Sequence analysis of polyribonucleotides by stepwise chemical degradation. Method for the introduction of radioactive label into nucleoside fragments after cleavage

Stepwise degradation of polyribonucleotides

“…Gel filtration on a column of beaded polyacrylamide fulfills the requirements of such a system in that it allows the rapid separation of the oligonucleotide together with the phosphatase from the other components of the degradation reaction: amine buffer, iodate, rhamnose, and its oxidation products, and the nucleoside fragment (Figure 2). The nucleoside fragment, at this stage, is presumed to be a morpholine derivative (Schwartz and Gilham, 1972) and, as such, tends to bind ionically to the column by virtue of the partial acidic character of the polyacrylamide gel. Thus, it is necessary to use elution with dilute acetic acid to recover the fragment, and the column then requires neutralization with sodium acetate solution prior to the next separation.…”

“…6 These products correspond to the dialcohol derivatives formed by treating the nucleotide with sodium periodate at 0°and then with sodium borohydride in the absence of a primary amine. 6 These products correspond to the methylene dialcohol derivatives (Schwartz and Gilham, 1972) formed by oxidation with periodate and the removal of the excess with rhamnose, followed by treatment of the reaction mixture at 45 °in the absence of primary amines and the subsequent reduction of the product with sodium borohydride.…”

“…A cycle of degradation using this procedure consists of (i) the periodate oxidation of the polynucleotide at 0°, (ii) the removal of the excess periodate at 0°with rhamnose, (iii) the amine-catalyzed removal of the oxidized nucleoside moiety and the enzymatic dephosphorylation of the polynucleotide in the same reaction mixture at 45°, (iv) the separation of the dephosphorylated polynucleotide from the nucleoside fragment, and (v) the identification of the purine or pyrimidine base contained in the nucleoside fragment. This degradation procedure has several advantages over the earlier approach in that (i) the total reaction time is reduced, (ii) the polynucleotide is not exposed to periodate at the high temperature during the /3-elimination step thus avoiding possible side reactions, (iii) the released nucleoside fragment contains all of the original carbon atoms of the ribose moiety thus permitting the introduction of radioactive label into the fragment to increase sensitivity in sequence analysis applications (Schwartz and Gilham, 1972), and (iv) the cycles of degradation are controlled by the presence or absence of periodate and by the two temperatures used, and this allows a number of degradation cycles to be specifically carried out in one reaction mixture. The present work contains the details of this degradation procedure and its application to adenosine oligonucleotides and tRNA fragments.…”

Continuous directional degradation — a novel method for sequence analysis of polyribonucleotides

“…Nucleoside trialcohols were prepared as described [5] . Nucleoside methylene dialcohols [9] were prepared as follows: Nucleotide dialdehydes were obtained by treating ribonucleoside-5' monophosphates (0.01 M) with NaI04 (0.02 M) at 23" for 30 min. Ethylene glycol was added (final cont.…”

“…Nucleoside trialcohols were prepared as described [5] . Nucleoside methylene dialcohols [9] were prepared as follows: Nucleotide dialdehydes were obtained by treating ribonucleoside-5' monophosphates (0.01 M) with NaI04 (0.02 M) at 23" for 30 min. (see Experimental), three radioactive compounds were detected on chromatograrns by fluorographic visualization; these compounds co-chromatographed with G", U" and A'.…”