JBC, Vol. 251, Issue 3, 651-657, Feb, 1976
Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 9. Enzymatic joining of chemically synthesized deoxyribopolynucleotide segments corresponding to nucleotide sequence 57-94
A. Panet, R. Kleppe, K. Kleppe and H. G. Khorana
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.
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