Abstract
The DNA duplex (designated [B] and shown in Fig. 1), corresponding to the middle part (nucleotide sequence 17 to 50) of the major yeast alanine transfer RNA has been synthesized. The duplex contained short single-stranded regions at both ends for subsequent joining to the [A](paper CXII) and [C](paper CXIV) parts of the total DNA duplex. The T4 ligase was shown to bring about the joining of d-(5′- 32P)-C-T-A-A-G (segment 9) to the icosanucleotide (segment 6) in the presence of the complementary segment 8 (Fig. 1); similarly, d-(5′- 32P)-A-G-A-G-T-C-T (segment 7) could be joined efficiently to segment 8 in the presence of segment 6. The synthesis of [B] containing the five components (segments 5 to 9) was carried out as follows: the joining of segment 9 to segment 6 and that of segment 7 to segment 8 was carried out in one step at 15 °C. Without isolation of the resulting duplex, 5′- 32P-labeled segment 5 was added and the incubation continued at 25 °C. The joining of segment 7 was complete at this temperature. As required for joining [B] to the two terminal parts, [A] and [C] of the DNA, [B] as obtained above was enzymically phosphorylated at the two 5′-terminal deoxyguanosine units using γ-labeled ATP and polynucleotide kinase and the product was isolated by gel-filtration.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.