the gene. The DNA duplex consisting of segments 13 to 19, which possesses both terminal 5'-OH groups at protruding single-stranded ends, was readily phosphorylated and used successfully in the total synthesis of the gene as described in an accompanying paper.